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Dummy builder generates no files." diff --git a/docs/OvlpHists.rst b/docs/OvlpHists.rst new file mode 100644 index 00000000..50f93ccf --- /dev/null +++ b/docs/OvlpHists.rst @@ -0,0 +1,34 @@ +.. _OvlpHists: + +.. image:: media/OvlpHist.png + :height: 500px + :width: 1000 px + :alt: Pread Ovlp Histogram + :align: left + + +Example R Code +-------------- + +.. code-block:: R + + library(ggplot2) + o<-read.table("ovlp.stats", header=F) + colnames(o)<-c("pread","length","fivePrimeOvlps","threePrimeOvlps") + + pdf(file="OvlpHist.pdf", width=11, height=5) + par(oma=c(3,3,2,0), cex=1.6, las=1, mar=c(4,4,2,2), mfrow=c(1,2)) + hist(o$fivePrimeOvlps, breaks=100, + xlab="number of overlaps between preads", + ylab="count", main="Five Prime") + hist(o$threePrimeOvlps, breaks=100, + xlab="number of overlaps between preads", + ylab="count", main="Three Prime") + dev.off() + + + +Example Input Files +------------------- + +:download:`ovlp.stats ` diff --git a/docs/Rhists.rst b/docs/Rhists.rst new file mode 100644 index 00000000..6df6cc8a --- /dev/null +++ b/docs/Rhists.rst @@ -0,0 +1,46 @@ +.. _RHists: + +.. image:: media/RawReadHist.png + :height: 700px + :width: 700 px + :alt: Raw Read Histogram + :align: center + +.. image:: media/PreadHist.png + :height: 700px + :width: 700 px + :alt: PRead Histogram + :align: center + + +Example R Code +-------------- + +.. code-block:: R + + setwd("my/dir") + raw<-read.table("raw_reads.stats.txt", header=T) + preads<-read.table("preads.stats.txt",header=T) + + pdf(file="RawReadHist.pdf", width=11, height=8.5) + par(oma=c(4,4,2,0), cex=1.6, las=1, mar=c(4,4,2,2)) + plot(data=raw, Count~Bin, type="h",col="DeepSkyBlue", lwd=5, + ylab="", xlab="Read Length", main="Raw Reads") + mtext("Read Count", side=2, cex=1.7, las=3, line=4) + dev.off() + + pdf(file="PreadHist.pdf", width=11, height=8.5) + par(oma=c(4,4,2,0), cex=1.6, las=1, mar=c(4,4,2,2)) + plot(data=preads, Count~Bin, type="h",col="ForestGreen", lwd=5, + ylab="", xlab="Read Length", main="Preassembled Reads") + mtext("Read Count", side=2, cex=1.7, las=3, line=4) + dev.off() + + + +Example Input Files +------------------- + +:download:`raw_reads.stats.txt ` + +:download:`preads.stats.txt ` diff --git a/docs/about.rst b/docs/about.rst new file mode 100644 index 00000000..21c21950 --- /dev/null +++ b/docs/about.rst @@ -0,0 +1,132 @@ +.. caution:: These documents refer to an obsolete way of installing and running FALCON. They will remain up for historical context and for individuals still using the older version of FALCON/FALCON_unzip. + +.. attention:: The current PacBio Assembly suite documentation which includes new bioconda instructions for installing FALCON, FALCON_unzip and their associated dependencies can be found here `pb_assembly `_ + + +.. image:: media/falcon_icon2.png + :height: 200px + :width: 200 px + :alt: FALCON Assembler + :align: right + + +.. _about: + +About FALCON +============ + +Overview +-------- + +``FALCON`` and ``FALCON-Unzip`` are *de novo* genome assemblers for PacBio long reads, also known as +single-molecule real-time (SMRT) sequences. ``FALCON`` is a diploid-aware assembler +which follows the hierarchical genome assembly process (HGAP_) and is optimized for +large genome assembly (e.g. non-microbial). ``FALCON`` produces a set of :term:`primary contigs `(p-contigs), +which represent the backbone of the genome sequence, as well as :term:`associate contigs ` (a-contigs), +which represent divergent allelic variants. Each a-contig is associated with a homologous +genomic region on an p-contig. + +``FALCON-Unzip`` is a true diploid assembler. It takes the contigs from +``FALCON`` and phases the reads based on heterozygous SNPs identified in the initial +assembly. It then produces a set of partially phased :term:`primary contigs ` and fully phased +:term:`haplotigs ` which represent divergent haplotyes. + + +Detailed Description +-------------------- + +The hierarchical genome assembly process proceeds in two rounds. The first round of assembly involves the selection of seed reads, +or the longest reads in the dataset (user-defined :ref:`length_cutoff `). All shorter reads are aligned to +the seed reads, in +order to generate consensus sequences with high accuracy. We refer to these as pre-assembled reads but they can also be +thought of as +“error corrected” reads. During the pre-assembly process, seed reads may be split or trimmed at regions of low read +coverage (user-defined `min_cov` for :ref:`falcon_sense_option `). The performance of the pre-assembly +process is captured in the `pre-assembly stats file. +`_ + +In the next round of HGAP, the :term:`preads `, are aligned to each other and assembled into +genomic contigs. + +.. image:: media/HGAP.png + +.. image:: media/Fig1.png + +For more complex genomes assembled with ``FALCON``, +"bubbles" in the contig-assembly graph that result from structural variation between haplotypes may be resolved as associate +and primary contigs. The unzip process will extend haplotype phasing beyond "bubble" regions, increasing the amount of phased +contig sequence. It is important to note that +while individual haplotype blocks are phased, phasing does not extend **between** haplotigs. Thus, in part **C)** of the +figure above, **haplotig_1** and **haplotig_2** may originate from different parental haplotypes. Additional information is +needed to phase the haplotype blocks with each other. + +Associate contig IDs contain the name of their primary contig but the precise location of alignment must be determined with third party +tools such as NUCmer_. For example, in a ``FALCON`` assembly, `000123F-010-01` is an associated contig to primary contig +`000123F`. In a ``FALCON-Unzip`` assembly, `000123F_001` is a haplotig of primary contig `000123F`. + +Below are examples of alignments between associate and primary contigs from ``FALCON``, and haplotigs and primary contigs +from ``FALCON-Unzip``. Alignments were built with NUCmer_ and visualized with Assemblytics_. Precise coordinates +may be obtained with the show-coords_ utilty from MUMmer_. + +.. image:: media/dotplots.png + + +Choosing an Assembler: HGAP4 vs FALCON vs FALCON-Unzip +------------------------------------------------------ + +HGAP4 +~~~~~ + +We recommend ``HGAP4``, part of the SMRT Link web-based analysis suite, for genomes of known complexity, no larger than +human (3Gb or +smaller), +although underlying +compute resources for your SMRT Link instance will influence performance and feasibility. The assembly +process for ``HGAP4`` in the SMRT Link GUI (graphical user interface) is identical to ``FALCON`` at the command line, besides +differences in +compute resource configuration and minor differences in directory structure. The ``HGAP4`` pipeline by default includes a round of +genome "polishing" +which employs the ``resequencing`` pipeline. + +``HGAP4`` RESULTS ARE NOT COMPATIBLE WITH ``FALCON-Unzip`` AT THIS TIME! + + +``HGAP4`` inputs are a PacBio subread BAM_ dataset, either Sequel or RSII. The FASTA and FASTQ files output from ``HGAP4`` are a concatenation of the primary +and associate contigs, which are output from ``FALCON`` as separate files. + + +Command Line +~~~~~~~~~~~~ + +Users more comfortable at the command line may use ``FALCON`` for genomes of any size +or complexity. Command line inputs are FASTA files of Sequel or RSII subreads. Command-line ``FALCON`` does not automatically polish the assembly. If a user +wishes, assembly polishing may +be run using the ``resequencing`` pipeline of pbsmrtpipe_ (available for command-line installation using the SMRT_Link_ download, see +SMRT_Tools_Reference_Guide_ for +installation instructions). Resequencing requires PacBio subread BAM_ inputs. + +We recommend the ``FALCON-Unzip`` module for heterozygous or outbred organisms that are diploid or higher ploidy. Users wishing to run +``FALCON-Unzip`` must do so only after running ``FALCON`` on the +command line. ``HGAP4`` IS NOT COMPATIBLE WITH ``FALCON-UNZIP``! The ``FALCON-Unzip`` module requires both FASTA and PacBio BAM_ inputs for subreads. + + +References +---------- + +`Chin et al. (2016). Phased diploid genome assembly with single-molecule real-time sequencing. Nature Methods. 13(12), 1050. +`_ + +`Chin, et al. (2013). Nonhybrid, finished microbial genome assemblies from long-read SMRT sequencing data. Nature Methods. 10(6), 563. +`_ + + +.. _HGAP: http://www.nature.com/nmeth/journal/v10/n6/full/nmeth.2474.html +.. _NUCmer: http://mummer.sourceforge.net/manual/#nucmer +.. _assemblytics: http://qb.cshl.edu/assemblytics/ +.. _MUMmer: http://mummer.sourceforge.net/manual/ +.. _show-coords: http://mummer.sourceforge.net/manual/#coords +.. _pbsmrtpipe: http://pbsmrtpipe.readthedocs.io/en/master/getting_started.html +.. _SMRT_Link: http://www.pacb.com/support/software-downloads/ +.. _SMRT_Tools_Reference_Guide: http://programs.pacificbiosciences.com/l/1652/2017-02-01/3rzxn6/184345/SMRT_Tools_Reference_Guide__v4.0.0_.pdf +.. _BAM: http://pacbiofileformats.readthedocs.io/en/3.0/BAM.html diff --git a/docs/cfgs/fc_run_arabidopsis.cfg b/docs/cfgs/fc_run_arabidopsis.cfg new file mode 100644 index 00000000..b68da509 --- /dev/null +++ b/docs/cfgs/fc_run_arabidopsis.cfg @@ -0,0 +1,34 @@ +[General] +input_fofn = input2.fofn + +input_type = raw + +# The length cutoff used for seed reads used for initial mapping +length_cutoff = 5000 + +# The length cutoff used for seed reads usef for pre-assembly +length_cutoff_pr = 5000 + + +sge_option_da = -pe smp 4 -q bigmem +sge_option_la = -pe smp 20 -q bigmem +sge_option_pda = -pe smp 6 -q bigmem +sge_option_pla = -pe smp 16 -q bigmem +sge_option_fc = -pe smp 24 -q bigmem +sge_option_cns = -pe smp 8 -q bigmem + +pa_concurrent_jobs = 192 +cns_concurrent_jobs = 192 +ovlp_concurrent_jobs = 192 + +pa_HPCdaligner_option = -v -dal128 -e0.75 -M24 -l2500 -k18 -h1250 -w8 -s100 +ovlp_HPCdaligner_option = -v -dal128 -M24 -k24 -h1250 -e.96 -l1500 -s100 + +pa_DBsplit_option = -a -x500 -s200 +ovlp_DBsplit_option = -s200 + + +falcon_sense_option = --output_multi --output_dformat --min_idt 0.70 --min_cov 4 --max_n_read 400 --n_core 8 +falcon_sense_skip_contained = False + +overlap_filtering_setting = --max_diff 120 --max_cov 120 --min_cov 4 --n_core 12 diff --git a/docs/cfgs/fc_run_bird.cfg b/docs/cfgs/fc_run_bird.cfg new file mode 100644 index 00000000..6f2ff6a7 --- /dev/null +++ b/docs/cfgs/fc_run_bird.cfg @@ -0,0 +1,29 @@ +[General] +input_fofn = input.fofn + +input_type = raw + +length_cutoff = 10000 + +length_cutoff_pr = 10000 + +sge_option_da = -pe smp 4 -q bigmem +sge_option_la = -pe smp 20 -q bigmem +sge_option_pda = -pe smp 6 -q bigmem +sge_option_pla = -pe smp 16 -q bigmem +sge_option_fc = -pe smp 24 -q bigmem +sge_option_cns = -pe smp 8 -q bigmem + +pa_concurrent_jobs = 192 +cns_concurrent_jobs = 192 +ovlp_concurrent_jobs = 192 + +pa_HPCdaligner_option = -v -dal128 -t16 -e0.75 -M24 -l3200 -k18 -h480 -w8 -s100 +ovlp_HPCdaligner_option = -v -dal128 -M24 -k24 -h1024 -e.96 -l2500 -s100 + +pa_DBsplit_option = -a -x500 -s200 +ovlp_DBsplit_option = -s200 + +falcon_sense_option = --output_multi --output_dformat --min_idt 0.70 --min_cov 4 --max_n_read 200 --n_core 8 + +overlap_filtering_setting = --max_diff 120 --max_cov 120 --min_cov 2 --n_core 12 diff --git a/docs/cfgs/fc_run_dipteran.cfg b/docs/cfgs/fc_run_dipteran.cfg new file mode 100644 index 00000000..ffce35ff --- /dev/null +++ b/docs/cfgs/fc_run_dipteran.cfg @@ -0,0 +1,38 @@ +[General] +input_fofn = input.fofn + +input_type = raw + +#openending = True + +length_cutoff = -1 + +genome_size = 1800000000 +seed_coverage = 30 + +length_cutoff_pr = 1000 + +sge_option_da = -pe smp 5 -q bigmem +sge_option_la = -pe smp 20 -q bigmem +sge_option_pda = -pe smp 6 -q bigmem +sge_option_pla = -pe smp 16 -q bigmem +sge_option_fc = -pe smp 24 -q bigmem +sge_option_cns = -pe smp 12 -q bigmem + +pa_concurrent_jobs = 96 +cns_concurrent_jobs = 96 +ovlp_concurrent_jobs = 96 + + +pa_HPCdaligner_option = -v -B128 -t16 -M32 -e.70 -l6400 -s100 -k18 -h480 -w8 +ovlp_HPCdaligner_option = -v -B128 -M32 -h1024 -e.96 -l2400 -s100 -k24 + + +pa_DBsplit_option = -x500 -s400 +ovlp_DBsplit_option = -s400 + + +falcon_sense_option = --output_multi --min_idt 0.70 --min_cov 2 --max_n_read 200 --n_core 8 +falcon_sense_skip_contained = True + +overlap_filtering_setting = --max_diff 100 --max_cov 100 --min_cov 2 --n_core 12 diff --git a/docs/cfgs/fc_run_ecoli.cfg b/docs/cfgs/fc_run_ecoli.cfg new file mode 100644 index 00000000..1a870d63 --- /dev/null +++ b/docs/cfgs/fc_run_ecoli.cfg @@ -0,0 +1,64 @@ +[General] +#job_type = SGE + +# list of fasta files +input_fofn = input.fofn + +# input type, raw or pre-assembled reads (preads, error corrected reads) +input_type = raw +#input_type = preads + +# The length cutoff used for seed reads used for error correction. +# "-1" indicates FALCON should calculate the cutoff using +# the user-defined genome length and coverage cut off +# otherwise, user can specify length cut off in bp (e.g. 2000) +length_cutoff = 15000 +genome_size = 4652500 +#seed_coverage = 30 + +# The length cutoff used for overalpping the preassembled reads +length_cutoff_pr = 12000 + +## resource usage ## +jobqueue = bigmem +# grid settings for... +# daligner step of raw reads +sge_option_da = -pe smp 5 -q %(jobqueue)s +# las-merging of raw reads +sge_option_la = -pe smp 20 -q %(jobqueue)s +# consensus calling for preads +sge_option_cns = -pe smp 12 -q %(jobqueue)s +# daligner on preads +sge_option_pda = -pe smp 6 -q %(jobqueue)s +# las-merging on preads +sge_option_pla = -pe smp 16 -q %(jobqueue)s +# final overlap/assembly +sge_option_fc = -pe smp 24 -q %(jobqueue)s + +# job concurrency settings for... +# preassembly +pa_concurrent_jobs = 48 +# consensus calling of preads +cns_concurrent_jobs = 48 +# overlap detection +ovlp_concurrent_jobs = 48 + +# daligner parameter options for... +# https://dazzlerblog.wordpress.com/command-guides/daligner-command-reference-guide/ +# initial overlap of raw reads +pa_HPCdaligner_option = -v -B4 -t16 -e.70 -l1000 -s1000 +# overlap of preads +ovlp_HPCdaligner_option = -v -B4 -t32 -h60 -e.96 -l500 -s1000 + +# parameters for creation of dazzler database of... +# https://dazzlerblog.wordpress.com/command-guides/dazz_db-command-guide/ +# raw reads +pa_DBsplit_option = -x500 -s50 +# preads +ovlp_DBsplit_option = -x500 -s50 + +# settings for consensus calling for preads +falcon_sense_option = --output_multi --min_idt 0.70 --min_cov 4 --max_n_read 200 --n_core 6 + +# setting for filtering of final overlap of preads +overlap_filtering_setting = --max_diff 100 --max_cov 100 --min_cov 20 --bestn 10 --n_core 24 diff --git a/docs/cfgs/fc_run_ecoli_local.cfg b/docs/cfgs/fc_run_ecoli_local.cfg new file mode 100644 index 00000000..61e72d02 --- /dev/null +++ b/docs/cfgs/fc_run_ecoli_local.cfg @@ -0,0 +1,67 @@ +[General] +job_type = local + +# list of fasta files +input_fofn = input.fofn + +# input type, raw or pre-assembled reads (preads, error corrected reads) +input_type = raw +#input_type = preads + +# The length cutoff used for seed reads used for error correction. +# "-1" indicates FALCON should calculate the cutoff using +# the user-defined genome length and coverage cut off +# otherwise, user can specify length cut off in bp (e.g. 2000) +length_cutoff = -1 +genome_size = 4652500 +seed_coverage = 30 + +# The length cutoff used for overalpping the preassembled reads +length_cutoff_pr = 12000 + +## resource usage ## +# need this line even if running local +jobqueue = your_queue +# grid settings for... +# daligner step of raw reads +sge_option_da = -pe smp 5 -q %(jobqueue)s +# las-merging of raw reads +sge_option_la = -pe smp 20 -q %(jobqueue)s +# consensus calling for preads +sge_option_cns = -pe smp 12 -q %(jobqueue)s +# daligner on preads +sge_option_pda = -pe smp 6 -q %(jobqueue)s +# las-merging on preads +sge_option_pla = -pe smp 16 -q %(jobqueue)s +# final overlap/assembly +sge_option_fc = -pe smp 24 -q %(jobqueue)s + +# job concurrency settings for... +# all jobs +default_concurrent_jobs = 30 +# preassembly +pa_concurrent_jobs = 30 +# consensus calling of preads +cns_concurrent_jobs = 30 +# overlap detection +ovlp_concurrent_jobs = 30 + +# daligner parameter options for... +# https://dazzlerblog.wordpress.com/command-guides/daligner-command-reference-guide/ +# initial overlap of raw reads +pa_HPCdaligner_option = -v -B4 -t16 -e.70 -l1000 -s1000 +# overlap of preads +ovlp_HPCdaligner_option = -v -B4 -t32 -h60 -e.96 -l500 -s1000 + +# parameters for creation of dazzler database of... +# https://dazzlerblog.wordpress.com/command-guides/dazz_db-command-guide/ +# raw reads +pa_DBsplit_option = -x500 -s50 +# preads +ovlp_DBsplit_option = -x500 -s50 + +# settings for consensus calling for preads +falcon_sense_option = --output_multi --min_idt 0.70 --min_cov 4 --max_n_read 200 --n_core 6 + +# setting for filtering of final overlap of preads +overlap_filtering_setting = --max_diff 100 --max_cov 100 --min_cov 20 --bestn 10 --n_core 24 diff --git a/docs/cfgs/fc_run_fungal.cfg b/docs/cfgs/fc_run_fungal.cfg new file mode 100644 index 00000000..1a507921 --- /dev/null +++ b/docs/cfgs/fc_run_fungal.cfg @@ -0,0 +1,31 @@ +[General] +input_fofn = input.fofn +input_type = raw + + +length_cutoff = 6000 + +length_cutoff_pr = 8000 + +jobqueue = default +sge_option_da = -pe smp 4 -q %(jobqueue)s +sge_option_la = -pe smp 20 -q %(jobqueue)s +sge_option_pda = -pe smp 6 -q %(jobqueue)s +sge_option_pla = -pe smp 16 -q %(jobqueue)s +sge_option_fc = -pe smp 24 -q %(jobqueue)s +sge_option_cns = -pe smp 8 -q %(jobqueue)s + +pa_concurrent_jobs = 192 +cns_concurrent_jobs = 192 +ovlp_concurrent_jobs = 192 + +pa_HPCdaligner_option = -v -dal128 -t16 -e0.75 -M24 -l3200 -k18 -h480 -w8 -s100 +ovlp_HPCdaligner_option = -v -dal128 -M24 -k24 -h1024 -e.96 -l2500 -s100 + +pa_DBsplit_option = -a -x500 -s200 +ovlp_DBsplit_option = -s200 + +falcon_sense_option = --output_multi --min_idt 0.70 --min_cov 4 --max_n_read 200 --n_core 8 + +overlap_filtering_setting = --max_diff 120 --max_cov 120 --min_cov 2 --n_core 12 + diff --git a/docs/cfgs/fc_run_human.cfg b/docs/cfgs/fc_run_human.cfg new file mode 100644 index 00000000..bda98f2e --- /dev/null +++ b/docs/cfgs/fc_run_human.cfg @@ -0,0 +1,32 @@ +[General] +input_fofn = input.fofn + +input_type = raw + +length_cutoff = 23000 + +length_cutoff_pr = 23000 + +jobqueue = default +sge_option_da = -pe smp 4 -q %(jobqueue)s +sge_option_la = -pe smp 20 -q %(jobqueue)s +sge_option_pda = -pe smp 6 -q %(jobqueue)s +sge_option_pla = -pe smp 16 -q %(jobqueue)s +sge_option_fc = -pe smp 24 -q %(jobqueue)s +sge_option_cns = -pe smp 8 -q %(jobqueue)s + +pa_concurrent_jobs = 384 +cns_concurrent_jobs = 384 +ovlp_concurrent_jobs = 384 + +pa_HPCdaligner_option = -v -dal128 -t16 -e0.75 -M24 -l4800 -k18 -h480 -w8 -s100 +ovlp_HPCdaligner_option = -v -dal128 -M24 -k24 -h1024 -e.96 -l2500 -s100 + +pa_DBsplit_option = -x500 -s400 +ovlp_DBsplit_option = -s400 + +falcon_sense_option = --output_multi --min_idt 0.70 --min_cov 4 --max_n_read 200 --n_core 8 +falcon_sense_skip_contained = False + +overlap_filtering_setting = --max_diff 60 --max_cov 60 --min_cov 0 --n_core 12 + diff --git a/docs/cfgs/fc_run_mammal.cfg b/docs/cfgs/fc_run_mammal.cfg new file mode 100644 index 00000000..d7c1808c --- /dev/null +++ b/docs/cfgs/fc_run_mammal.cfg @@ -0,0 +1,33 @@ +[General] +input_fofn = input.fofn + +input_type = raw + +openending = True + +length_cutoff = 10000 + +length_cutoff_pr = 8000 + + +sge_option_da = -pe smp 5 -q bigmem +sge_option_la = -pe smp 20 -q bigmem +sge_option_pda = -pe smp 6 -q bigmem +sge_option_pla = -pe smp 16 -q bigmem +sge_option_fc = -pe smp 24 -q bigmem +sge_option_cns = -pe smp 8 -q bigmem + +pa_concurrent_jobs = 96 +cns_concurrent_jobs = 96 +ovlp_concurrent_jobs = 96 + +pa_HPCdaligner_option = -v -dal128 -t16 -M32 -e.70 -l4800 -s100 -k18 -h480 -w8 -H15000 +ovlp_HPCdaligner_option = -v -dal128 -t32 -M32 -h1024 -e.96 -l2400 -s100 -k24 -H15000 + +pa_DBsplit_option = -x500 -s400 +ovlp_DBsplit_option = -s400 + +falcon_sense_option = --output_multi --output_dformat --min_idt 0.70 --min_cov 2 --max_n_read 200 --n_core 8 +falcon_sense_skip_contained = True + +overlap_filtering_setting = --max_diff 100 --max_cov 100 --min_cov 2 --n_core 12 diff --git a/docs/cfgs/fc_run_mammalSequel.cfg b/docs/cfgs/fc_run_mammalSequel.cfg new file mode 100644 index 00000000..21380a5e --- /dev/null +++ b/docs/cfgs/fc_run_mammalSequel.cfg @@ -0,0 +1,46 @@ +[General] + +# list of files of the initial subread fasta files +input_fofn = fa.fofn + +input_type = raw +#input_type = preads + +# The length cutoff used for seed reads used for initial mapping +#genome_size = 2900000000 +#seed_coverage = 30 +length_cutoff = 5000 + +# The length cutoff used for seed reads usef for pre-assembly +length_cutoff_pr = 10000 + +use_tmpdir = /scratch +job_queue = bigmem +sge_option_da = -pe smp 4 +sge_option_la = -pe smp 20 +sge_option_pda = -pe smp 6 +sge_option_pla = -pe smp 16 +sge_option_fc = -pe smp 24 +sge_option_cns = -pe smp 8 + +# concurrency setting +default_concurrent_jobs = 288 +pa_concurrent_jobs = 288 +cns_concurrent_jobs = 288 +ovlp_concurrent_jobs = 288 + +# overlapping options for Daligner +# SEQUEL DATA +pa_HPCdaligner_option = -v -dal128 -e0.75 -M24 -l1200 -k14 -h256 -w8 -s100 -t16 +ovlp_HPCdaligner_option = -v -dal128 -M24 -k24 -h600 -e.95 -l1800 -s100 + +pa_DBsplit_option = -x500 -s400 +ovlp_DBsplit_option = -s400 + +# error correction consensus option +falcon_sense_option = --output_multi --min_idt 0.70 --min_cov 4 --max_n_read 200 --n_core 24 + +# overlap filtering options +overlap_filtering_setting = --max_diff 120 --max_cov 120 --min_cov 2 --n_core 12 + + diff --git a/docs/cfgs/fc_run_plant.cfg b/docs/cfgs/fc_run_plant.cfg new file mode 100644 index 00000000..9d11c63b --- /dev/null +++ b/docs/cfgs/fc_run_plant.cfg @@ -0,0 +1,37 @@ +[General] +input_fofn = input.fofn + +input_type = raw + +stop_all_jobs_on_failure = False + +length_cutoff = 5000 + +genome_size = 1400000000 +seed_coverage = 30 + +length_cutoff_pr = 4000 + +sge_option_da = -pe smp 5 -q bigmem +sge_option_la = -pe smp 20 -q bigmem +sge_option_pda = -pe smp 6 -q bigmem +sge_option_pla = -pe smp 16 -q bigmem +sge_option_fc = -pe smp 24 -q bigmem +sge_option_cns = -pe smp 12 -q bigmem + +pa_concurrent_jobs = 96 +cns_concurrent_jobs = 96 +ovlp_concurrent_jobs = 96 + + +pa_HPCdaligner_option = -v -B128 -M32 -e.70 -l4800 -s100 -k18 -h480 -w8 +ovlp_HPCdaligner_option = -v -B128 -M32 -h1024 -e.96 -l2400 -s100 -k18 + + +pa_DBsplit_option = -a -x500 -s400 +ovlp_DBsplit_option = -s400 + +falcon_sense_option = --output_multi --min_idt 0.70 --min_cov 2 --max_n_read 200 --n_core 8 +falcon_sense_skip_contained = True + +overlap_filtering_setting = --max_diff 100 --max_cov 100 --min_cov 2 --n_core 12 diff --git a/docs/cfgs/fc_run_yeast.cfg b/docs/cfgs/fc_run_yeast.cfg new file mode 100644 index 00000000..4fdf3161 --- /dev/null +++ b/docs/cfgs/fc_run_yeast.cfg @@ -0,0 +1,35 @@ +[General] +input_fofn = input.fofn + +input_type = raw + +openending = True + +length_cutoff = -1 +genome_size = 12000000 + +length_cutoff_pr = 1000 + + +sge_option_da = -pe smp 5 -q bigmem +sge_option_la = -pe smp 20 -q bigmem +sge_option_pda = -pe smp 6 -q bigmem +sge_option_pla = -pe smp 16 -q bigmem +sge_option_fc = -pe smp 24 -q bigmem +sge_option_cns = -pe smp 8 -q bigmem + +pa_concurrent_jobs = 192 +cns_concurrent_jobs = 192 +ovlp_concurrent_jobs = 192 + +pa_HPCdaligner_option = -v -dal128 -M32 -e.70 -l2000 -s100 -k18 -h240 -w8 +ovlp_HPCdaligner_option = -v -dal128 -M32 -h240 -e.96 -l1000 -s100 -k24 + +pa_DBsplit_option = -x500 -s400 +ovlp_DBsplit_option = -s400 + +falcon_sense_option = --output_multi --min_idt 0.70 --min_cov 2 --max_n_read 200 --n_core 8 +falcon_sense_skip_contained = True + +overlap_filtering_setting = --max_diff 100 --max_cov 100 --min_cov 2 --n_core 12 + diff --git a/docs/cfgs/fc_unzip.cfg b/docs/cfgs/fc_unzip.cfg new file mode 100644 index 00000000..5a4643f2 --- /dev/null +++ b/docs/cfgs/fc_unzip.cfg @@ -0,0 +1,20 @@ +[General] +job_type = SGE +#job_type = local +job_queue = default + +[Unzip] +input_fofn= input.fofn +input_bam_fofn= input_bams.fofn + +smrt_bin=/path/to/smrtcmds/bin/ + +sge_phasing= -pe smp 12 +sge_quiver= -pe smp 24 +sge_track_reads= -pe smp 12 +sge_blasr_aln= -pe smp 24 +sge_hasm= -pe smp 48 +unzip_blasr_concurrent_jobs = 80 +unzip_phasing_concurrent_jobs = 500 +quiver_concurrent_jobs = 64 + diff --git a/docs/changelog.rst b/docs/changelog.rst new file mode 100644 index 00000000..90c00404 --- /dev/null +++ b/docs/changelog.rst @@ -0,0 +1,64 @@ +.. _changelog: + +.. caution:: These documents refer to an obsolete way of installing and running FALCON. They will remain up for historical context and for individuals still using the older version of FALCON/FALCON_unzip. + +.. attention:: The current PacBio Assembly suite documentation which includes new bioconda instructions for installing FALCON, FALCON_unzip and their associated dependencies can be found here `pb_assembly `_ + + +Changelog +========= + + +.. _3122018tarball: + +3/12/2018 +--------- + + +.. _8082018tarball: + +8/08/2018 +-------- + +FALCON Updates: ++++++++++++++++ + +Repeat Masking + +- Integration of DAMASKER: Tandem repeat masking (done) and general repeat masking (in progress) + +Improved default settings for microbial assembly + +- Use one longest read per ZMW: reduced chimerism, coverage bias +- Retuned parameters to increase contiguity + +New! GFA-2 support + +- Assembly graphs now written in both GFA-1 and GFA-2 formats +- Placement coordinates of associate contigs now available in a new "contig.gfa2" file + +Performance Improvements + +- General workflow and resource specification improvements +- Easier integration of future features with Pbsmrtpipe + + +FALCON-Unzip Updates: ++++++++++++++++++++++ + +Improved Haplotig Extraction + +- Algorithm and data structure improvements reduce haplotype switching and improve extraction (resolved nested and overlapping haplotigs) +- Can now handle circular contigs! + +New! Placement Files + +- Haplotig placement (PAF format) generated after Unzip +- Easier integration with FALCON-Phase + +Performance Improvements + +- Use of Minimap2 instead of BLASR for phasing in Unzip reduces time requirements +- Significantly reduced memory consumption of the final stage of Unzip (preads no longer have to be loaded in memory) +- Unzipping and polishing are now combined in the same workflow and run consecutively. + diff --git a/docs/cmds/DB2Falcon.rst b/docs/cmds/DB2Falcon.rst new file mode 100644 index 00000000..06da6f20 --- /dev/null +++ b/docs/cmds/DB2Falcon.rst @@ -0,0 +1,7 @@ +######### +DB2Falcon +######### + +.. code-block:: bash + + Usage: DB2fasta [-U] [-w] diff --git a/docs/cmds/fc_consensus.rst b/docs/cmds/fc_consensus.rst new file mode 100644 index 00000000..d27b3962 --- /dev/null +++ b/docs/cmds/fc_consensus.rst @@ -0,0 +1,56 @@ +.. _fc_consensus: + + +############### +fc_consensus.py +############### + +.. code-block:: bash + + usage: fc_consensus.py [-h] [--n_core N_CORE] [--min_cov MIN_COV] + [--min_cov_aln MIN_COV_ALN] [--max_cov_aln MAX_COV_ALN] + [--min_len_aln MIN_LEN_ALN] [--min_n_read MIN_N_READ] + [--max_n_read MAX_N_READ] [--trim] [--output_full] + [--output_multi] [--min_idt MIN_IDT] + [--edge_tolerance EDGE_TOLERANCE] + [--trim_size TRIM_SIZE] + + a simple multi-processor consensus sequence generator + + optional arguments: + -h, --help show this help message and exit + --n_core N_CORE number of processes used for generating consensus; 0 + for main process only (default: 24) + --min_cov MIN_COV minimum coverage to break the consensus (default: 6) + --min_cov_aln MIN_COV_ALN + minimum coverage of alignment data; a seed read with + less than MIN_COV_ALN average depth of coverage will + be completely ignored (default: 10) + --max_cov_aln MAX_COV_ALN + maximum coverage of alignment data; a seed read with + more than MAX_COV_ALN average depth of coverage of the + longest alignments will be capped, excess shorter + alignments will be ignored (default: 0) + --min_len_aln MIN_LEN_ALN + minimum length of a sequence in an alignment to be + used in consensus; any shorter sequence will be + completely ignored (default: 0) + --min_n_read MIN_N_READ + 1 + minimum number of reads used in generating the + consensus; a seed read with fewer alignments will be + completely ignored (default: 10) + --max_n_read MAX_N_READ + 1 + maximum number of reads used in generating the + consensus (default: 500) + --trim trim the input sequence with k-mer spare dynamic + programming to find the mapped range (default: False) + --output_full output uncorrected regions too (default: False) + --output_multi output multi correct regions (default: False) + --min_idt MIN_IDT minimum identity of the alignments used for correction + (default: 0.7) + --edge_tolerance EDGE_TOLERANCE + for trimming, the there is unaligned edge leng > + edge_tolerance, ignore the read (default: 1000) + --trim_size TRIM_SIZE + the size for triming both ends from initial sparse + aligned region (default: 50) diff --git a/docs/cmds/fc_get_read_hctg_map.rst b/docs/cmds/fc_get_read_hctg_map.rst new file mode 100644 index 00000000..e2c61709 --- /dev/null +++ b/docs/cmds/fc_get_read_hctg_map.rst @@ -0,0 +1,18 @@ +.. _fc_get_read_hctg_map + +####################### +fc_get_read_hctg_map.py +####################### + +.. code-block:: bash + + usage: fc_get_read_hctg_map.py [-h] [--basedir BASEDIR] + + generate `3-unzip/read_maps/read_to_contig_map` that contains the information + from the chain of mapping: (contig id, last col) -> (internal p-read id) -> + (internal raw-read id) -> (original read id) it assumes the 2-asm- + falcon/read_maps/raw_read_ids and pread_ids are already generated + + optional arguments: + -h, --help show this help message and exit + --basedir BASEDIR the base working dir of a FALCON assembly (default: ./) \ No newline at end of file diff --git a/docs/cmds/fc_graph_to_contig.rst b/docs/cmds/fc_graph_to_contig.rst new file mode 100644 index 00000000..e0a1b64d --- /dev/null +++ b/docs/cmds/fc_graph_to_contig.rst @@ -0,0 +1,19 @@ +.. graph_to_contig: + +##################### +fc_graph_to_contig.py +##################### + +The final step in the generation of draft contigs is to find a single path for each contig graph and to generate +sequence accordingly. In the case that a contig graph is not a :term:`simple path`, we find the end-to-end path that +has the most overlapped bases. This is called as the :term:`primary contig`. For each :ref:`compound path` within the +graph, if an alternative path different from primary one is possible, we will construct the :ref:`associated contig`. +In the case where the :term:`associated contigs` are induced by sequencing error, the identity of the +alternative contig and the :term:`primary contig` will be high ( > 99% identity most of time). +In the case where there are true structural variations, there are typically bigger differences between the +:term:`associated contigs ` and the :term:`primary contigs `. + +Essentially, the script ``fc_graph_to_contig`` generates contigs given sequence data and the final assembly graph. +Currently it generates :ref:`primary contigs ` as well as all +:ref:`associated contigs ` without any filtering. Some post-processing to remove duplicate +:ref:`associated contigs ` induced by errors will generally be necessary. diff --git a/docs/cmds/fc_ovlp_filter.rst b/docs/cmds/fc_ovlp_filter.rst new file mode 100644 index 00000000..b76ba494 --- /dev/null +++ b/docs/cmds/fc_ovlp_filter.rst @@ -0,0 +1,75 @@ +.. ovlp_filter: + +################# +fc_ovlp_filter.py +################# + + +.. code:: bash + + usage: fc_ovlp_filter [-h] [--n_core N_CORE] [--fofn FOFN] [--db DB_FN] + [--max_diff MAX_DIFF] [--max_cov MAX_COV] + [--min_cov MIN_COV] [--min_len MIN_LEN] [--bestn BESTN] + [--stream] [--debug] [--silent] + + a simple multi-processes LAS ovelap data filter + + optional arguments: + -h, --help show this help message and exit + --n_core N_CORE number of processes used for generating consensus; 0 + for main process only (default: 4) + --fofn FOFN file contains the path of all LAS file to be processed + in parallel (default: None) + --db DB_FN read db file path (default: None) + --max_diff MAX_DIFF max difference of 5' and 3' coverage (default: None) + --max_cov MAX_COV max coverage of 5' or 3' coverage (default: None) + --min_cov MIN_COV min coverage of 5' or 3' coverage (default: None) + --min_len MIN_LEN min length of the reads (default: 2500) + --bestn BESTN output at least best n overlaps on 5' or 3' ends if + possible (default: 10) + --stream stream from LA4Falcon, instead of slurping all at once; + can save memory for large data (default: False) + --debug, -g single-threaded, plus other aids to debugging (default: + False) + --silent suppress cmd reporting on stderr (default: False) + + +Not all overlaps are "independent", so it is possible to impose some filtering step to reduce computation and +assembly graph complexity. For example, if a read is fully contained in another read, the overlap information +between these two reads does not provide extra information for re-constructing the genome. Also, due to the +transitive property of the overlapping relations, a lot of overlap information can be simply inferred. In fact, +the first stage for constructing contigs are to remove the transitive reducible edges. It means that we might +just needs the "best n overlaps" in the ``5'`` or ``3'`` ends. The ``--bestn`` parameter in :ref:`overlap_filtering_setting` +option can be used to control the maximum overlap reported for each read. + +Another useful heuristics is to only keep reads that have average ``5'`` and ``3'`` coverage. That's because if a read +ends in a repeat, it might have higher than normal coverage at the end which is a repeat. And such reads do not +provide much value for uniquely resolving the related repeats. We can filter them out and hopefully there are +reads which span through the repeats and have "normal" unique anchors on both ends. Also, if the coverage is too +low on one end of a read, it could be just too many errors or sequencing artifacts over there. Such reads create +"spurs" in the assembly graph which are typically filtered out anyway. The --max_cov and --min_cov are used for +filtering reads that have too high or too low overlaps. + +The filtering scripts also allows filtering out some "split" reads. If a read have very unequal coverage between +the ``5'`` and ``3'`` ends, it can be also a signal that one end is a repeat. The ``--max_diff`` parameter can be used to +filter out the reads where one ends has much more coverage than the other end. + +What is the right numbers used for these parameters? These parameters may the most tricky ones to be set right. +If the overall coverage of the error corrected reads longer than the length cut off is known and reasonable high +(e.g. greater than 20x), it might be safe to set ``--min_cov`` to be 5, max_cov to be three times of the average +coverage and the max_diff to be twice of the average coverage. However, in low coverage case, it might better +to set ``--min_cov`` to be one or two. A helper script called :doc:`fc_ovlp_stats`` can help to dump the number of the +``3'`` and ``5'`` overlap of a given length cutoff, you can plot the distribution of the number of overlaps to make a +better decision. + +One can also set the ``--max_diff`` and ``--max_cov`` to be really high to avoid any filtering if that is preferred +in some cases. + +This filtering process will certainly filter out information about high copy repeats. Namely, those repeats will +likely to be filtered out totally and do not appear in the final assembly. If you are interested in those repeats +even though they may not be able to placed within some longer contig, you will probably want to avoid filtering +them out or process them differently. In general, it might be more efficient and useful to process those repeats +separately. Including them in the assembly process typically does not help much for getting better contiguity and +maybe messy for post-processing with current algorithms. I think it is a very interesting but also very challenging +bioinformatics topic on how to process these repeats better for improving assembly beside understand the nature +of these repeats. \ No newline at end of file diff --git a/docs/cmds/fc_ovlp_to_graph.rst b/docs/cmds/fc_ovlp_to_graph.rst new file mode 100644 index 00000000..abf9d71d --- /dev/null +++ b/docs/cmds/fc_ovlp_to_graph.rst @@ -0,0 +1,104 @@ +.. _ovlp_to_graph: + + +################### +fc_ovlp_to_graph.py +################### + +Here is the usage information for running fc_ovlp_to_graph.py: + +.. code:: bash + + usage: fc_ovlp_to_graph.py [-h] [--min_len MIN_LEN] [--min_idt MIN_IDT] + [--lfc] + overlap_file + + a example string graph assembler that is desinged for handling diploid genomes + + positional arguments: + overlap_file a file that contains the overlap information. + + optional arguments: + -h, --help show this help message and exit + --min_len MIN_LEN minimum length of the reads to be considered for + assembling + --min_idt MIN_IDT minimum alignment identity of the reads to be considered + for assembling + --lfc use local flow constraint method rather than best overlap + method to resolve knots in string graph + +In some case, you might want to lower the min_idt to keep more overlap or increase min_len to reduce the number of +overlap used for constructing the contig after the overlap filtering step. The ``--lfc`` toggles the rule for resolving +local knots in the graph. If ``--lfc`` is not specified, "the best overlapped edge" will be kept when there are multiple +in- or out- edges from a node while the others will be removed. + +The first stage of the assembly is to construct the initial string graph and classify each edges in the string graph. +``sg_edges_list`` contained the information of the information of the edges in the full string graph and the +classification. For example, 5 edges are shown in the five lines of the file below + +.. code:: bash + + $ head -5 sg_edges_list + 000017363:B 000007817:E 000007817 10841 28901 10841 99.52 TR + 000015379:E 000004331:B 000004331 6891 0 18178 99.35 TR + 000006813:B 000000681:E 000000681 7609 23795 7616 99.72 TR + 000002258:E 000002505:B 000002505 5850 0 17215 99.62 TR + 000013449:B 000012565:B 000012565 3317 0 20570 99.72 G + + +The first two columns indicates the in and out node of the edge. The node notation contains two files operated by :. +The first field is the read identifier. The second field is either B or E. B is the ``5'`` end of the read and E is the +``3'`` end of the reads. The next three field indicates the corresponding sequences of the edges. In this example, the +edge in the first line contains the sequence from read ``000007817`` base ``[10841, 28901)``. If the second coordinate is +smaller than the first one, it means the corresponded sequence is reverse complimented. The next two column are the +number of overlapped base and the overlap identity. The final column is the classification. Currently, there are 4 +different types ``G``, ``TR``, ``R``, and ``S``. An edge with type ``G`` is used for the final string graph. A ``TR`` +means the edge is transitive reducible. ``R`` means the edge is removed during the local repeat resolution and ``S`` +means the edge is likely to be a "spur" which only one ends is connected. + +The initial string graph is further to be simplified into a set of "unitig" edges. The ``utg_data`` file contains the +details of each unitig. Each line in the file represents a unitig. The first three fields are "start node", "via node", +and "end node". Two untigs might have the same "start node" and "end node", so we need another "via node" to uniquely +identify the unitigs. Here is an example of the utg_data files: + + +.. code:: bash + + $ head -10 utg_data + 000015696:B 000009028:B 000016941:B contained 16438 134865 000015696:B~000006612:B~000002456:B~000014643:B~000007407:B~000015939:E~000009028:B~000016941:B + 000010623:B 000015633:B 000014991:B contained 30158 18666 000010623:B~000015633:B~000014991:B + 000015636:B 000002245:B 000010757:E contained 15402 40356 000015636:B~000002245:B~000010757:E + 000014184:E NA 000012028:E compound 14895 56928 000014184:E~000012765:E~000012028:E|000014184:E~000007953:B~000012028:E + 000010757:B NA 000015636:E compound 15402 40356 000010757:B~000002245:E~000015636:E|000010757:B~000014783:E~000015636:E + 000014184:E 000007953:B 000012028:E contained 14792 32932 000014184:E~000007953:B~000012028:E + 000010623:B NA 000014991:B compound 30148 163627 000010623:B~000015633:B~000014991:B|000010623:B~000001407:B~000014991:B + 000012028:B 000012765:B 000014184:B contained 19137 56928 000012028:B~000000382:E~000012765:B~000014184:B + 000016941:B 000003353:B 000008783:B simple 88381 615439 000016941:B~000003353:B~000010261:B~000011789:E~000017006:B~000016307:B~... + 000014991:B 000013790:E 000002926:B simple 392373 2274104 000014991:B~000013790:E~000004614:B~000003329:B~000004898:B~000000461:B~000017105:E~... + +The forth field indicates the type of the unitigs, the fifth field is the estimate length of the unitig and the six +field is the total number of overlapped bases in the unitig. There are three kinds of unitigs: "simple", "contained", +and "compound". "Simple" unitigs are those unitigs which are just a simple path (every node has one in- and one +out-edge except the begining and ending nodes of the path.) It is represented by a list of nodes which each node +is separated by ~ characters in the 7th column. The "contained" contigs are simple path but those unitigs are also +part of other "compound" paths. The "compound" unitigs represents bubble-like subgraph in the graph. While it is not +"simple", it has well defined in- and out- nodes and they are treated as a single unit when the contigs are +constructed. The structure inside a "compound" unitig can be from biological nature or sequencing/alignment errors. +Each edge in the "compound" unitig subgraph are encoded explicitly as a collection of simple contained unitigs in +the 7th column. The contained unitigs within a compound unitig are separated by the ``|`` character. + +The file ctg_paths encodes the graph for each contig after the unitigs are analyzed and put into contigs. Each line +has 7 columns. The first column is the contig ID. The contig ID are just the serial numbers followed by R or F. +Two contigs with same serial number but different endings are "dual" to each other. Namely, they are constructed +from "dual" edges and they are mostly reverse complemented to each other except near the ends of the contigs. +The second column is the type of contig. If a unitig is circular (the beginning node and the ending node are the +same), then it will be marked as ``ctg_circular``. Everything else will be ``ctg_linear``. In some case, even a contig +is marked as ``ctg_linear``, it can be still a circular contig if the beginning node and the ending node are the +same but it is not a "simple" path. One can detect that by checking the beginning and ending nodes if necessary. + +The third field indicates the first unitig in the contig in the form of begin_node~via_node~end_node. The fourth +field is the ending node of the contig. The 5th and 6th fields are the estimated length and the overlapped +based of the contig respectively. The final column are the unitigs in the contig. The three node format unitig +IDs are separated by ``|`` + + diff --git a/docs/cmds/fc_quiver.rst b/docs/cmds/fc_quiver.rst new file mode 100644 index 00000000..9f012567 --- /dev/null +++ b/docs/cmds/fc_quiver.rst @@ -0,0 +1,7 @@ +.. _fc_quiver: + +############ +fc_quiver.py +############ + +Run quiver on your completed unzipped assembly diff --git a/docs/cmds/fc_run.rst b/docs/cmds/fc_run.rst new file mode 100644 index 00000000..02b4476a --- /dev/null +++ b/docs/cmds/fc_run.rst @@ -0,0 +1,17 @@ +.. _fc_run.py: + + +######### +fc_run.py +######### + +.. code-block:: bash + + usage: fc_run.py [-h] config [logger] + + positional arguments: + config .cfg/.ini/.json + logger (Optional)JSON config for standard Python logging module + + optional arguments: + -h, --help show this help message and exit \ No newline at end of file diff --git a/docs/commands.rst b/docs/commands.rst new file mode 100644 index 00000000..01ec6fff --- /dev/null +++ b/docs/commands.rst @@ -0,0 +1,186 @@ +.. _commands: + +.. caution:: These documents refer to an obsolete way of installing and running FALCON. They will remain up for historical context and for individuals still using the older version of FALCON/FALCON_unzip. + +.. attention:: The current PacBio Assembly suite documentation which includes new bioconda instructions for installing FALCON, FALCON_unzip and their associated dependencies can be found here `pb_assembly `_ + + +Commands +======== + +FALCON Commands +--------------- + +.. _DB2dalcon: + +:doc:`DB2Falcon ` + Used to dump dazzler preads.db into FASTA format for subsequent :term:`String Graph` assembly + +.. _fc_run.py: + +:doc:`fc_run.py ` + This script drives the entire assembly process + +.. _fc_consensus.py: + +:doc:`fc_consensus.py ` + ``fc_consensus`` has many options. You can use the parameter :ref:`falcon_sense_option` to control it. + In most cases, the ``--min_cov`` and ``--max_n_read`` are the most important options. ``--min_cov`` controls + when a seed read gets trimmed or broken due to low coverage. ``--max_n_read`` puts a cap on the number of reads + used for error correction. In highly repetitive genome, you will need to make the value for ``--max_n_read`` + smaller to make sure the consensus code does not waste time aligning repeats. The longest proper overlaps are used + for correction to reduce the probability of collapsed repeats. + +.. _fc_dedup_a_tigs.py: + +:doc:`fc_dedup_a_tigs.py ` + remove duplicated :term:`associated contigs`, mostly induced by tandem repeat alignment + uncertainty + +.. _fc_graph_to_contig.py: + +:doc:`fc_graph_to_contig.py ` + Generate contigs based on assembly graph + +.. _fc_ovlp_to_graph.py: + +:doc:`fc_ovlp_to_graph.py ` + Generate an assembly graph given a list of overlapping preads. + +.. _fc_ovlp_filter.py: + +:doc:`fc_ovlp_filter.py ` + Filter overlaps based on given criteria + +FALCON_unzip commands +--------------------- + +:doc:`fc_get_read_hctg_map.py ` + Generate a read-to-contig map + +.. _fc_dedup_h_tigs.py: + +:doc:`fc_dedup_h_tigs.py ` + +.. _fc_graphs_to_h_tigs.py: + +:doc:`fc_graphs_to_h_tigs.py ` + +.. _fc_ovlp_filter_with_phase.py: + +:doc:`fc_ovlp_filter_with_phase.py ` + +.. _fc_phased_ovlp_to_graph.py: + +:doc:`fc_phased_ovlp_to_graph.py ` + +.. _fc_phasing.py: + +:doc:`fc_phasing.py ` + +.. _fc_phasing_readmap.py: + +:doc:`fc_phasing_readmap.py ` + +.. _fc_quiver.py: + +:doc:`fc_quiver.py ` + +.. _fc_rr_hctg_track.py: + +:doc:`fc_rr_hctg_track.py ` + +.. _fc_select_reads_from_bam.py: + +:doc:`fc_select_reads_from_bam.py ` + +.. _fc_track_reads_htigs.py: + +:doc:`fc_track_reads_htigs.py ` + +.. _fc_unzip.py: + +:doc:`fc_unzip.py ` + +Dazzler commands +---------------- + +These commands are part of Gene Meyer's Dazzler Suite of tools `Dazzler Blog `_ + +FALCON relies on a slightly modified version of Gene Meyer's code that can be found +`here `_, but is also bundled with the +`FALCON-integrate `_ github repository. + +.. _dazzdaligner: + +`daligner `_: + Compare subject sequences to target sequences + ``daligner`` is controlled by :ref:`pa_HPCdaligner_option ` and + :ref:`ovlp_HPCdaligner_option `. + + To limit memory, one can use the ``-M`` option. For human assembly, we've tested with ``-M 32`` for using 32G RAM for + each daligner. Other possibilities are under investigation. + + For more details on daligner options, see the `Dazzler Blog `_ + +.. _dazzDB2fasta: + +`DB2fasta `_: + The set of .fasta files for the given DB are recreated from the DB exactly as they were input. + +.. _dazzDBdump: + +`DBdump `_: + Like DBshow, DBdump allows one to display a subset of the reads in the DB and select which information to show + about them including any mask tracks. + +.. _dazzDBdust: + +`DBdust `_: + Runs the symmetric DUST algorithm over the reads in the untrimmed DB + +.. _dazzDBsplit: + +`DBsplit `_: + The total number of jobs that are run is determined by how one "splits" the sequence database. You should read + Gene Myers's blog `Dazzler Blog ` carefully to understand how the tuning options, + :ref:`pa_DBsplit_option ` and :ref:`pa_HPCdaligner_option ` work. Generally, for large genomes, you should use + ``-s400`` (400Mb sequence per block) in :ref:`pa_DBsplit_option `. This will make a smaller number of jobs but each + job will run longer. However, if you have a job scheduler which limits how long a job can run, it might be + desirable to have a smaller number for the ``-s`` option. + +.. _dazzDBstats: + +`DBstats `_: + Show overview statistics for all the reads in the trimmed data base .db + +.. _dazzfasta2DB: + +`fasta2DB `_: + Convert a fasta to a dazzler DB. + +.. _dazzHPC.daligner: + +`HPC.daligner `_: + Generates overlap script to run all necessary daligner, LAsort and LAmerge commands + +.. _dazzLA4Falcon: + +`LA4Falcon `_: + Output data from a Dazzler DB into fasta format for FALCON. You can supply the argument ``-H`` with an integer value + to filter reads below a given threshold. + +.. _dazzLAcheck: + +`LAcheck `_: + Check integrity of alignment files. + +.. _dazzLAmerge: + +`LAmerge `_: + Merge the .las files into a singled sorted file + +.. _dazzLAsort: + +`LAsort `_: + Sort alignment files diff --git a/docs/conf.py b/docs/conf.py new file mode 100644 index 00000000..51ba72b9 --- /dev/null +++ b/docs/conf.py @@ -0,0 +1,355 @@ +import sys +import os +# -*- coding: utf-8 -*- +# +# FALCON build configuration file, created by +# sphinx-quickstart on Wed Nov 2 13:50:34 2016. +# +# This file is execfile()d with the current directory set to its +# containing dir. +# +# Note that not all possible configuration values are present in this +# autogenerated file. +# +# All configuration values have a default; values that are commented out +# serve to show the default. + +# If extensions (or modules to document with autodoc) are in another directory, +# add these directories to sys.path here. If the directory is relative to the +# documentation root, use os.path.abspath to make it absolute, like shown here. +# +# import os +# import sys +# sys.path.insert(0, os.path.abspath('.')) + +# -- General configuration ------------------------------------------------ + +# If your documentation needs a minimal Sphinx version, state it here. +# +# needs_sphinx = '1.0' + +# Add any Sphinx extension module names here, as strings. They can be +# extensions coming with Sphinx (named 'sphinx.ext.*') or your custom +# ones. +extensions = [] + +# Add any paths that contain templates here, relative to this directory. +templates_path = ['_templates'] + +# The suffix(es) of source filenames. +# You can specify multiple suffix as a list of string: +# +# source_suffix = ['.rst', '.md'] +source_suffix = '.rst' + +# The encoding of source files. +# +# source_encoding = 'utf-8-sig' + +# The master toctree document. +master_doc = 'index' + +# General information about the project. +project = u'FALCON' +copyright = u'2016, Greg Concepcion, Sarah Kingan, Chris Dunn, Jason Chin' +author = u'Greg Concepcion, Sarah Kingan, Chris Dunn, Jason Chin' + +# The version info for the project you're documenting, acts as replacement for +# |version| and |release|, also used in various other places throughout the +# built documents. +# +# The short X.Y version. +version = u'0.5' +# The full version, including alpha/beta/rc tags. +release = u'0.5' + +# The language for content autogenerated by Sphinx. Refer to documentation +# for a list of supported languages. +# +# This is also used if you do content translation via gettext catalogs. +# Usually you set "language" from the command line for these cases. +language = None + +# There are two options for replacing |today|: either, you set today to some +# non-false value, then it is used: +# +# today = '' +# +# Else, today_fmt is used as the format for a strftime call. +# +# today_fmt = '%B %d, %Y' + +# List of patterns, relative to source directory, that match files and +# directories to ignore when looking for source files. +# This patterns also effect to html_static_path and html_extra_path +exclude_patterns = ['_build', 'Thumbs.db', '.DS_Store'] + +# The reST default role (used for this markup: `text`) to use for all +# documents. +# +# default_role = None + +# If true, '()' will be appended to :func: etc. cross-reference text. +# +# add_function_parentheses = True + +# If true, the current module name will be prepended to all description +# unit titles (such as .. function::). +# +# add_module_names = True + +# If true, sectionauthor and moduleauthor directives will be shown in the +# output. They are ignored by default. +# +# show_authors = False + +# The name of the Pygments (syntax highlighting) style to use. +pygments_style = 'sphinx' + +# A list of ignored prefixes for module index sorting. +# modindex_common_prefix = [] + +# If true, keep warnings as "system message" paragraphs in the built documents. +# keep_warnings = False + +# If true, `todo` and `todoList` produce output, else they produce nothing. +todo_include_todos = False + + +# -- Options for HTML output ---------------------------------------------- + +# The theme to use for HTML and HTML Help pages. See the documentation for +# a list of builtin themes. +# +html_theme = 'sphinx_rtd_theme' + +# Theme options are theme-specific and customize the look and feel of a theme +# further. For a list of options available for each theme, see the +# documentation. +# +# html_theme_options = {} + +# Add any paths that contain custom themes here, relative to this directory. +html_theme_path = ['./'] + + +# Build using the RTD theme, if not on RTD. +# https://read-the-docs.readthedocs.org/en/latest/theme.html +# https://github.com/snide/sphinx_rtd_theme +# +on_rtd = os.environ.get('READTHEDOCS', None) == 'True' + +if not on_rtd: # only import and set the theme if we're building docs locally + import sphinx_rtd_theme + html_theme = 'sphinx_rtd_theme' + html_theme_path = [ "/usr/local/lib/python2.7/site-packages", ] + + + + +# The name for this set of Sphinx documents. +# " v documentation" by default. +# +# html_title = u'FALCON v0.5' + +# A shorter title for the navigation bar. Default is the same as html_title. +# +# html_short_title = None + +# The name of an image file (relative to this directory) to place at the top +# of the sidebar. +# +# html_logo = None + +# The name of an image file (relative to this directory) to use as a favicon of +# the docs. This file should be a Windows icon file (.ico) being 16x16 or 32x32 +# pixels large. +# +# html_favicon = None + +# Add any paths that contain custom static files (such as style sheets) here, +# relative to this directory. They are copied after the builtin static files, +# so a file named "default.css" will overwrite the builtin "default.css". +html_static_path = ['sphinx_rtd_theme/static'] + +# Add any extra paths that contain custom files (such as robots.txt or +# .htaccess) here, relative to this directory. These files are copied +# directly to the root of the documentation. +# +# html_extra_path = [] + +# If not None, a 'Last updated on:' timestamp is inserted at every page +# bottom, using the given strftime format. +# The empty string is equivalent to '%b %d, %Y'. +# +# html_last_updated_fmt = None + +# If true, SmartyPants will be used to convert quotes and dashes to +# typographically correct entities. +# +# html_use_smartypants = True + +# Custom sidebar templates, maps document names to template names. +# +# html_sidebars = {} + +# Additional templates that should be rendered to pages, maps page names to +# template names. +# +# html_additional_pages = {} + +# If false, no module index is generated. +# +# html_domain_indices = True + +# If false, no index is generated. +# +# html_use_index = True + +# If true, the index is split into individual pages for each letter. +# +# html_split_index = False + +# If true, links to the reST sources are added to the pages. +# +# html_show_sourcelink = True + +# If true, "Created using Sphinx" is shown in the HTML footer. Default is True. +# +# html_show_sphinx = True + +# If true, "(C) Copyright ..." is shown in the HTML footer. Default is True. +# +# html_show_copyright = True + +# If true, an OpenSearch description file will be output, and all pages will +# contain a tag referring to it. The value of this option must be the +# base URL from which the finished HTML is served. +# +# html_use_opensearch = '' + +# This is the file name suffix for HTML files (e.g. ".xhtml"). +# html_file_suffix = None + +# Language to be used for generating the HTML full-text search index. +# Sphinx supports the following languages: +# 'da', 'de', 'en', 'es', 'fi', 'fr', 'hu', 'it', 'ja' +# 'nl', 'no', 'pt', 'ro', 'ru', 'sv', 'tr', 'zh' +# +# html_search_language = 'en' + +# A dictionary with options for the search language support, empty by default. +# 'ja' uses this config value. +# 'zh' user can custom change `jieba` dictionary path. +# +# html_search_options = {'type': 'default'} + +# The name of a javascript file (relative to the configuration directory) that +# implements a search results scorer. If empty, the default will be used. +# +# html_search_scorer = 'scorer.js' + +# Output file base name for HTML help builder. +htmlhelp_basename = 'FALCONdoc' + +# -- Options for LaTeX output --------------------------------------------- + +latex_elements = { + # The paper size ('letterpaper' or 'a4paper'). + # + # 'papersize': 'letterpaper', + + # The font size ('10pt', '11pt' or '12pt'). + # + # 'pointsize': '10pt', + + # Additional stuff for the LaTeX preamble. + # + # 'preamble': '', + + # Latex figure (float) alignment + # + # 'figure_align': 'htbp', +} + +# Grouping the document tree into LaTeX files. List of tuples +# (source start file, target name, title, +# author, documentclass [howto, manual, or own class]). +latex_documents = [ + (master_doc, 'FALCON.tex', u'FALCON', + u'Greg Concepcion, Sarah Kingan, Chris Dunn, Jason Chin', 'manual'), +] + +# The name of an image file (relative to this directory) to place at the top of +# the title page. +# +# latex_logo = None + +# For "manual" documents, if this is true, then toplevel headings are parts, +# not chapters. +# +# latex_use_parts = False + +# If true, show page references after internal links. +# +# latex_show_pagerefs = False + +# If true, show URL addresses after external links. +# +# latex_show_urls = False + +# Documents to append as an appendix to all manuals. +# +# latex_appendices = [] + +# It false, will not define \strong, \code, itleref, \crossref ... but only +# \sphinxstrong, ..., \sphinxtitleref, ... To help avoid clash with user added +# packages. +# +# latex_keep_old_macro_names = True + +# If false, no module index is generated. +# +# latex_domain_indices = True + + +# -- Options for manual page output --------------------------------------- + +# One entry per manual page. List of tuples +# (source start file, name, description, authors, manual section). +man_pages = [ + (master_doc, 'falcon', u'FALCON', + [author], 1) +] + +# If true, show URL addresses after external links. +# +# man_show_urls = False + + +# -- Options for Texinfo output ------------------------------------------- + +# Grouping the document tree into Texinfo files. List of tuples +# (source start file, target name, title, author, +# dir menu entry, description, category) +texinfo_documents = [ + (master_doc, 'FALCON', u'FALCON', + author, 'FALCON', 'Diploid aware PacBio Assembler.', + 'Miscellaneous'), +] + +# Documents to append as an appendix to all manuals. +# +# texinfo_appendices = [] + +# If false, no module index is generated. +# +# texinfo_domain_indices = True + +# How to display URL addresses: 'footnote', 'no', or 'inline'. +# +# texinfo_show_urls = 'footnote' + +# If true, do not generate a @detailmenu in the "Top" node's menu. +# +# texinfo_no_detailmenu = False diff --git a/docs/faq.rst b/docs/faq.rst new file mode 100644 index 00000000..3115b04c --- /dev/null +++ b/docs/faq.rst @@ -0,0 +1,246 @@ +.. _faq: + +.. caution:: These documents refer to an obsolete way of installing and running FALCON. They will remain up for historical context and for individuals still using the older version of FALCON/FALCON_unzip. + +.. attention:: The current PacBio Assembly suite documentation which includes new bioconda instructions for installing FALCON, FALCON_unzip and their associated dependencies can be found here `pb_assembly `_ + + +Frequently Asked Questions +========================== + +General +------- + +Can I start from corrected reads? +~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ + +Yes. The option :ref:`input_type ` can be set to either ``raw`` or ``preads``. In the case of the latter, +:ref:`fc_run.py` will assume the fasta files in :ref:`input_fofn ` are all error-corrected reads and it +will ignore any :term:`error correction` step and go directly into the final assembly overlapping step. + +How do I select a length cutoff? +~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ + +The option :ref:`length_cutoff ` controls the read length cutoff used during the +:term:`error correction` process and :ref:`length_cutoff_pr ` controls the cutoff used for the +final assembly overlapping steps. In the final assembly, more reads may not lead to a better assembly due to the +fact that some of the reads can be noisy and create false links in the assembly graph. Sometimes you might want +to re-run the final steps of the assembly pipeline in ``2-asm-falcon`` with different values for ``--min_len`` +in ``run_falcon_asm.sub.sh`` as this step is quick relative to the overlap detection steps in the earlier stages +of the pipeline. + +If you're not sure, and you are not compute resource limited, one strategy is to choose a smaller +:ref:`length_cutoff ` and do the computation once. Later, one can use a different +:ref:`length_cutoff_pr ` to achieve a more contiguous assembly + +In general we recommend that you tune the cutoff so that you're left with roughly 15x to 20x for final genome assembly. +If you set :ref:`length_cutoff ` equal to ``-1``, FALCON will attempt to autocalculate this cutoff +for you. + + +.. _primary_vs_associated: + +What's the difference between a Primary and an Associated contig? +~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ + +:term:`Primary contigs ` can be thought of as the longest continuous stretches of contiguously +assembled sequence, while :term:`associate contigs ` can be thought of mostly as structural +variants that occur over the length of the primary contigs. Thus, each alternate primary contig configuration +(associated contig) can be "associated" with it's primary based on it's ``XXXXXXF`` prefix. + +Some basic information about how the associated contigs are generated can be found +in `this speakerdeck `_ (pg.14) +, `here `_ +(pg.14-15) `and here `_. + +Conceptually, if a genome is haploid, then all contigs should be primary contigs. However, in general there will usually +still be some associated contigs generated. This is likely due to: + +1. Sequencing errors +2. Segmental duplications. + +For the first case, Quiver should help by filtering out low quality contigs. Since there is more sequence in +the set of primary contigs for blasr to anchor reads and there is no true unique region in the erroneous +associated contigs, the raw read coverage on them should be low. We can thus filter low quality +:term:`associated contig` consensus as there won't be much raw read data to support them. + +For the second case, one could potentially partition the reads into different haplotype groups and construct +an assembly graph for each haplotype and generate contigs accordingly. + +If a genome is a diploid genome, then most of the associated contigs will be locally alternative alleles. +Typically, when there are big structural variations between homologous chromosomes, there will be alternative +paths in the assembly graph and the alternative paths correspond to the associated contigs. In such case, +the primary contigs are “fused contigs” from both haplotypes. + +FALCON_unzip is currently being developed to resolve the haplotypes so :term:`haplotigs ` can +be generated. Two videos illustrating the concept - (`Video 1 `_ , +`Video 2 `_) + +A `slide `_ illustrating the method on a synthetic genome. + +What are the differences between a_ctg.fasta and a_ctg_base.fasta ++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++ + +The file ``a_ctg_base.fasta`` contains the sequences in the primary contigs fasta that correspond to the associated +contigs inside ``a_ctg.fasta``. Namely, each sequence of a_ctg_base.fasta is a contiguous sub-sequence of a primary +contig. For each sequence inside `a_ctg_base.fasta, there are one or more associated contigs in a_ctg.fasta. + + +For a given contig in a_ctg.fa, how can I find it's primary contig map coordinates? ++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++ + +The 2nd field and the 3rd field of the sequence header inside ``a_ctg.fa`` indicate the begin node and the end node of +the contig. For example, if we have a header like + +.. code-block:: bash + + >000000F-001-01 000941458:E 000486369:E 15593 47559 5 0.9969 0.8447 + +It means the associated contig 000000F-001-01 starts from node 000941458:E and ends at 000486369:E. Thsee two nodes +should be also in the path of the corresponding primary contig. The path of the primary contig is fully specified in +the file p_ctg_tiling_path, you can find exact beginning and ending points where the associated contig are attached +to the primary contigs. However, the coordinates are not conserved after the :ref:`Quiver ` consensus step, +it might be necessary to do some quite alignment to recalibrate the attaching points after quiver consensus. +In some case, you can even just do quick sequence alignment to find the homologous region in the primary contig of +an associated contigs. + + +How does FALCON avoid chimeras given homologous repeat regions on different chromosomes? +~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ + +Such repeats are typically called as “segmental duplications”. Yes, Falcon will collapse these regions if the +overlapper can not distinguish the repeats. As discussed above in some case, it is just +like the case of a diploid genome, we can potentially resolve the two distinct haplotypes. In other cases, +the repeat is more complicated, such as if there are more than 2 copies, (e.g. the middle part of contigs 4006 in +page 21 of +`this slide deck `_. +To resolve these regions, we'll need to do more investigation to separate the reads into more than two groups +to resolve them. + + + +Can Falcon handle X-ploid genome data? +~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ + +Falcon, in its current form, is a “diploid or polyploid aware assembler”. I believe there is no fully specific +definition what a “diploid or polyploid assembler” should deliver yet at the moment of this writing. +From the point of the genome assembly research field, it is still quite new. There were a couple of papers published +before for diploid assemblies. However, the general strategy is the phasing adding reads on top on earlier assembly +step. + +To some degree, the current Falcon assembler provides a better way to build that foundation for a full diploid / +polyploid assembler. Please refer to this slide deck +https://speakerdeck.com/jchin/string-graph-assembly-for-diploid-genomes-with-long-reads for some detail. Some +technical details of the deck are already obsoleted for a little bit, but the general concept is still applied to +most recent code in Falcon. + +For a tetraploid genome, depending on the genome structure, I would argue one will get better continuity from +the primary contigs if you use Falcon for assembling the genome. However, you will need to do good analysis +on both primary and associated contigs (or better, the assembly graph directly) after running Falcon to +interpret the results correctly. The primary contigs will be “fused” contigs from all haplotypes unless +the differences between haplotypes are big such that the assembler’s overlap segregate them apart already. + +There are some prototype work to fully segregate the “fused primary contigs” for diploid case. I just +presented the ideas in #SFAF2015 conference. For tetraploid case, it will need some hard-code non-trivial +mathematics research work to get it work right. + + +Why don't I have two perfectly phased haplotypes after FALCON_unzip? +~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ + +It's useful to first understand that not all genomes are alike. Haploid genomes are the holy grail of genome assembly +as there is only one haplotype phase present and assembly is trivial if you have reads long enough to span repeats. +Diploid and (allo/auto)polyploid genomes become difficult as there are two or more haplotype phases present. This fact, +coupled with widely varying levels of heterozygosity and structural variation lead to complications during the assembly +process. To understand your FALCON output, it's useful to look at this supplemental figure from the FALCON_unzip_ paper: + +.. _FALCON_unzip: http://www.nature.com/nmeth/journal/vaop/ncurrent/full/nmeth.4035.html + +.. image:: media/heterozygosity.jpg + +Consider the first line as a cartoon illustrating 3 ranges of heterozygosity (low/medium/high). +In general, all genomes will have regions that fall into each of these three categories depending on organismal +biology. During the first step of the FALCON assembly process, a diploid aware assembly graph is generated. +At this point, in medium heterozygosity regions structural variation information is captured as bubbles or +alternative pathways in the assembly graph whereas at high levels of heterozygosity the haplotype phases assemble into +distinct primary assembly graphs. + +The ``FALCON_unzip`` add-on module to the FALCON pipeline is an attempt to leverage the heterozygous SNP information to +phase the medium level heterozygosity regions of the genome. Low heterozygosity regions have insufficient SNP +density for phasing, while high heterozygosity regions will likely have already been assembled as distinct haplotypes +in the primary contigs. + +FALCON_unzip yields two fasta files. One containing primary contigs, and one containing haplotigs. The primary contigs +fasta file is the main output that most people consider first and should consist of the majority of your genome. Primary +contigs are considered ``partially-phased``. What this means is that even after the unzipping process, certain regions +with insufficient SNP density are unable to be phased and are thus represented as ``collapsed haplotypes``. The presence +of these regions of low heterozygosity makes it impossible to maintain phase across the entire primary contig. Thus +primary contigs may contain phase-switches between unzipped regions. The haplotigs file will consist of the ``unzippapble`` +or ``phaseable`` regions of the genome and are considered fully phased. This means there should be no phase switching within +a haplotig and each haplotig should represent only one phase. See this figure for reference: + +.. image:: media/phaseswitch.png + +It's also important to note that in high heterozygosity situations, we often see the primary contig fasta file +approaching 1.5X+ the expected haploid genome size, due to the assembly of both phases of certain chromosomes or +chromosomal regions in the primary assembly. + +Also, one needs to consider that FALCON_unzip was designed to phase the plant and fungal genomes in the 2016 Nature Methods +paper above, but many people have successfully used it to help phase their genome of interest. But as always with +free software on the internet, your mileage may vary. + + +How much haplotype divergence can FALCON-Unzip handle? +~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ + +The magnitude of haplotype divergence determines the structure of the resulting FALCON-Unzip assembly. Genomic regions with low +heterozygisty will be assembled as collapsed haplotype on a single primary contig. Haplotypes up to ~5% diverged will be Unzipped, +while highly divergent haplotypes will be assembled on different primary contigs. In the latter case, it is up to the user to +identify these contigs as homologous using gene annotation or sequence alignment. + +For a variety of FALCON-Unzip assemblies, here is the distribution of haplotype divergence for unzipped regions. Each haplotig +was aligned to the corresponding primary contig with `nucmer `_, filtered with delta-filter and +divergence was estimated with show-choords. (Data credits to John Williams, Tim Smith, Paolo Ajmone-Marsan, David Hume, Erich Jarvis, +John Henning, Dave Hendrix, Carlos Machado, and Iago Hale). + +.. image:: media/unzippedHapDiv.png + + +Why does FALCON have trouble assembling my amplicon data? +~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ + +FALCON was designed for whole genome shot gun assembly rather than amplicon assembly. In whole genome shotgun +assembly we suppress repetitive high copy regions to assemble less repetitive regions first. +When you assemble PCR product of a short region in a genome, FALCON sees the whole thing as a high copy repeat +and filters alot of the data out. + +You can try to down sample your data and make the daligner block size even smaller ( reduce -s50 in +pa_DBsplit_option and ovlp_concurrent_jobs ) and increase the overlap filter thresholds (--max_diff 100 +--max_cov 100 in overlap_filtering_setting) to try to make it work, however it's not really within the scope of +the FALCON algorithm. + +Workflow +-------- + +How do I restart a failed workflow? +~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ + +Often times restarting a FALCON job due to unexplained failure is an easy process. It's typically just a matter +of removing any sentinel files and re-invoking ``fc_run.py fc_run.cfg`` from the FALCON root directory. Read +`this `_ section of the wiki for details. +If your job failed due to +`quota or other disk full issues `_ +, you may need to wipe the directory and start over again due to corrupt DB's + + +How do I turn on logging? +~~~~~~~~~~~~~~~~~~~~~~~~~ + +See `this `_ github issue. + +In short, you should pass ``logging.ini`` as the 2nd argument to :ref:`fc_run.py ` + +.. code-block:: bash + + $ fc_run.py fc_run.cfg logging.ini + diff --git a/docs/fc_graph_to_contig.rst b/docs/fc_graph_to_contig.rst new file mode 100644 index 00000000..e0a1b64d --- /dev/null +++ b/docs/fc_graph_to_contig.rst @@ -0,0 +1,19 @@ +.. graph_to_contig: + +##################### +fc_graph_to_contig.py +##################### + +The final step in the generation of draft contigs is to find a single path for each contig graph and to generate +sequence accordingly. In the case that a contig graph is not a :term:`simple path`, we find the end-to-end path that +has the most overlapped bases. This is called as the :term:`primary contig`. For each :ref:`compound path` within the +graph, if an alternative path different from primary one is possible, we will construct the :ref:`associated contig`. +In the case where the :term:`associated contigs` are induced by sequencing error, the identity of the +alternative contig and the :term:`primary contig` will be high ( > 99% identity most of time). +In the case where there are true structural variations, there are typically bigger differences between the +:term:`associated contigs ` and the :term:`primary contigs `. + +Essentially, the script ``fc_graph_to_contig`` generates contigs given sequence data and the final assembly graph. +Currently it generates :ref:`primary contigs ` as well as all +:ref:`associated contigs ` without any filtering. Some post-processing to remove duplicate +:ref:`associated contigs ` induced by errors will generally be necessary. diff --git a/docs/fc_ovlp_filter.rst b/docs/fc_ovlp_filter.rst new file mode 100644 index 00000000..b76ba494 --- /dev/null +++ b/docs/fc_ovlp_filter.rst @@ -0,0 +1,75 @@ +.. ovlp_filter: + +################# +fc_ovlp_filter.py +################# + + +.. code:: bash + + usage: fc_ovlp_filter [-h] [--n_core N_CORE] [--fofn FOFN] [--db DB_FN] + [--max_diff MAX_DIFF] [--max_cov MAX_COV] + [--min_cov MIN_COV] [--min_len MIN_LEN] [--bestn BESTN] + [--stream] [--debug] [--silent] + + a simple multi-processes LAS ovelap data filter + + optional arguments: + -h, --help show this help message and exit + --n_core N_CORE number of processes used for generating consensus; 0 + for main process only (default: 4) + --fofn FOFN file contains the path of all LAS file to be processed + in parallel (default: None) + --db DB_FN read db file path (default: None) + --max_diff MAX_DIFF max difference of 5' and 3' coverage (default: None) + --max_cov MAX_COV max coverage of 5' or 3' coverage (default: None) + --min_cov MIN_COV min coverage of 5' or 3' coverage (default: None) + --min_len MIN_LEN min length of the reads (default: 2500) + --bestn BESTN output at least best n overlaps on 5' or 3' ends if + possible (default: 10) + --stream stream from LA4Falcon, instead of slurping all at once; + can save memory for large data (default: False) + --debug, -g single-threaded, plus other aids to debugging (default: + False) + --silent suppress cmd reporting on stderr (default: False) + + +Not all overlaps are "independent", so it is possible to impose some filtering step to reduce computation and +assembly graph complexity. For example, if a read is fully contained in another read, the overlap information +between these two reads does not provide extra information for re-constructing the genome. Also, due to the +transitive property of the overlapping relations, a lot of overlap information can be simply inferred. In fact, +the first stage for constructing contigs are to remove the transitive reducible edges. It means that we might +just needs the "best n overlaps" in the ``5'`` or ``3'`` ends. The ``--bestn`` parameter in :ref:`overlap_filtering_setting` +option can be used to control the maximum overlap reported for each read. + +Another useful heuristics is to only keep reads that have average ``5'`` and ``3'`` coverage. That's because if a read +ends in a repeat, it might have higher than normal coverage at the end which is a repeat. And such reads do not +provide much value for uniquely resolving the related repeats. We can filter them out and hopefully there are +reads which span through the repeats and have "normal" unique anchors on both ends. Also, if the coverage is too +low on one end of a read, it could be just too many errors or sequencing artifacts over there. Such reads create +"spurs" in the assembly graph which are typically filtered out anyway. The --max_cov and --min_cov are used for +filtering reads that have too high or too low overlaps. + +The filtering scripts also allows filtering out some "split" reads. If a read have very unequal coverage between +the ``5'`` and ``3'`` ends, it can be also a signal that one end is a repeat. The ``--max_diff`` parameter can be used to +filter out the reads where one ends has much more coverage than the other end. + +What is the right numbers used for these parameters? These parameters may the most tricky ones to be set right. +If the overall coverage of the error corrected reads longer than the length cut off is known and reasonable high +(e.g. greater than 20x), it might be safe to set ``--min_cov`` to be 5, max_cov to be three times of the average +coverage and the max_diff to be twice of the average coverage. However, in low coverage case, it might better +to set ``--min_cov`` to be one or two. A helper script called :doc:`fc_ovlp_stats`` can help to dump the number of the +``3'`` and ``5'`` overlap of a given length cutoff, you can plot the distribution of the number of overlaps to make a +better decision. + +One can also set the ``--max_diff`` and ``--max_cov`` to be really high to avoid any filtering if that is preferred +in some cases. + +This filtering process will certainly filter out information about high copy repeats. Namely, those repeats will +likely to be filtered out totally and do not appear in the final assembly. If you are interested in those repeats +even though they may not be able to placed within some longer contig, you will probably want to avoid filtering +them out or process them differently. In general, it might be more efficient and useful to process those repeats +separately. Including them in the assembly process typically does not help much for getting better contiguity and +maybe messy for post-processing with current algorithms. I think it is a very interesting but also very challenging +bioinformatics topic on how to process these repeats better for improving assembly beside understand the nature +of these repeats. \ No newline at end of file diff --git a/docs/fc_ovlp_to_graph.rst b/docs/fc_ovlp_to_graph.rst new file mode 100644 index 00000000..abf9d71d --- /dev/null +++ b/docs/fc_ovlp_to_graph.rst @@ -0,0 +1,104 @@ +.. _ovlp_to_graph: + + +################### +fc_ovlp_to_graph.py +################### + +Here is the usage information for running fc_ovlp_to_graph.py: + +.. code:: bash + + usage: fc_ovlp_to_graph.py [-h] [--min_len MIN_LEN] [--min_idt MIN_IDT] + [--lfc] + overlap_file + + a example string graph assembler that is desinged for handling diploid genomes + + positional arguments: + overlap_file a file that contains the overlap information. + + optional arguments: + -h, --help show this help message and exit + --min_len MIN_LEN minimum length of the reads to be considered for + assembling + --min_idt MIN_IDT minimum alignment identity of the reads to be considered + for assembling + --lfc use local flow constraint method rather than best overlap + method to resolve knots in string graph + +In some case, you might want to lower the min_idt to keep more overlap or increase min_len to reduce the number of +overlap used for constructing the contig after the overlap filtering step. The ``--lfc`` toggles the rule for resolving +local knots in the graph. If ``--lfc`` is not specified, "the best overlapped edge" will be kept when there are multiple +in- or out- edges from a node while the others will be removed. + +The first stage of the assembly is to construct the initial string graph and classify each edges in the string graph. +``sg_edges_list`` contained the information of the information of the edges in the full string graph and the +classification. For example, 5 edges are shown in the five lines of the file below + +.. code:: bash + + $ head -5 sg_edges_list + 000017363:B 000007817:E 000007817 10841 28901 10841 99.52 TR + 000015379:E 000004331:B 000004331 6891 0 18178 99.35 TR + 000006813:B 000000681:E 000000681 7609 23795 7616 99.72 TR + 000002258:E 000002505:B 000002505 5850 0 17215 99.62 TR + 000013449:B 000012565:B 000012565 3317 0 20570 99.72 G + + +The first two columns indicates the in and out node of the edge. The node notation contains two files operated by :. +The first field is the read identifier. The second field is either B or E. B is the ``5'`` end of the read and E is the +``3'`` end of the reads. The next three field indicates the corresponding sequences of the edges. In this example, the +edge in the first line contains the sequence from read ``000007817`` base ``[10841, 28901)``. If the second coordinate is +smaller than the first one, it means the corresponded sequence is reverse complimented. The next two column are the +number of overlapped base and the overlap identity. The final column is the classification. Currently, there are 4 +different types ``G``, ``TR``, ``R``, and ``S``. An edge with type ``G`` is used for the final string graph. A ``TR`` +means the edge is transitive reducible. ``R`` means the edge is removed during the local repeat resolution and ``S`` +means the edge is likely to be a "spur" which only one ends is connected. + +The initial string graph is further to be simplified into a set of "unitig" edges. The ``utg_data`` file contains the +details of each unitig. Each line in the file represents a unitig. The first three fields are "start node", "via node", +and "end node". Two untigs might have the same "start node" and "end node", so we need another "via node" to uniquely +identify the unitigs. Here is an example of the utg_data files: + + +.. code:: bash + + $ head -10 utg_data + 000015696:B 000009028:B 000016941:B contained 16438 134865 000015696:B~000006612:B~000002456:B~000014643:B~000007407:B~000015939:E~000009028:B~000016941:B + 000010623:B 000015633:B 000014991:B contained 30158 18666 000010623:B~000015633:B~000014991:B + 000015636:B 000002245:B 000010757:E contained 15402 40356 000015636:B~000002245:B~000010757:E + 000014184:E NA 000012028:E compound 14895 56928 000014184:E~000012765:E~000012028:E|000014184:E~000007953:B~000012028:E + 000010757:B NA 000015636:E compound 15402 40356 000010757:B~000002245:E~000015636:E|000010757:B~000014783:E~000015636:E + 000014184:E 000007953:B 000012028:E contained 14792 32932 000014184:E~000007953:B~000012028:E + 000010623:B NA 000014991:B compound 30148 163627 000010623:B~000015633:B~000014991:B|000010623:B~000001407:B~000014991:B + 000012028:B 000012765:B 000014184:B contained 19137 56928 000012028:B~000000382:E~000012765:B~000014184:B + 000016941:B 000003353:B 000008783:B simple 88381 615439 000016941:B~000003353:B~000010261:B~000011789:E~000017006:B~000016307:B~... + 000014991:B 000013790:E 000002926:B simple 392373 2274104 000014991:B~000013790:E~000004614:B~000003329:B~000004898:B~000000461:B~000017105:E~... + +The forth field indicates the type of the unitigs, the fifth field is the estimate length of the unitig and the six +field is the total number of overlapped bases in the unitig. There are three kinds of unitigs: "simple", "contained", +and "compound". "Simple" unitigs are those unitigs which are just a simple path (every node has one in- and one +out-edge except the begining and ending nodes of the path.) It is represented by a list of nodes which each node +is separated by ~ characters in the 7th column. The "contained" contigs are simple path but those unitigs are also +part of other "compound" paths. The "compound" unitigs represents bubble-like subgraph in the graph. While it is not +"simple", it has well defined in- and out- nodes and they are treated as a single unit when the contigs are +constructed. The structure inside a "compound" unitig can be from biological nature or sequencing/alignment errors. +Each edge in the "compound" unitig subgraph are encoded explicitly as a collection of simple contained unitigs in +the 7th column. The contained unitigs within a compound unitig are separated by the ``|`` character. + +The file ctg_paths encodes the graph for each contig after the unitigs are analyzed and put into contigs. Each line +has 7 columns. The first column is the contig ID. The contig ID are just the serial numbers followed by R or F. +Two contigs with same serial number but different endings are "dual" to each other. Namely, they are constructed +from "dual" edges and they are mostly reverse complemented to each other except near the ends of the contigs. +The second column is the type of contig. If a unitig is circular (the beginning node and the ending node are the +same), then it will be marked as ``ctg_circular``. Everything else will be ``ctg_linear``. In some case, even a contig +is marked as ``ctg_linear``, it can be still a circular contig if the beginning node and the ending node are the +same but it is not a "simple" path. One can detect that by checking the beginning and ending nodes if necessary. + +The third field indicates the first unitig in the contig in the form of begin_node~via_node~end_node. The fourth +field is the ending node of the contig. The 5th and 6th fields are the estimated length and the overlapped +based of the contig respectively. The final column are the unitigs in the contig. The three node format unitig +IDs are separated by ``|`` + + diff --git a/docs/glossary.rst b/docs/glossary.rst new file mode 100644 index 00000000..796d5311 --- /dev/null +++ b/docs/glossary.rst @@ -0,0 +1,77 @@ +.. caution:: These documents refer to an obsolete way of installing and running FALCON. They will remain up for historical context and for individuals still using the older version of FALCON/FALCON_unzip. + +.. attention:: The current PacBio Assembly suite documentation which includes new bioconda instructions for installing FALCON, FALCON_unzip and their associated dependencies can be found here `pb_assembly `_ + +.. image:: media/falcon_icon2.png + :height: 200px + :width: 200 px + :alt: Falcon Assembler + :align: right + +.. _glossary: + +Glossary +======== + +.. glossary:: + + associated contig + Alternate configuration (phase) of a portion of a primary contig*. See :ref:`this ` + discussion on primary vs associated contigs + + compound path + multi-paths from a single source to a single sink in a graph + + contig + contiguous sequence output from a genome assembler + + error correction + The process of combining data from multiple raw sequences with random error profile + together to eliminate the errors. + + full-pass subread + A subread that begins at one adapter sequence and ends at another adapter sequence. A + full-pass subread does not begin or end in the middle of an insert sequence. + + haplotig + Contig from specific haplotype + + pread + Pre-assembled Reads, error corrected reads through the pre-assembly process. + + pre-assembly + Error correction process assembling raw sequences to generate high qualityy consensus for the + final step of assembly. + + primary contig + contig which captures a contiguous part of a genome regardless the variations due to the + variation between haplotypes associated contig generated by alternative paths from a portion + in the primary contig + + proper overlap + read overlaps without unaligned overhangs: + + Quiver + A highly accurate consensus and variant caller that can generate 99.999% accurate consensus + sequences using local realignment and the full range of quality scores associated with + Pacific Biosciences reads. Part of the SMRT® Analysis suite. + + rawreads + Uncorrected raw SMRTcell movie data + + simple path + a path without any branches in the assembly graph + + string graph + see `The fragment assembly string graph `_ + by Eugene W. Myers, 2005 + + subread + Each polymerase read is partitioned to form one or more subreads, which contain sequence + from a single pass of a polymerase on a single strand of an insert within a SMRTbell™ + template and no adapter sequences. The subreads contain the full set of quality values and + kinetic measurements. Subreads are useful for applications like de novo assembly, + resequencing, base modification analysis, and so on. + + + diff --git a/docs/hgap4_adapt.rst b/docs/hgap4_adapt.rst new file mode 100644 index 00000000..6eedb812 --- /dev/null +++ b/docs/hgap4_adapt.rst @@ -0,0 +1,143 @@ +.. _hgap4_adapt: + +.. caution:: These documents refer to an obsolete way of installing and running FALCON. They will remain up for historical context and for individuals still using the older version of FALCON/FALCON_unzip. + +.. attention:: The current PacBio Assembly suite documentation which includes new bioconda instructions for installing FALCON, FALCON_unzip and their associated dependencies can be found here `pb_assembly `_ + + +============ +Running Unzip on HGAP4 output +============ + +Overview +-------- + +``HGAP4`` is a ``FALCON``-based assembly pipeline, available through the SMRT Link interface. The pipeline itself encapsulates *de novo* assembly and polishing of the resulting contigs, but *not* the ``FALCON-unzip`` process as well. ``FALCON-unzip`` is currently available as a standalone tool, runnable only via command line. + +Although ``HGAP4`` runs ``FALCON`` under the hood, the folder structure it generates is different than that of ``FALCON``. The ``FALCON-unzip``, however, requires the assembly folders to be formatted in the ``FALCON``-style. + +This tutorial describes the necessary steps required to adjust the HGAP4 output to be compatible with a form required by ``FALCON-unzip``. + + +In brief, the majority of work required to adjust the ``HGAP4`` output to a ``FALCON``-compatible directory structure is implemented in a script called ``hgap4_adapt``. This script lives in the ``FALCON`` repository. + +The complete process is composed of the following steps: + +1. Installing ``FALCON`` and ``FALCON-unzip``. +2. Running ``hgap4_adapt``. +3. Creating the ``fc_unzip.cfg`` configuration file for ``FALCON-unzip``. +4. Creating the ``input.fofn`` and ``input_bam.fofn``. +5. Running ``FALCON-unzip``. + +**IMPORTANT:** ``FALCON-unzip`` can only be run on HGAP4 jobs which had the ``Save Output for Unzip`` option turned on. It is not possible to run ``FALCON-unzip`` otherwise, because critical files will be missing from your job's output. + + +1. Installing ``FALCON`` and ``FALCON-unzip`` +--------------------------------------------- + +The latest versions of ``FALCON`` and ``FALCON-unzip`` are available as precompiled Linux binaries. The easiest approach to installing them is through a wrapper script, described here: + +:ref:`Quick Start` + +Follow this approach to set-up the environment before moving on to step 2. + +Alternatively, one can install the binaries manually by following the instructions here: +https://github.com/PacificBiosciences/FALCON_unzip/wiki/Binaries + + +2. Running ``hgap4_adapt`` +-------------------------- + +Once the ``FALCON`` installation was successful, one needs to activate the installation environment to make the ``hgap4_adapt`` script available. This will also activate ``FALCON`` and ``FALCON-unzip``. To verify the installation, run the following: + +.. code-block:: bash + + source /path/to/your/install/dir/fc_env/bin/activate + python -m falcon_kit.mains.hgap4_adapt --help + +.. + +If everything was successful, this should output verbose usage information to screen. After this is set-up and working, adapting an existing HGAP4 run is as simple as the following example (take note of the dummy path, and replace it with a real one): + +.. code-block:: bash + + source /path/to/your/install/dir/fc_env/bin/activate + + job_dir=/path/to/your/hgap4/job/123/123456/ + mkdir –p example1 + cd example1 + python -m falcon_kit.mains.hgap4_adapt --job-output-dir=${job_dir} + +.. + +The result should be visible in the ``example1`` directory - it should now be populated to folders resembling a typical ``FALCON`` assembly run. + + +3. Creating the ``fc_unzip.cfg`` configuration file for ``FALCON-unzip`` +------------------------------------------------------------------------ + +For help on .cfg files, please take a look at these Wiki pages: + +- https://github.com/PacificBiosciences/FALCON/wiki +- https://github.com/PacificBiosciences/FALCON_unzip/wiki +- https://github.com/PacificBiosciences/FALCON-integrate/wiki + + +4. Creating the ``input.fofn`` and ``input_bam.fofn`` +----------------------------------------------------- + +The ``input.fofn`` file ("file of file names") contains the paths to files containing plain FASTA sequences of your raw reads, one file per row. All raw reads in the FASTA format should be available in your job dir: + +.. code-block:: bash + + job_dir=/path/to/your/hgap4/job/123/123456/ + mkdir –p example1 + cd example1 + + echo "${job_dir}/tasks/pbcoretools.tasks.gather_fasta-1/file.fasta" > input.fofn + +.. + + +The ``input_bam.fofn`` is required for the polishing step. This file is composed of a list of all BAM files from the input dataset which was provided to the initial HGAP4 run: + +.. code-block:: bash + + source /path/to/your/install/dir/fc_env/bin/activate + + job_dir=/path/to/your/hgap4/job/123/123456/ + mkdir –p example1 + cd example1 + + dataset summarize ${job_dir}/tasks/pbcoretools.tasks.filterdataset-0/filtered.subreadset.xml | grep -E "*.bam$" > input_bam.fofn + +.. + +5. Running ``FALCON-unzip`` +--------------------------- + +Before running ``FALCON-unzip``, the adapted folder structure should be similar to the following: + +.. code-block:: bash + + $ cd example1 + $ ls | xargs -n 1 + 0-rawreads + 1-preads_ovl + 2-asm-falcon + fc_unzip.cfg + input_bam.fofn + input.fofn + +.. + +Finally, to run ``FALCON-unzip``, do the following: + +.. code-block:: bash + + source /path/to/your/install/dir/fc_env/bin/activate + cd example1 + fc_unzip.py fc_unzip.cfg + fc_quiver.py fc_unzip.cfg + +.. diff --git a/docs/index.rst b/docs/index.rst new file mode 100644 index 00000000..a2857dbe --- /dev/null +++ b/docs/index.rst @@ -0,0 +1,48 @@ +.. FALCON \ documentation master file, created by + sphinx-quickstart on Wed Nov 2 13:50:34 2016. + You can adapt this file completely to your liking, but it should at least + contain the root `toctree` directive. + +.. caution:: These documents refer to an obsolete way of installing and running FALCON. They will remain up for historical context and for individuals still using the older version of FALCON/FALCON_unzip. + +.. attention:: The current PacBio Assembly suite documentation which includes new bioconda instructions for installing FALCON, FALCON_unzip and their associated dependencies can be found here `pb_assembly `_ + +.. image:: media/falcon_icon2.png + :height: 200px + :width: 200 px + :alt: Falcon Assembler + :align: right + + +FALCON Assembler +================ + + +.. toctree:: + :hidden: + + about + quick_start + pipeline + commands + tutorial + parameters + glossary + faq + changelog + resources + hgap4_adapt + +`FALCON `_ is a diploid aware genome assembler designed for Pacific Biosciences long read data. + +* :ref:`About ` - About FALCON +* :ref:`Quick Start ` - Want to start using FALCON immediately? +* :ref:`Tutorial ` - Follow an example on how to run FALCON +* :ref:`Pipeline ` - How does a FALCON job work? +* :ref:`Commands ` - Information on the different commands used +* :ref:`Parameters ` - Descriptions of parameters found in fc_run.cfg +* :ref:`Glossary ` - Glossary of FALCON jargon +* :ref:`Frequently Asked Questions ` - Frequently asked questions +* :ref:`Changelog ` - Changes +* :ref:`Resources ` - Talks and slides about PacBio +* :ref:`Running Unzip on HGAP4 output ` - How to run apply Unzip on finished HGAP4 jobs? diff --git a/docs/make.bat b/docs/make.bat new file mode 100644 index 00000000..afd6e6db --- /dev/null +++ b/docs/make.bat @@ -0,0 +1,281 @@ +@ECHO OFF + +REM Command file for Sphinx documentation + +if "%SPHINXBUILD%" == "" ( + set SPHINXBUILD=sphinx-build +) +set BUILDDIR=_build +set ALLSPHINXOPTS=-d %BUILDDIR%/doctrees %SPHINXOPTS% . +set I18NSPHINXOPTS=%SPHINXOPTS% . +if NOT "%PAPER%" == "" ( + set ALLSPHINXOPTS=-D latex_paper_size=%PAPER% %ALLSPHINXOPTS% + set I18NSPHINXOPTS=-D latex_paper_size=%PAPER% %I18NSPHINXOPTS% +) + +if "%1" == "" goto help + +if "%1" == "help" ( + :help + echo.Please use `make ^` where ^ is one of + echo. html to make standalone HTML files + echo. dirhtml to make HTML files named index.html in directories + echo. singlehtml to make a single large HTML file + echo. pickle to make pickle files + echo. json to make JSON files + echo. htmlhelp to make HTML files and a HTML help project + echo. qthelp to make HTML files and a qthelp project + echo. devhelp to make HTML files and a Devhelp project + echo. epub to make an epub + echo. epub3 to make an epub3 + echo. latex to make LaTeX files, you can set PAPER=a4 or PAPER=letter + echo. text to make text files + echo. man to make manual pages + echo. texinfo to make Texinfo files + echo. gettext to make PO message catalogs + echo. changes to make an overview over all changed/added/deprecated items + echo. xml to make Docutils-native XML files + echo. pseudoxml to make pseudoxml-XML files for display purposes + echo. linkcheck to check all external links for integrity + echo. doctest to run all doctests embedded in the documentation if enabled + echo. coverage to run coverage check of the documentation if enabled + echo. dummy to check syntax errors of document sources + goto end +) + +if "%1" == "clean" ( + for /d %%i in (%BUILDDIR%\*) do rmdir /q /s %%i + del /q /s %BUILDDIR%\* + goto end +) + + +REM Check if sphinx-build is available and fallback to Python version if any +%SPHINXBUILD% 1>NUL 2>NUL +if errorlevel 9009 goto sphinx_python +goto sphinx_ok + +:sphinx_python + +set SPHINXBUILD=python -m sphinx.__init__ +%SPHINXBUILD% 2> nul +if errorlevel 9009 ( + echo. + echo.The 'sphinx-build' command was not found. Make sure you have Sphinx + echo.installed, then set the SPHINXBUILD environment variable to point + echo.to the full path of the 'sphinx-build' executable. Alternatively you + echo.may add the Sphinx directory to PATH. + echo. + echo.If you don't have Sphinx installed, grab it from + echo.http://sphinx-doc.org/ + exit /b 1 +) + +:sphinx_ok + + +if "%1" == "html" ( + %SPHINXBUILD% -b html %ALLSPHINXOPTS% %BUILDDIR%/html + if errorlevel 1 exit /b 1 + echo. + echo.Build finished. The HTML pages are in %BUILDDIR%/html. + goto end +) + +if "%1" == "dirhtml" ( + %SPHINXBUILD% -b dirhtml %ALLSPHINXOPTS% %BUILDDIR%/dirhtml + if errorlevel 1 exit /b 1 + echo. + echo.Build finished. The HTML pages are in %BUILDDIR%/dirhtml. + goto end +) + +if "%1" == "singlehtml" ( + %SPHINXBUILD% -b singlehtml %ALLSPHINXOPTS% %BUILDDIR%/singlehtml + if errorlevel 1 exit /b 1 + echo. + echo.Build finished. The HTML pages are in %BUILDDIR%/singlehtml. + goto end +) + +if "%1" == "pickle" ( + %SPHINXBUILD% -b pickle %ALLSPHINXOPTS% %BUILDDIR%/pickle + if errorlevel 1 exit /b 1 + echo. + echo.Build finished; now you can process the pickle files. + goto end +) + +if "%1" == "json" ( + %SPHINXBUILD% -b json %ALLSPHINXOPTS% %BUILDDIR%/json + if errorlevel 1 exit /b 1 + echo. + echo.Build finished; now you can process the JSON files. + goto end +) + +if "%1" == "htmlhelp" ( + %SPHINXBUILD% -b htmlhelp %ALLSPHINXOPTS% %BUILDDIR%/htmlhelp + if errorlevel 1 exit /b 1 + echo. + echo.Build finished; now you can run HTML Help Workshop with the ^ +.hhp project file in %BUILDDIR%/htmlhelp. + goto end +) + +if "%1" == "qthelp" ( + %SPHINXBUILD% -b qthelp %ALLSPHINXOPTS% %BUILDDIR%/qthelp + if errorlevel 1 exit /b 1 + echo. + echo.Build finished; now you can run "qcollectiongenerator" with the ^ +.qhcp project file in %BUILDDIR%/qthelp, like this: + echo.^> qcollectiongenerator %BUILDDIR%\qthelp\FALCONDocumentation.qhcp + echo.To view the help file: + echo.^> assistant -collectionFile %BUILDDIR%\qthelp\FALCONDocumentation.ghc + goto end +) + +if "%1" == "devhelp" ( + %SPHINXBUILD% -b devhelp %ALLSPHINXOPTS% %BUILDDIR%/devhelp + if errorlevel 1 exit /b 1 + echo. + echo.Build finished. + goto end +) + +if "%1" == "epub" ( + %SPHINXBUILD% -b epub %ALLSPHINXOPTS% %BUILDDIR%/epub + if errorlevel 1 exit /b 1 + echo. + echo.Build finished. The epub file is in %BUILDDIR%/epub. + goto end +) + +if "%1" == "epub3" ( + %SPHINXBUILD% -b epub3 %ALLSPHINXOPTS% %BUILDDIR%/epub3 + if errorlevel 1 exit /b 1 + echo. + echo.Build finished. The epub3 file is in %BUILDDIR%/epub3. + goto end +) + +if "%1" == "latex" ( + %SPHINXBUILD% -b latex %ALLSPHINXOPTS% %BUILDDIR%/latex + if errorlevel 1 exit /b 1 + echo. + echo.Build finished; the LaTeX files are in %BUILDDIR%/latex. + goto end +) + +if "%1" == "latexpdf" ( + %SPHINXBUILD% -b latex %ALLSPHINXOPTS% %BUILDDIR%/latex + cd %BUILDDIR%/latex + make all-pdf + cd %~dp0 + echo. + echo.Build finished; the PDF files are in %BUILDDIR%/latex. + goto end +) + +if "%1" == "latexpdfja" ( + %SPHINXBUILD% -b latex %ALLSPHINXOPTS% %BUILDDIR%/latex + cd %BUILDDIR%/latex + make all-pdf-ja + cd %~dp0 + echo. + echo.Build finished; the PDF files are in %BUILDDIR%/latex. + goto end +) + +if "%1" == "text" ( + %SPHINXBUILD% -b text %ALLSPHINXOPTS% %BUILDDIR%/text + if errorlevel 1 exit /b 1 + echo. + echo.Build finished. The text files are in %BUILDDIR%/text. + goto end +) + +if "%1" == "man" ( + %SPHINXBUILD% -b man %ALLSPHINXOPTS% %BUILDDIR%/man + if errorlevel 1 exit /b 1 + echo. + echo.Build finished. The manual pages are in %BUILDDIR%/man. + goto end +) + +if "%1" == "texinfo" ( + %SPHINXBUILD% -b texinfo %ALLSPHINXOPTS% %BUILDDIR%/texinfo + if errorlevel 1 exit /b 1 + echo. + echo.Build finished. The Texinfo files are in %BUILDDIR%/texinfo. + goto end +) + +if "%1" == "gettext" ( + %SPHINXBUILD% -b gettext %I18NSPHINXOPTS% %BUILDDIR%/locale + if errorlevel 1 exit /b 1 + echo. + echo.Build finished. The message catalogs are in %BUILDDIR%/locale. + goto end +) + +if "%1" == "changes" ( + %SPHINXBUILD% -b changes %ALLSPHINXOPTS% %BUILDDIR%/changes + if errorlevel 1 exit /b 1 + echo. + echo.The overview file is in %BUILDDIR%/changes. + goto end +) + +if "%1" == "linkcheck" ( + %SPHINXBUILD% -b linkcheck %ALLSPHINXOPTS% %BUILDDIR%/linkcheck + if errorlevel 1 exit /b 1 + echo. + echo.Link check complete; look for any errors in the above output ^ +or in %BUILDDIR%/linkcheck/output.txt. + goto end +) + +if "%1" == "doctest" ( + %SPHINXBUILD% -b doctest %ALLSPHINXOPTS% %BUILDDIR%/doctest + if errorlevel 1 exit /b 1 + echo. + echo.Testing of doctests in the sources finished, look at the ^ +results in %BUILDDIR%/doctest/output.txt. + goto end +) + +if "%1" == "coverage" ( + %SPHINXBUILD% -b coverage %ALLSPHINXOPTS% %BUILDDIR%/coverage + if errorlevel 1 exit /b 1 + echo. + echo.Testing of coverage in the sources finished, look at the ^ +results in %BUILDDIR%/coverage/python.txt. + goto end +) + +if "%1" == "xml" ( + %SPHINXBUILD% -b xml %ALLSPHINXOPTS% %BUILDDIR%/xml + if errorlevel 1 exit /b 1 + echo. + echo.Build finished. The XML files are in %BUILDDIR%/xml. + goto end +) + +if "%1" == "pseudoxml" ( + %SPHINXBUILD% -b pseudoxml %ALLSPHINXOPTS% %BUILDDIR%/pseudoxml + if errorlevel 1 exit /b 1 + echo. + echo.Build finished. The pseudo-XML files are in %BUILDDIR%/pseudoxml. + goto end +) + +if "%1" == "dummy" ( + %SPHINXBUILD% -b dummy %ALLSPHINXOPTS% %BUILDDIR%/dummy + if errorlevel 1 exit /b 1 + echo. + echo.Build finished. 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19405 74 58 +000032623 18254 66 66 +000032624 28690 73 56 +000032633 13441 65 58 +000032634 13012 67 59 +000032636 19104 66 62 +000032637 15725 70 80 +000032638 17136 81 85 +000032643 14286 95 82 +000032644 17063 90 71 +000032645 13729 84 77 diff --git a/docs/parameters.rst b/docs/parameters.rst new file mode 100644 index 00000000..9ccc1edc --- /dev/null +++ b/docs/parameters.rst @@ -0,0 +1,325 @@ +.. caution:: These documents refer to an obsolete way of installing and running FALCON. They will remain up for historical context and for individuals still using the older version of FALCON/FALCON_unzip. + +.. attention:: The current PacBio Assembly suite documentation which includes new bioconda instructions for installing FALCON, FALCON_unzip and their associated dependencies can be found here `pb_assembly `_ + + +.. image:: media/falcon_icon2.png + :height: 200px + :width: 200 px + :alt: Falcon Assembler + :align: right + + +.. _parameters: + +########## +Parameters +########## + + +.. _configuration: + +Configuration +============= + +Here are some example ``fc_run.cfg`` and ``fc_unzip.cfg`` files. We make no guarantee that they will work with your +dataset and cluster configuration. We merely provide them as starting points that have proven themselves on internal +datasets. A lot of your success will depend purely on the quality of the input data prior to even engaging the FALCON +pipeline. Also, these particular configs were designed to work in our SGE compute cluster, so some tuning will likely +be necessary on your part. You should consult with your HPC administrator to assist in tuning to your cluster. + +FALCON Parameter sets +--------------------- + +:download:`fc_run_fungal.cfg ` - Has worked well on a 40Mb fungal genome + +:download:`fc_run_human.cfg ` - Has worked well on at least one human dataset + +:download:`fc_run_bird.cfg ` - Has worked well on at least one avian dataset + +:download:`fc_run_yeast.cfg ` - Has worked well on at least one yeast dataset + +:download:`fc_run_dipteran.cfg ` - Has worked well on at least one dipteran (insect) dataset + +:download:`fc_run_mammal.cfg ` - Has worked well on at least one mammalian dataset + +:download:`fc_run_mammalSequel.cfg ` - Has worked well on at least one mammalian Sequel dataset + +:download:`fc_run_plant.cfg ` - Has worked well on at least one plant (Ranunculales) dataset + +:download:`fc_run_arabidopsis.cfg ` - Configuration for arabidopsis assembly in Chin et al. +2016 + +:download:`fc_run_ecoli.cfg ` - Configuration for test *E. coli* dataset + +:download:`fc_run_ecoli_local.cfg ` - Configuration for test *E. coli* dataset run locally + + +FALCON_unzip Parameter sets +--------------------------- + +:download:`fc_unzip.cfg ` - General all purpose unzip config + + +Available Parameters +==================== + +.. _fc_run.cfg: + +fc_run.cfg +---------- + +.. _input_fofn: + +input_fofn + filename for the file-of-filenames (fofn) + Each line is fasta filename. + Any relative paths are relative to the location of the input_fofn. + +.. _input_type: + +input_type + "raw" or "preads" + + +.. _genome_size: + +genome_size + estimated number of base-pairs in haplotype + +.. _seed_coverage: + +seed-coverage + requested coverage for auto-calculated cutoff + +.. _length_cutoff: + +length_cutoff + Raw reads shorter than this cutoff won't be considered in the assembly process. + If '-1', then auto-calculate the cutoff based on genome_size and seed_coverage. + +.. _length_cutoff_pr: + +length_cutoff_pr + minimum length of seed-reads used after pre-assembly, for the "overlap" stage + +.. _target: + +target + "assembly" or "preads" + If "preads", then pre-assembly stage is skipped and input is assumed to be preads. + + +.. _default_concurrent_jobs: + +default_concurrent_jobs + maximum concurrency + This applies even to "local" (non-distributed) jobs. + +.. _pa_concurrent_jobs: + +pa_concurrent_jobs + Concurrency settings for pre-assembly + +.. _cns_concurrent_jobs: + +cns_concurrent_jobs + Concurrency settings for consensus calling + + One can use cns_concurrent_jobs to control the maximum number of concurrent consensus jobs submitted to the + job management system. The ``out.XXXXX.fasta`` files produced are used as input for the next step in the pipeline. + + +.. _ovlp_concurrent_jobs: + +ovlp_concurrent_jobs + Concurrency settings for Overlap detection + +.. _job_type: + +job_type + grid submission system, or "local" + Supported types include: "sge", "lsf", "pbs", "torque", "slurm", "local" + case-insensitive + +.. _job_queue: + +job_queue + grid job-queue name + Can be overridden with section-specific sge_option_* + +.. _sge_option_da: + +sge_option_da + Grid concurrency settings for initial daligner steps ``0-rawreads/`` + +.. _sge_option_la: + +sge_option_la + Grid concurrency settings for initial las-merging ``0-rawreads/`` + +.. _sge_option_cns: + +sge_option_cns + Grid concurrency settings for error correction consensus calling + +.. _sge_option_pda: + +sge_option_pda + Grid concurrency settings for daligner on preads ``1-preads_ovl/`` + +.. _sge_option_pla: + +sge_option_pla + Grid concurrency settings for las-merging on preads in ``1-preads_ovl/`` + +.. _sge_option_fc: + +sge_option_fc + Grid concurrency settings for stage 2 in ``2-asm-falcon/`` + +.. _pa_DBdust_option: + +pa_DBdust_option + Passed to ``DBdust``. Used only if ``dust = true``. + +.. _pa_DBsplit_option: + +pa_DBsplit_option + Passed to ``DBsplit`` during pre-assembly stage. + + +.. _pa_HPCdaligner_option: + +pa_HPCdaligner_option + Passed to ``HPC.daligner`` during pre-assembly stage. + We will add ``-H`` based on``length_cutoff``. + + The ``-dal`` option also controls the number of jobs being spawned. The number + for the ``-dal`` option determines how many blocks are compared to each in single jobs. Having a larger number + will spawn a fewer number of larger jobs, while the opposite will give you a larger number of small jobs. This + will depend on your on your compute resources available. + + In this workflow, the trace point generated by ``daligner`` is not used. ( Well, to be efficient, one should use the trace + points but one have to know how to pull them out correctly first. ) The ``-s1000`` argument makes the trace points + sparse to save some disk space (not much though). We can also ignore all reads below a certain + threshold by specifying a length cutoff with ``-l1000``. + + The biggest difference between this parameter and the ``ovlp_HPCdaligner_option`` parameter is that the latter needs + to have a relaxed error rate switch ``-e`` as the alignment is being performed on uncorrected reads. + +.. _pa_dazcon_option: + +pa_dazcon_option + Passed to ``dazcon``. Used only if ``dazcon = true``. + +.. _falcon_sense_option: + +falcon_sense_option + Passed to ``fc_consensus``. + Ignored if ``dazcon = true``. + +.. _falcon_sense_skip_contained: + +falcon_sense_skip_contained + Causes ``-s`` to be passed to ``LA4Falcon``. Rarely needed. + +.. _ovlp_DBsplit_option: + +ovlp_DBsplit_option + Passed to ``DBsplit`` during overlap stage. + +.. _ovlp_HPCdaligner_option: + +ovlp_HPCdaligner_option + Passed to ``HPC.daligner`` during overlap stage. + +.. _overlap_filtering_setting: + +overlap_filtering_setting + Passed to ``fc_ovlp_filter`` during assembly stage. + +.. _fc_ovlp_to_graph_option: + +fc_ovlp_to_graph_option + Passed to ``fc_ovlp_to_graph``. + +.. _skip_checks: + +skip_check + If "true", then skip ``LAcheck`` during ``LAmerge``/``LAsort``. + (Actually, ``LAcheck`` is run, but failures are ignored.) + When ``daligner`` bugs are finally fixed, this will be unnecessary. + + +.. _dust: + +dust + If true, then run ``DBdust`` before pre-assembly. + +.. _dazcon: + +dazcon + If true, then use ``dazcon`` (from pbdagcon repo). + + +.. _stop_all_jobs_on_failure: + +stop_all_jobs_on_failure + DEPRECATED + This was used for the old pypeFLOW refresh-loop, used by ``run0.py``. + (This is *not* the option to let jobs currently in SGE (etc) to keep running, which is still TODO.) + +.. _use_tmpdir: + +use_tmpdir + (boolean string) whether to run each job in ``TMPDIR`` and copy results back to nfs + If "true", use ``TMPDIR``. (Actually, ``tempfile.tmpdir``. See standard Python docs: https://docs.python.org/2/library/tempfile.html ) + If the value looks like a path, then it is used instead of ``TMPDIR``. + + +.. _fc_unzip.cfg: + +fc_unzip.cfg +------------ + +job_type + same as above. + grid submission system, or "local" + Supported types include: "sge", "lsf", "pbs", "torque", "slurm", "local" + case-insensitive + +input_fofn + This will be the same input file you used in your :ref:`fc_run.cfg` + +.. _input_bam_fofn: + +input_bam_fofn + List of movie bam files. Only necessary if performing consensus calling step at the end. + +smrt_bin + path to ``bin`` directory containing samtools, blasr, and various GenomicConsensus utilities + +jobqueue + Queue to submit SGE jobs to. + +sge_phasing + Phasing grid settings. Example: ``-pe smp 12 -q %(jobqueue)s`` + +sge_quiver + Consensus calling grid settings. Example ``-pe smp 24 -q %(jobqueue)s`` + +sge_track_reads + Read tracking grid settings. Example ``-pe smp 12 -q %(jobqueue)s`` + +sge_blasr_aln + ``blasr`` alignment grid settings. Example ``-pe smp 24 -q %(jobqueue)s`` + +sge_hasm + Final haplotyped assemble grid settings Example ``-pe smp 48 -q %(jobqueue)s`` + +unzip_concurrent_jobs + Number of concurrent unzip jobs to run at a time + +quiver_concurrent_jobs + Number of concurrent consensus calling jobs to run diff --git a/docs/pipeline.rst b/docs/pipeline.rst new file mode 100644 index 00000000..f15fc8f5 --- /dev/null +++ b/docs/pipeline.rst @@ -0,0 +1,282 @@ +.. _pipeline: + +.. caution:: These documents refer to an obsolete way of installing and running FALCON. They will remain up for historical context and for individuals still using the older version of FALCON/FALCON_unzip. + +.. attention:: The current PacBio Assembly suite documentation which includes new bioconda instructions for installing FALCON, FALCON_unzip and their associated dependencies can be found here `pb_assembly `_ + +.. image:: media/falcon_icon2.png + :height: 200px + :width: 200 px + :alt: Falcon Assembler + :align: right + + +######## +Pipeline +######## + +FALCON +====== + +A FALCON job can be broken down into 3 steps: + +1. :ref:`Overlap detection and error correction of rawreads ` +2. :ref:`Overlap detection between corrected reads ` +3. :ref:`String Graph assembly of corrected reads ` + +Each step is performed in it's own subdirectory within the FALCON job + +.. code-block:: bash + + falcon_job/ + ├── 0-rawreads/ # Raw read error correction directory + ├── 1-preads_ovl/ # Corrected read overlap detection + ├── 2-asm-falcon/ # String Graph Assembly + ├── mypwatcher/ # Job scheduler logs + ├── scripts/ + └── sge_log/ # deprecated + +The assembly process is driven by the script :ref:`fc_run.py` which should be sent to the scheduler or run on a head node +as it needs to persist throughout the entire assembly process. +It takes as input a single :ref:`config ` file typically named :ref:`fc_run.cfg`, which references a list +of fasta input files. The config file can be configured to +run locally, or submit to a job scheduler. However, if your dataset is anything larger than a bacterial sized +genome and unless you've tuned your system specifically for the organism you're trying to assemble, then most likely you +should be running on a cluster in order to more effectively leverage your computational resources. + +The configuration file also allows you to control other aspects of your job such as how your compute resources are +distributed as well as set many parameters to help you reach an "optimized" assembly according to the nature of +your input data. Unfortunately at this point there is no "magic" way to auto-tune the parameters so you should +probably spend some time in the :ref:`configuration` section to understand what options are available to you. +Some example configuration files can be found :ref:`here ` + + +.. _step_one: + +Step 1: Overlap detection and error correction of raw reads +----------------------------------------------------------- + +The first step of the pipeline is to identify all overlaps in the raw reads. Currently this is performed with +a modified version of Gene Myers' DALIGNER_. + +In order to identify overlaps, your :term:`raw reads` must first be converted from fasta format into a dazzler +database. This is a very I/O intensive process and will be run from the node where ``fc_run.py`` was executed. If this +is an issue, you should submit the command with a wrapper script to your grid directly. + +Once the database has been created and partitioned according to the parameters set in your +:ref:`fc_run.cfg `, an all vs all comparison of the reads must be performed. Accordingly, due to the +all vs all nature of the search this is the most time consuming step in the assembly process. To walk through the +actual steps of this part of the pipeline you should take a look at ``0-rawreads/prepare_rdb.sub.sh``. +Essentially it consists of running: + +1. :ref:`fasta2DB ` to format the database +2. :ref:`DBsplit ` to partition the database +3. :ref:`HPC.daligner ` to generate the :ref:`daligner` commands necessary for all-vs-all comparison + +After overlaps have been detected, you will be left with many ``job_*`` directories full of alignment files ``*.las`` +containing the information about the overlaps. After merging the alignment files (see ``m_*`` directories), the +next step is to error correct the reads leveraging the overlap information. In the ``0-rawreads/preads`` directory you +will find a series of scripts for +performing the error correction. The process basically consists of using ``LA4Falcon`` with a length cutoff and piping the +output to :ref:`fc_consensus.py ` to generate a fasta file with corrected reads. + + +.. code-block:: bash + + 0-rawreads/ + ├── job_* # dirs for all of the daligner jobs + ├── m_*/ # dirs for all of the LA4Merge jobs + ├── preads/ # sub-dir for preads generation + ├── report/ # pre-assembly stats + ├── cns-scatter/ # dir of scripts for falcon-consensus jobs + ├── daligner-scatter/ # dir of scripts for daligner jobs + ├── merge-scatter/ # dir of scripts for LAMerge jobs + ├── merge-gather/ # dir of scripts for gathering LAMerge inputs + ├── raw-gather/ # dir of scripts for gathering daligner jobs for merging + ├── input.fofn # list if your input *.fasta files + ├── length_cutoff # text file with length cutoff for seed reads + ├── pwatcher.dir # dir of individual pipeline jobs stderr and stdout + ├── prepare_rdb.sh # env wrapper script + ├── raw_reads.db # dazzler DB file + ├── raw-fofn-abs # dir of scripts for gathering raw reads inputs + ├── rdb_build_done # database construction sentinel file + ├── run_jobs.sh # listing of all overlap step commands + ├── run.sh # masker job script + ├── run.sh.done # sentinel file for all jobs + ├── task.json # json file specifying inputs, outputs, and params + └── task.sh # script to run json file + + + +The following parameters affect this step directly: + +* :ref:`sge_option_da ` +* :ref:`sge_option_la ` +* :ref:`pa_concurrent_jobs ` +* :ref:`cns_concurrent_jobs ` +* :ref:`pa_DBsplit_option ` +* :ref:`falcon_sense_option ` + +.. _DALIGNER: http://dazzlerblog.wordpress.com +.. _Dazzler: https://dazzlerblog.wordpress.com/2014/06/01/the-dazzler-db/ + + +.. _step_two: + +Step 2: Overlap detection of corrected reads +-------------------------------------------- + +The only conceptual difference between the first and second overlap steps is that consensus calling is +not performed in the second step. After :term:`pread` overlap detection, it's simply a +matter of extracting the information from the corrected reads database with ``DB2Falcon -U preads``. + +Depending on how well the error-correction step proceeded as well as the how much +initial coverage was fed into the pipeline (e.g. :ref:`length_cutoff `), the input data for this +step should be significantly reduced and thus, the second overlap detection step +will proceed significantly faster. + +The commands in this step of the pipeline are very similar to before albeit with different parameter settings to account +for the reduced error-rate of the :term:`preads `. See the driver script ``prepare_pdb.sub.sh`` for +details on actual parameter settings used. + +.. code-block:: bash + + 1-preads_ovl/ + ├── job_*/ # directories for daligner jobs + ├── m_*/ # directories for LA4Merge jobs + ├── db2falcon/ # dir of scripts for formatting preads for falcon + ├── gathered-las/ # dir of scripts for gathering daligner jobs + ├── merge-gather/ # dir of scripts for gathering LAMerge inputs + ├── merge-scatter/ # dir of scripts for LAMerge jobs + ├── daligner-scatter/ # dir of scripts for daligner jobs + ├── pdb_build_done # sentinel file for pread DB building + ├── preads.db # preads dazzler DB + ├── prepare_pdb.sh # env wrapper script + ├── pwatcher.dir # dir of individual pipeline jobs stderr and stdout + ├── run_jobs.sh # listing of all pread overlap job commands + ├── run.sh # masker job script + ├── run.sh.done # sentinel file for all jobs + ├── task.json # json file specifying inputs, outputs, and params + └── task.sh # script to run json file + +The following parameters affect this step directly: + +* :ref:`sge_option_pda ` +* :ref:`sge_option_pla ` +* :ref:`ovlp_concurrent_jobs ` +* :ref:`ovlp_DBsplit_option ` +* :ref:`ovlp_HPCdaligner_option ` + + +.. _step_three: + +Step 3: String Graph assembly +----------------------------- + +The final step of the FALCON Assembly pipeline is generation of the final :term:`String Graph` assembly and +output of contig sequences in fasta format. Four commands are run in the final phase of FALCON: + +1. :ref:`fc_ovlp_filter ` - Filters overlaps based on the criteria provided in :ref:`fc_run.cfg` +2. :ref:`fc_ovlp_to_graph ` - Constructs an overlap graph of reads larger than the length cutoff +3. :ref:`fc_graph_to_contig ` - Generates fasta files for contigs from the overlap graph. +4. :ref:`fc_dedup_a_tigs ` - Removes duplicate associated contigs + +You can see the details on the parameters used by inspecting ``2-asm_falcon/run_falcon_asm.sub.sh`` +This step of the pipeline is very fast relative to the overlap detection steps. Sometimes it may be useful to run +several iterations of this step with different parameter settings in order to identify a "best" assembly. + +The final output of this step is a fasta file of all of the primary contigs, ``p_ctg.fa`` as well as an associated contig +fasta file, ``a_ctg.fa`` that consists of all of the structural variants from the primary contig assembly. + +.. code-block:: bash + + 2-asm-falcon/ + ├── a_ctg_all.fa # all associated contigs, including duplicates + ├── a_ctg_base.fa # + ├── a_ctg_base_tiling_path # + ├── a_ctg.fa # De-duplicated associated fasta file + ├── a_ctg_tiling_path # tiling path informaiton for each associated contig + ├── falcon_asm_done # FALCON Assembly sentinal file + ├── p_ctg.fa # Fasta file of all primary contigs + ├── p_ctg_tiling_path # Tiling path of preads through each primary contig + ├── c_path # + ├── ctg_paths # corrected read paths for each contig + ├── fc_ovlp_to_graph.log # logfile for process of converting overlaps to assembly graph + ├── utg_data # + ├── sg_edges_list # list of all edges + ├── chimers_nodes # + ├── preads.ovl # List of all overlaps between preads + ├── run_falcon_asm.sh # env wrapper script + ├── task.json # json file specifying inputs, outputs, and params + ├── task.sh # script to run json file + ├── run.sh.done # sentinel file for all jobs + └── run.sh # Assembly driver script + + +The following parameters affect this step directly: + +* :ref:`sge_option_fc ` +* :ref:`overlap_filtering_setting ` +* :ref:`length_cutoff_pr ` + + +FALCON_unzip +============ + +`FALCON_unzip`_ operates from a completed FALCON job directory. After tracking the raw reads to contig, +A FALCON_unzip job can be broken down into 3 steps + +1. :ref:`Identify SNPs and assign phases ` +2. :ref:`Annotate Assembly graph with Phases ` +3. :ref:`Graph building ` + +.. code-block:: bash + + 3-unzip/ + ├── 0-phasing/ # Contig phasing jobs + ├── 1-hasm/ # Contig Graph assembly information + ├── read_maps/ # rawread_to_contigs; read_to_contig_map + ├── reads/ # raw read fastas for each contig + ├── all_p_ctg.fa # partially phased primary contigs + ├── all_h_ctg.fa # phased haplotigs + ├── all_p_ctg_edges # primary contig edge list + ├── all_h_ctg_edges # haplotig edge list + ├── all_h_ctg_ids # haplotig id index + └── all_phased_reads # table of all phased raw reads + + +.. _FALCON_unzip:: https://github.com/PacificBiosciences/FALCON_unzip + +.. _unzip_step_one: + +Step 1: Identify SNPs and assign phases +--------------------------------------- + +Inside of ``0-phasing/`` you vill find a number of directories for each contig. Each contains the scripts +to map the raw reads to the contigs and subsequently identify SNPs. The generated SNP tables can +subsequently be used to assign phases to reads. + + +.. _unzip_step_two: + +Step 2: Graph annotation and haplotig +------------------------------------- + +Inside of ``1-hasm/`` you can find the driver script ``hasm.sh`` which contains the commands necessary to +filter overlaps and traverse the assembly graph paths and subsequently output phased contig sequence. +Assembly Graphs for each contig as well as fasta files for the partially phased primary contigs and fully phased +haplotigs can be found in each ``1-hasm/XXXXXXF`` directory. + + +.. _unzip_step_three: + +Step 3: Call Consensus (Optional) +--------------------------------- + +Finally, the ``FALCON_unzip`` pipeline can optionally be used to run quiver and call high quality consensus. This step +takes as input the primary contig and haplotig sequences output in the previous step. For convenience, these files +have all been concatenated together into ``3-unzip/all_p_ctg.fa`` and ``3-unzip/all_h_ctg.fa`` respectively. +The final consensus output can be found in ``falcon_jobdir/4-quiver/cns_output/*.fast[a|q]``. +In order to run the consensus step as part of the FALCON_unzip pipeline, You need to provide the :ref:`input_bam_fofn` +:ref:`fc_unzip.cfg` option in order for this to work. + diff --git a/docs/preads.stats.txt b/docs/preads.stats.txt new file mode 100644 index 00000000..e1b0e263 --- /dev/null +++ b/docs/preads.stats.txt @@ -0,0 +1,39 @@ +Bin Count PercReads PercBases Average +36000 3 0.0 0.0 36272 +35000 3 0.0 0.1 35807 +34000 11 0.1 0.2 34888 +33000 8 0.1 0.2 34448 +32000 21 0.1 0.4 33498 +31000 27 0.2 0.7 32745 +30000 66 0.4 1.3 31660 +29000 71 0.6 1.9 30903 +28000 113 1.0 2.8 30056 +27000 142 1.4 3.9 29265 +26000 176 2.0 5.2 28501 +25000 228 2.7 6.9 27702 +24000 290 3.6 8.9 26896 +23000 375 4.7 11.4 26060 +22000 436 6.0 14.2 25268 +21000 598 7.9 17.9 24388 +20000 747 10.2 22.2 23509 +19000 1005 13.2 27.8 22571 +18000 1271 17.1 34.5 21636 +17000 1545 21.9 42.2 20737 +16000 1879 27.6 51.1 19851 +15000 1854 33.3 59.3 19108 +14000 1710 38.5 66.4 18489 +13000 925 41.4 70.0 18148 +12000 837 43.9 73.0 17819 +11000 906 46.7 75.9 17444 +10000 1003 49.8 78.9 17016 +9000 1128 53.2 82.0 16528 +8000 1167 56.8 84.8 16022 +7000 1297 60.8 87.6 15463 +6000 1461 65.3 90.3 14848 +5000 1695 70.4 92.9 14157 +4000 1793 75.9 95.2 13458 +3000 1995 82.0 97.2 12716 +2000 2075 88.4 98.7 11982 +1000 2294 95.4 99.7 11208 +0 1491 100.0 100.0 10730 + diff --git a/docs/quick_start.rst b/docs/quick_start.rst new file mode 100644 index 00000000..f296ad0f --- /dev/null +++ b/docs/quick_start.rst @@ -0,0 +1,46 @@ +.. _quick_start: + +.. caution:: These documents refer to an obsolete way of installing and running FALCON. They will remain up for historical context and for individuals still using the older version of FALCON/FALCON_unzip. + +.. attention:: The current PacBio Assembly suite documentation which includes new bioconda instructions for installing FALCON, FALCON_unzip and their associated dependencies can be found here `pb_assembly `_ + + +Quick Start Guide +================= + +Installation +------------ + +The quickest way to install FALCON + FALCON_unzip is to download and run this install script: + + +============================================================== ====================== ======= +Install script Tarball date Status +============================================================== ====================== ======= +:download:`install_unzip.sh ` :ref:`3122018tarball` +:download:`install_unzip.sh ` :ref:`8082018tarball` Beta +============================================================== ====================== ======= + + +.. code-block:: bash + + $ bash -ex install_unzip.sh /path/to/your/install/dir + + +.. NOTE:: + + This will clone the FALCON-integrate repository, FALCON_unzip binaries, build a virtualenv and launch a small test case assembly to ensure successful installation. + + samtools_ and minimap2_ must be installed separately and in your $PATH. + + If you don't see any errors, you will have installed FALCON/FALCON_unzip and successfully assembled and unzipped a small test dataset. At this point you should be ready to confidently launch a larger genome assembly. + +To activate your FALCON_unzip virtualenv in the future: + +.. code-block:: bash + + $ source /path/to/your/install/dir/fc_env/bin/activate + + +.. _samtools: http://www.htslib.org/download/ +.. _minimap2: https://github.com/lh3/minimap2 diff --git a/docs/raw_reads.stats.txt b/docs/raw_reads.stats.txt new file mode 100644 index 00000000..331c9085 --- /dev/null +++ b/docs/raw_reads.stats.txt @@ -0,0 +1,48 @@ +Bin Count PercReads PercBases Average +45000 1 0.0 0.0 45611 +44000 0 0.0 0.0 45611 +43000 0 0.0 0.0 45611 +42000 1 0.0 0.0 43824 +41000 0 0.0 0.0 43824 +40000 1 0.0 0.0 42680 +39000 4 0.0 0.0 40739 +38000 6 0.0 0.1 39750 +37000 8 0.0 0.1 38874 +36000 22 0.0 0.2 37641 +35000 31 0.1 0.3 36745 +34000 49 0.1 0.5 35823 +33000 69 0.2 0.7 34976 +32000 89 0.3 1.0 34183 +31000 144 0.5 1.5 33277 +30000 219 0.7 2.2 32329 +29000 280 1.0 3.0 31465 +28000 349 1.4 4.0 30643 +27000 428 1.9 5.3 29848 +26000 530 2.5 6.7 29046 +25000 689 3.2 8.5 28204 +24000 829 4.1 10.6 27383 +23000 986 5.2 13.0 26572 +22000 1286 6.6 16.0 25696 +21000 1529 8.3 19.5 24843 +20000 1821 10.3 23.3 23993 +19000 2216 12.8 27.8 23131 +18000 2461 15.5 32.5 22318 +17000 2858 18.6 37.7 21501 +16000 3004 21.9 42.8 20746 +15000 3149 25.4 47.9 20029 +14000 3438 29.2 53.1 19310 +13000 3641 33.2 58.2 18607 +12000 3999 37.6 63.3 17890 +11000 4357 42.4 68.5 17165 +10000 4713 47.6 73.7 16438 +9000 5007 53.1 78.6 15717 +8000 5363 59.0 83.3 14993 +7000 5268 64.8 87.4 14322 +6000 5196 70.6 90.9 13687 +5000 5024 76.1 93.8 13092 +4000 4907 81.5 96.1 12522 +3000 4826 86.8 97.8 11969 +2000 4910 92.2 99.1 11413 +1000 4754 97.5 99.8 10881 +0 2285 100.0 100.0 10626 + diff --git a/docs/references.rst b/docs/references.rst new file mode 100644 index 00000000..3deda124 --- /dev/null +++ b/docs/references.rst @@ -0,0 +1,6 @@ +.. _references: + +References +========== + +TODO \ No newline at end of file diff --git a/docs/repeatOvlp.rst b/docs/repeatOvlp.rst new file mode 100644 index 00000000..b1b58e99 --- /dev/null +++ b/docs/repeatOvlp.rst @@ -0,0 +1,8 @@ +.. _RepeatOvlp: + +.. image:: media/repeatOvlps.png + :height: 500px + :width: 500 px + :alt: Pread Ovlp Histogram with Repeats + :align: left + diff --git a/docs/resources.rst b/docs/resources.rst new file mode 100644 index 00000000..f4033e76 --- /dev/null +++ b/docs/resources.rst @@ -0,0 +1,103 @@ +.. _resources: + +Relevant Talks and Poster Presentations +======================================= + +Sequencing, Finishing and Analysis in the Future +------------------------------------------------ +May 23rd 2018, Sante Fe, NM + + +*FALCON-Phase talk by Sarah Kingan* +----------------------------------- + +:download:`FALCON-Phase ` + + +SMRT Informatics Developers Conference +-------------------------------------- +Jan 17th 2018, San Diego, CA + +:download:`Agenda ` + +*Keynote by Tim Smith* +---------------------- + +:download:`TimSmith_The_evolution_of_reference_assembly ` + + +*Software Updates from PacBio* +------------------------------ + +:download:`JimDrakePacBio_SMRTAnalysisUpdates ` + +*Lightning Talks* +----------------- + +:download:`ZevKronenberg_PhaseGenomics_HighQualityGenomesPacBioHiC ` + +:download:`SKoren_NHGRI_TrioBinning ` + +:download:`ETseng_PacBio_IsoPhase ` + +:download:`AConesa_UF-PF_IsoSeq_SQUANTI_TAPPAS ` + +:download:`BBowman_PacBio_RepeatAnalysisNoAmp ` + +:download:`AWenger_PacBio_PBSV +` + + +PAGXXVI in San Diego, January 2018 +---------------------------------- + +*PacBio Presented Posters* +-------------------------- + +:download:`Concepcion-PAG-2018-Maize_Soy_SV.pdf ` + +:download:`Concepcion-PAG-2018-S.californicum_de novo_assembly.pdf ` + +:download:`Kingan-PAG-2018-BestPracticesforDiploidAssemblyofComplexGenomesUsingPacBioHops.pdf +` + +:download:`Tseng-PAG-2018-Haplotyping-of-full-length-transcripts.pdf +` + + +*Talks* +------- + +Zev Kronenberg (Phase Genomics, fmr Eichler Lab, UW) work on great ape genomes +:download:`ZKronenberg_GreatApeComparativeGenomics_PAG2018.pdf ` + +Lloyd Low (Williams Lab, Adelaide) work on waterbuffalo, includes issue of haplotype switching and scaffolding +:download:`LLow_WaterBuffalo_PAG2018.pdf ` + + +Tools for Polyploids Meeting +---------------------------- +San Diego Botanical Garden, Jan 12th, 2018 + +:download:`KinganPacBio_ToolsForPolyploidsPAG2018.pdf ` + +PacBio East Coast User Group Meeting +------------------------------------ +Baltimore, June 27th, 2017 + +:download:`Kingan_DiploidGenome_ECUGM2017_BFX.pdf ` + +:download:`Fritz_Sedlazeck_SVswithPacB.pdf ` + +:download:`Ghurye_PacBHiC_Asm.pdf ` + +:download:`Wenger_pbsv_BFXwrkshop.pdf ` + +:download:`Goodwin_SamplePrep_ExtraLongLibraries.pdf ` + +:download:`Smith_SamplePrep_BestPracLargeInsertLib.pdf ` + +:download:`HamidAshrafi_BlueberryAsmIsoSeq.pdf ` + + diff --git a/docs/scripts/ReadHist.R b/docs/scripts/ReadHist.R new file mode 100644 index 00000000..009fd7c5 --- /dev/null +++ b/docs/scripts/ReadHist.R @@ -0,0 +1,18 @@ +setwd("my/dir") +raw<-read.table("raw_reads.stats.txt", header=T) +preads<-read.table("preads.stats.txt",header=T) + +pdf(file="RawReadHist.pdf", width=11, height=8.5) +par(oma=c(4,4,2,0), cex=1.6, las=1, mar=c(4,4,2,2)) +plot(data=raw, Count~Bin, type="h",col="DeepSkyBlue", lwd=5, + ylab="", xlab="Read Length", main="Raw Reads") +mtext("Read Count", side=2, cex=1.7, las=3, line=4) +dev.off() + +pdf(file="PreadHist.pdf", width=11, height=8.5) +par(oma=c(4,4,2,0), cex=1.6, las=1, mar=c(4,4,2,2)) +plot(data=preads, Count~Bin, type="h",col="ForestGreen", lwd=5, + ylab="", xlab="Read Length", main="Preassembled Reads") +mtext("Read Count", side=2, cex=1.7, las=3, line=4) +dev.off() + diff --git a/docs/scripts/countFasta.pl b/docs/scripts/countFasta.pl new file mode 100644 index 00000000..8b24a91e --- /dev/null +++ b/docs/scripts/countFasta.pl @@ -0,0 +1,106 @@ +#!/usr/bin/perl -w +# count_fasta.pl +# AUTHOR: Joseph Fass (modified from script by Brad Sickler) +# LAST REVISED: November 2010 +# +# The Bioinformatics Core at UC Davis Genome Center +# http://bioinformatics.ucdavis.edu +# Copyright (c) 2009 The Regents of University of California, Davis Campus. +# All rights reserved. +use strict; +use POSIX; +use Getopt::Std; + +my $usage = "\n\nusage: $0 [-i ] \n". + "Produces a histogram of sequence lengths and other measures to standard out.\n\n". + "-i # specify bin size for histogram (default 100)\n\n"; +our($opt_i); # histogram interval +getopts('i:') or die $usage; +if (!defined($opt_i) or !($opt_i =~ m/^[0-9]+$/)) {$opt_i = 100;} + +if( ( $#ARGV + 1 ) < 1 ) { + die $usage; +} + +# Read in sequences from one or more fasta files +my @data_files; +for(my $i = 0; $i < ($#ARGV + 1); $i++){ + $data_files[$i] = $ARGV[$i]; +} +my $Id; +my %seq; +foreach my $file (@data_files){ + open(FASTA, $file) or die"Can't open file $file\n"; + while () { +# if (/^>(.+)\s/) { $Id = $1; } # overwrites if id up to 1st whitespace is non-unique in file + if (/^>(.*)$/) { $Id = $1; } +# if (/(\w+)/) { + elsif (/^(\S+)$/) { $seq{$Id} .= $1 if $Id; } + } + close (FASTA); +} + +# Count the number of sequences in the file and create a histogram of the distribution +my $n = 0; +my $int; +my $totalLength = 0; +my $gcCount = 0; +my %len= (); +my @seqLengths; +foreach my $id (keys %seq) { + push @seqLengths, length($seq{$id}); # record length for N50 calc's + $n++; + $int = floor( length($seq{$id})/$opt_i ); + $totalLength += length($seq{$id}); + $gcCount += ($seq{$id} =~ tr/gGcC/gGcC/); + if( !defined($len{$int}) ) { + $len{$int} = 1; + } else { + $len{$int}++; + } +} + +# Calculate N25, N50, and N75 and counts +my $N25; my $N50; my $N75; +my $N25count=0; my $N50count=0; my $N75count=0; +my $frac_covered = $totalLength; +@seqLengths = reverse sort { $a <=> $b } @seqLengths; +$N25 = $seqLengths[0]; +while ($frac_covered > $totalLength*3/4) { + $N25 = shift(@seqLengths); + $N25count++; $N50count++; $N75count++; + $frac_covered -= $N25; +} +$N50 = $N25; +while ($frac_covered > $totalLength/2) { + $N50 = shift(@seqLengths); + $N50count++; $N75count++; + $frac_covered -= $N50; +} +$N75 = $N50; +while ($frac_covered > $totalLength/4) { + $N75 = shift(@seqLengths); + $N75count++; + $frac_covered -= $N75; +} + +# Print out the results +print "\n"; +my @ints = sort { $a <=> $b } keys(%len); +for(my $i=$ints[0]; $i <= $ints[-1]; $i++) { + $len{$i} = 0 if(!defined($len{$i})); + printf "%d:%d \t$len{$i}\n", ( ($i*$opt_i), ($i*$opt_i+$opt_i-1) ); +} +print "\nTotal length of sequence:\t$totalLength bp\n"; +print "Total number of sequences:\t$n\n"; +# not sure if these right wrt N25 and N75 .. +print "N25 stats:\t\t\t25% of total sequence length is contained in the ".$N25count." sequences >= ".$N25." +bp\n"; +print "N50 stats:\t\t\t50% of total sequence length is contained in the ".$N50count." sequences >= ".$N50." +bp\n"; +print "N75 stats:\t\t\t75% of total sequence length is contained in the ".$N75count." sequences >= ".$N75." +bp\n"; +print "Total GC count:\t\t\t$gcCount bp\n"; +printf "GC %%:\t\t\t\t%.2f %%\n", ($gcCount/$totalLength * 100); +print "\n"; + diff --git a/docs/scripts/install_unzip_180312.sh b/docs/scripts/install_unzip_180312.sh new file mode 100644 index 00000000..eefb9a61 --- /dev/null +++ b/docs/scripts/install_unzip_180312.sh @@ -0,0 +1,97 @@ +#!/bin/bash +# This is a simple script to install FALCON-integrate + FALCON_unzip. +# contact: gconcepcion@pacificbiosciences.com + +###Install script dependencies +##This script should work on both Ubuntu/CentOS as long as the following dependencies are installed and +##available in your $PATH + +#source /mnt/software/Modules/current/init/bash +#module load python/2.7.9 +#module load virtualenv/13.0.1 + +###These three are necessary for Unzip +#module load gcc/6.4.0 +#module load samtools + +###Variables +INPUT=$1 +if [ -z ${INPUT} ]; then + ROOT=$(pwd) +else + ROOT=$(readlink -f ${INPUT}) +fi + +SRC=${ROOT}/src +VENV_BASE=${ROOT}/fc_env +SLUG=$(date +%y%m%d) +VENV=${VENV_BASE}_${SLUG} +FALCON_PATH=${SRC}/FALCON-integrate +FALCON_REPO="https://github.com/PacificBiosciences/FALCON-integrate.git" +DL_CLOUD="https://downloads.pacbcloud.com/public/falcon/" + +###Test whether you need ucs4 or ucs2 tarball with this command: +UCS_VER=$(python2.7 -c 'import sysconfig,pprint; pprint.pprint(sysconfig.get_config_vars()["Py_UNICODE_SIZE"])') +UNZIP_TARBALL="falcon-2018.03.12-04.00-py2.7-ucs${UCS_VER}.tar.gz" + +###Cleanup +if [ ! -d ${ROOT} ]; then + mkdir ${ROOT} +fi + +if [ -d ${SRC} ]; then + rm -rf ${SRC} + mkdir ${SRC} +else + mkdir ${SRC} +fi + +if [ -d "${VENV}" ]; then + echo "Removing previous build from today" + rm -rf $VENV +fi + +if [ -d "${FALCON_PATH}" ]; then + echo "Removing old FALCON-integrate repo" + rm -rf ${FALCON_PATH} +fi + +if [ -L "${VENV_BASE}" ]; then + unlink ${VENV_BASE} +fi + +###Make python virtualenv +virtualenv --python=$(which python) --no-site-packages ${VENV} +echo "export LD_LIBRARY_PATH=${VENV}/lib:\${LD_LIBRARY_PATH}" >>${VENV}/bin/activate +source ${VENV}/bin/activate + +###Install FALCON and FALCON_unzip + +cd ${SRC} +curl -O ${DL_CLOUD}/${UNZIP_TARBALL} +tar zxvf ${UNZIP_TARBALL} -C ${VENV} + +ln -s ${VENV} ${VENV_BASE} + +###Install MUMmer 4.0.0 - for FALCON_unzip +MUMMER_400='https://github.com/mummer4/mummer/releases/download/v4.0.0beta2/mummer-4.0.0beta2.tar.gz' +cd ${SRC} +wget ${MUMMER_400} -P ${SRC} --no-check-certificate +tar zxvf mummer-4.0.0beta2.tar.gz +cd mummer-4.0.0beta2 +./configure --prefix=${VENV} +make && make install + +###Link samtools +ln -s $(which samtools) ${VENV}/bin/samtools + +###Test falcon_unzip pipeline +cd ${SRC} && git clone ${FALCON_REPO} +cd ${FALCON_PATH} && git submodule update --init +cd ${FALCON_PATH}/FALCON-examples && ../git-sym/git-sym update run/greg200k-sv2 +cd run/greg200k-sv2 && fc_run fc_run.cfg +fc_unzip.py fc_unzip.cfg + +echo "FALCON & FALCON_unzip have been successfully installed into a virtualenv." +echo "To activate the FALCON_unzip environment:" +echo "$ source ${VENV}/bin/activate" diff --git a/docs/scripts/install_unzip_180808.sh b/docs/scripts/install_unzip_180808.sh new file mode 100644 index 00000000..61b50b52 --- /dev/null +++ b/docs/scripts/install_unzip_180808.sh @@ -0,0 +1,97 @@ +#!/bin/bash +# This is a simple script to install FALCON-integrate + FALCON_unzip. +# contact: gconcepcion@pacificbiosciences.com + +###Install script dependencies +##This script should work on both Ubuntu/CentOS as long as the following dependencies are installed and +##available in your $PATH + +#source /mnt/software/Modules/current/init/bash + +#module load python/2.7.14-UCS4 +#module load virtualenv/13.0.1 +###These three are necessary for Unzip +#module load samtools +#module load minimap2 + +###Variables +INPUT=$1 +if [ -z ${INPUT} ]; then + ROOT=$(pwd) +else + ROOT=$(readlink -f ${INPUT}) +fi + +SRC=${ROOT}/src +VENV_BASE=${ROOT}/fc_env +SLUG=$(date +%y%m%d) +VENV=${VENV_BASE}_${SLUG} +FALCON_PATH=${SRC}/FALCON-integrate +FALCON_REPO="https://github.com/PacificBiosciences/FALCON-integrate.git" +DL_CLOUD="https://downloads.pacbcloud.com/public/falcon" + +python --version +###Test whether you need ucs4 or ucs2 tarball with this command: +UCS_VER=$(python2.7 -c 'import sysconfig,pprint; pprint.pprint(sysconfig.get_config_vars()["Py_UNICODE_SIZE"])') +UNZIP_TARBALL="falcon-2018.08.08-21.41-py2.7-ucs${UCS_VER}-beta.tar.gz" + +###Cleanup +if [ ! -d ${ROOT} ]; then + mkdir ${ROOT} +fi + +if [ -d ${SRC} ]; then + rm -rf ${SRC} + mkdir ${SRC} +else + mkdir ${SRC} +fi + +if [ -d "${VENV}" ]; then + echo "Removing previous build from today" + rm -rf $VENV +fi + +if [ -d "${FALCON_PATH}" ]; then + echo "Removing old FALCON-integrate repo" + rm -rf ${FALCON_PATH} +fi + +if [ -L "${VENV_BASE}" ]; then + unlink ${VENV_BASE} +fi + +###Make python virtualenv +virtualenv --python=$(which python) --no-site-packages ${VENV} +echo "export LD_LIBRARY_PATH=${VENV}/lib:\${LD_LIBRARY_PATH}" >>${VENV}/bin/activate +source ${VENV}/bin/activate + +###Install FALCON and FALCON_unzip + +cd ${SRC} +curl -v -k -O ${DL_CLOUD}/${UNZIP_TARBALL} +tar zxvf ${UNZIP_TARBALL} -C ${VENV} + +#ln -s ${VENV} ${VENV_BASE} + +###Install MUMmer 3.23 - for FALCON_unzip + +MUMMER_323='https://downloads.sourceforge.net/project/mummer/mummer/3.23/MUMmer3.23.tar.gz' +cd ${SRC} +wget ${MUMMER_323} -P ${SRC} --no-check-certificate +tar zxvf MUMmer3.23.tar.gz +cd MUMmer3.23 +make install +find . -maxdepth 1 -perm -111 -type f -exec cp {} ${VENV}/bin \; + +###Test falcon_unzip pipeline +cd ${SRC} && git clone ${FALCON_REPO} +cd ${FALCON_PATH} && git submodule update --init +cd ${FALCON_PATH}/FALCON-examples && ../git-sym/git-sym update run/greg200k-sv2 +git checkout 7aa47ddd99a6c2c8a87e76bce83eaa320b3d9474 +cd run/greg200k-sv2 && fc_run fc_run.cfg +fc_unzip.py fc_unzip.cfg + +echo "FALCON & FALCON_unzip have been successfully installed into a virtualenv." +echo "To activate the FALCON_unzip environment:" +echo "$ source ${VENV}/bin/activate" diff --git a/docs/tertiary.rst b/docs/tertiary.rst new file mode 100644 index 00000000..2e6d6ef5 --- /dev/null +++ b/docs/tertiary.rst @@ -0,0 +1,28 @@ +.. _tertiary: + +Tertiary +======== + +Slides presented at the East Coast UGM in Baltimore, June 27th, 2017 + +:download:`Kingan_DiploidGenome_ECUGM2017_BFX.pdf ` + + +:download:`Fritz_Sedlazeck_SVswithPacB.pdf ` + + +:download:`Ghurye_PacBHiC_Asm.pdf ` + + +:download:`Wenger_pbsv_BFXwrkshop.pdf ` + + +:download:`Goodwin_SamplePrep_ExtraLongLibraries.pdf ` + + +:download:`Smith_SamplePrep_BestPracLargeInsertLib.pdf ` + + +:download:`HamidAshrafi_BlueberryAsmIsoSeq.pdf ` + + diff --git a/docs/tutorial.rst b/docs/tutorial.rst new file mode 100644 index 00000000..cb65012e --- /dev/null +++ b/docs/tutorial.rst @@ -0,0 +1,388 @@ +.. caution:: These documents refer to an obsolete way of installing and running FALCON. They will remain up for historical context and for individuals still using the older version of FALCON/FALCON_unzip. + +.. attention:: The current PacBio Assembly suite documentation which includes new bioconda instructions for installing FALCON, FALCON_unzip and their associated dependencies can be found here `pb_assembly `_ + +.. image:: media/falcon_icon2.png + :height: 200px + :width: 200 px + :alt: FALCON Assembler + :align: right + +.. _tutorial: + + +Tutorial +======== + +In this section we will run the ``FALCON`` pipeline on an *E. coli* dataset. +We will work through the commands and results and give you ideas of how to assess +the perfomance of ``FALCON`` on your dataset so you can modify parameters and trouble-shoot more +effectively. This tutorial is a beginners guide to ``FALCON`` but assumes bioinformatics fluency. + +Input Files +----------- + +You will need three types of files to get started, your PacBio data in fasta format (can be one or many files), a +text file telling ``FALCON`` where to find your fasta files, and a :ref:`configuration ` file. +All files except the fasta files must be in your job directory. + + +1. Download *E. coli* dataset +~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ + +An example *E. coli* dataset can be download from here_. and then unpacked. e.g.: + +.. code-block:: bash + + $ wget https://downloads.pacbcloud.com/public/data/git-sym/ecoli.m140913_050931_42139_c100713652400000001823152404301535_s1_p0.subreads.tar.gz + $ tar -xvzf ecoli.m140913_050931_42139_c100713652400000001823152404301535_s1_p0.subreads.tar.gz + +.. _here: https://downloads.pacbcloud.com/public/data/git-sym/ + +You should find three fasta files of ~350 Mb each in the newly created subdirectory. + + +2. Create FOFN +~~~~~~~~~~~~~~ + +Next, create a "file-of-file-names", ("fofn") with the full path of each fasta file, one per line. + +.. code-block:: bash + + /my/path/to/data/ecoli.1.subreads.fasta + /my/path/to/data/ecoli.2.subreads.fasta + /my/path/to/data/ecoli.3.subreads.fasta + + + +3. Download configuration file +~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ + +If you are running on a cluster with a scheduler use this a starting point: +:download:`fc_run_ecoli.cfg ` +If you are running your job locally, try this file: +:download:`fc_run_ecoli_local.cfg ` + +These config files are meant to be starting points only! You will need to make adjustments according +to your particular compute setup. Please refer to these :ref:`config ` files as you plan your run. + + +Note that I have manually specified a seed read length cutoff of +15kb rather than using an automated cut off (:ref:`length_cutoff ` = -1, with calculated +length cut off = 22486). The +raw read coverage is very high (>200X); by reducing the seed read length cutoff, we avoid enriching +our seed reads for erroneous chimeric (and very long) reads. Try running the assembly using +the automated seed read length cut off, you should get a fragmented (28 contigs) and +incomplete assembly (< 900Mb). + + +Running FALCON +-------------- + +I send all of my ``FALCON`` jobs to the scheduler for ease of tracking job progress. Here is an example +bash script ``run_falcon.sh`` that submits to an SGE_ cluster: + +.. code-block:: bash + + #!/bin/bash + #$ -S /bin/bash + #$ -N myJob + #$ -cwd + #$ -q myqueue + + # load dependencies + module load python/2.7.9 gcc/4.9.2 + + # source build + cd /path_to_build/src/FALCON-integrate/ + source env.sh + + # navigate to job directory, directory containing input.fofn + cd /path/to/my/job_dir + + # run it! + fc_run fc_run.cfg + + +To initiate the ``FALCON`` run, I just submit my job to the scheduler with a qsub command: + +.. code-block:: bash + + $ qsub run_falcon.sh + + +Alternatively, you can add the ``fc_env/bin`` directory to your +``$PATH`` and invoke ``fc_run`` at the command line with your ``fc_run.cfg`` as the argument. +Note that this shell needs to persist through the entire assembly process so you may need +to use a window manager like screen_ to maintain your connection. + +.. code-block:: bash + + falcon_jobdir$ export PYTHONUSERBASE=/path_to_build/fc_env/ + falcon_jobdir$ export PATH=$PYTHONUSERBASE/bin:$PATH + falcon_jobdir$ fc_run fc_run.cfg + + +.. _SGE: http://gridscheduler.sourceforge.net/htmlman/manuals.html +.. _screen: https://www.gnu.org/software/screen/manual/screen.html + + +Assessing Run Progress +---------------------- + +Refer to the pipeline document for detailed summary of ``FALCON`` job directory structure, +sequence of commands, and output files created. + +Counting Completed Jobs +~~~~~~~~~~~~~~~~~~~~~~ + +The majority of run-time is spent during the daligner phases, performing the alignments and +then sorting and merging them. To determine how many jobs are performed for each step, refer to ``0-rawreads/run_jobs.sh``. + +.. code-block:: bash + + $ grep '^#' 0-rawreads/run_jobs.sh + + # Daligner jobs (60) + # Initial sort jobs (400) + # Check initial .las files jobs (80) (optional but recommended) + # Remove initial .las files + # Level 1 merge jobs (20) + # Check level 2 .las files jobs (20) (optional but recommended) + # Remove level 1 .las files (optional) + +To determine how many jobs have completed, count the sentinel files that indicate a job is complete. +For example: + +.. code-block:: bash + + $ find 0-rawreads/ -name "job*done" | wc -l + 60 + + $ find 0-rawreads/ -name "m_*done" | wc -l + 20 + + +Assessing Run Performance +------------------------- + +Raw and Pread Coverage and Quality +~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ + +The *E. coli* subreads +are a total of 1.01 Gb of data in 105,451 reads. :download:`countFasta.pl ` +is a useful script by Joseph Fass and Brad Sickler at UC Davis for calculating total sequence +length and other assembly metrics). + +You can confirm that your dazzler database was correctly constructed using a utility from the dazzler_ suite: + +.. _dazzler: https://dazzlerblog.wordpress.com/command-guides/dazz_db-command-guide/ + +.. code-block:: bash + + $ DBstats raw_reads.db > raw_reads.stats + $ head raw_reads.stats -n 17 + + Statistics for all reads of length 500 bases or more + + 90,747 reads out of 105,451 ( 86.1%) + 964,281,429 base pairs out of 1,013,118,375 ( 95.2%) + + 10,626 average read length + 6,805 standard deviation + + Base composition: 0.248(A) 0.242(C) 0.263(G) 0.246(T) + + Distribution of Read Lengths (Bin size = 1,000) + + Bin: Count % Reads % Bases Average + 45,000: 1 0.0 0.0 45611 + + +You can see that we discarded 13.9% of the raw bases and 4.8% of the reads by employing a +raw read length cut off of 500bp in the :ref:`DBsplit ` options. This file can +also be used to plot a :ref:`histogram ` of raw read lengths. + +The genome of this *E. coli* strain is 4.65 Mb long for a raw read coverage of ~207 fold. +Confirm this with the preassembly report: + +.. code-block:: bash + + $ cat 0-rawreads/report/pre_assembly_stats.json + + "genome_length": 4652500, + "length_cutoff": 15000, + "preassembled_bases": 350302363, + "preassembled_coverage": 75.293, + "preassembled_mean": 10730.33, + "preassembled_n50": 16120, + "preassembled_p95": 22741, + "preassembled_reads": 32646, + "preassembled_seed_fragmentation": 1.451, # number split preads / seed reads + "preassembled_seed_truncation": 4453.782, # ave bp lost per pread due to low cov + "preassembled_yield": 0.758, # total pread bp / seed read bp + "raw_bases": 964281429, + "raw_coverage": 207.261, + "raw_mean": 10626.042, + "raw_n50": 14591, + "raw_p95": 23194, + "raw_reads": 90747, + "seed_bases": 461851093, + "seed_coverage": 99.269, # raw base coverage depth on seed reads + "seed_mean": 20029.103, + "seed_n50": 19855, + "seed_p95": 28307, + "seed_reads": 23059 + +A note on these statistics: in the process of created preads, seeds reads with insufficient +raw read coverage (usually due to base errors) will be split or truncated. The preassembled seed +fragmentation, truncation, and yield stats summarize the quality of pread assembly. +A good preassembled yield should be greater than 50%. Note that if an automated seed read length +is used for this data (22486), preassembled seed read truncation is ~6kb, indicating that many of the longest +raw reads are not supported by the rest of the data. + +You can similarly summarize the contents of the dazzler database for preads using DBstats +and plotting in :ref:`R `. + +Contig Stats +~~~~~~~~~~~~ + +When your run is complete, you can summarize your assembly stats using the :download:`countFasta.pl ` +script: + + .. code-block:: bash + + $ countFasta.pl p_ctg.fa > p_ctg.stats + $ countFasta.pl a_ctg.fa > a_ctg.stats + $ tail p_ctg.stats + + Total length of sequence: 4635395 bp + Total number of sequences: 1 + N25 stats: 25% of total sequence length is contained in the 1 sequences >= 4635395 bp + N50 stats: 50% of total sequence length is contained in the 1 sequences >= 4635395 bp + N75 stats: 75% of total sequence length is contained in the 1 sequences >= 4635395 bp + Total GC count: 2352187 bp + GC %: 50.74 % + + +Assembly Graph and Pread Overlaps +~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ + +Assembly contiguity can be enhanced by adjusting a few parameters in the last stage of the +assembly process. You can try a grid of :ref:`pread length cut offs ` for +the filtering of the final overlaps in the assembly graph. In a general sense, longer pread length cut offs will increase the +contiguity (contig N50) in your assembly, but may result in shorter over all assembly length. +To try different length cut off, rename your 2-asm-falcon dir, +modify the config file, rename the log and mypwatcher directory, and restart ``FALCON``: + +.. code-block:: bash + + $ mv 2-asm-falcon 2-asm-falcon_12kb + $ mv mypwatcher/ mypwatcher0/ + $ mv all.log all0.log + $ qsub run_falcon.sh + + +The other parameter to adjust is the number of overlaps in the assembly graph. First, look +at a histogram of the number of overlaps on the 5' and 3' end of each read. Run the falcon utility: + +.. code-block:: bash + + # make sure utility is in $PATH + $ export PYTHONUSERBASE=/path_to_build/fc_env/ + $ export PATH=$PYTHONUSERBASE/bin:$PATH + + # navigate to directory + $ cd 2-asm-falcon + $ fc_ovlp_stats --fofn ../1-preads_ovl/merge-gather/las.fofn > ovlp.stats + +Then plot :ref:`histograms ` of the number of 5' and 3' overlaps between preads in R. +This can inform your parameters for :ref:`sge_option_fc ` where ``min_cov`` and ``max_cov`` +should flank the bulk of the distribution. For repetative genomes, a second mode in the :ref:`distribution ` +may appear, containing preads ending or begining in repetative material. It is best to choose a ``max_cov`` +to the left of the repeat mode that removes these repetative overlaps. + + + +Troubleshooting Run +------------------- + +If you find your run has died here are some suggestions of how to restart, +in order of increasing difficulty: + +Simple Restart +~~~~~~~~~~~~~~ + +Simply rename your log file and ``mypwatcher`` directory and restart the pipeline. Renaming these +files preserves them for you reference, and by removing the original mypwatcher directory +the pipeline, when restarted, will scan your job directory for completed jobs and pick up where it left off: + +.. code-block:: bash + + $ mv mypwatcher/ mypwatcher0/ + $ mv all.log all0.log + $ qsub run_falcon.sh + + +Directory Cleanup and Restart +~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ + +First, determine which job caused the run to fail. For example: + +.. code-block:: bash + + $ grep 'ERROR' all.log + + 2016-11-21 03:21:39,482 - pypeflow.simple_pwatcher_bridge - ERROR - Task Node(0-rawreads/m_00210) failed with exit-code=99 + 2016-11-21 03:21:39,482 - pypeflow.simple_pwatcher_bridge - ERROR - Failed to clean-up FakeThread: jobid=Pcfbdb8b3c50d5e status='EXIT ' + +Delete all directories that failed, then rename the log file and ``mypwatcher`` as above: + +.. code-block:: bash + + $ rm -rf 0-rawreads/m_00210 + $ mv mypwatcher/ mypwatcher0/ + $ mv all.log all0.log + $ qsub run_falcon.sh + +You can find out more details about the failed jobs in ``mypwatcher/`` to diagnose the problem. + +.. code-block:: bash + + $ less mypwatcher/jobs/Pcfbdb8b3c50d5e/stderr + $ less mypwatcher/jobs/Pcfbdb8b3c50d5e/stdout + + +Manual Running of Failed Jobs +~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ + +If your job still fails, try manually running the problematic jobs. Search in the job +directory for the shell script containing the individual tasks and try manually running +the shell script or individual tasks: + +.. code-block:: bash + + $ ls job_0000 + + job_0000_done L1.19.5.las L1.19.7.las L1.5.19.las L1.7.19.las raw_reads.db run.sh task.json + L1.19.4.las L1.19.6.las L1.4.19.las L1.6.19.las pwatcher.dir rj_0000.sh run.sh.done task.sh + + $ head job_0000/rj_0000.sh -n 12 + + #!/bin/bash + set -vex + + db_dir=/lustre/hpcprod/skingan/FALCON_tutorial/ecoli/0-rawreads + ln -sf ${db_dir}/.raw_reads.bps . + ln -sf ${db_dir}/.raw_reads.idx . + ln -sf ${db_dir}/raw_reads.db . + ln -sf ${db_dir}/.raw_reads.dust.anno . + ln -sf ${db_dir}/.raw_reads.dust.data . + daligner -v -t16 -H22486 -e0.7 -s1000 raw_reads.19 raw_reads.4 raw_reads.5 raw_reads.6 raw_reads.7 + LAcheck -v raw_reads *.las + LAsort -v raw_reads.4.raw_reads.19.C0 raw_reads.4.raw_reads.19.N0 raw_reads.4.raw_reads.19.C1 raw_reads.4.raw_reads.19.N1 raw_reads.4.raw_reads.19.C2 raw_reads.4.raw_reads.19.N2 raw_reads.4.raw_reads.19.C3 raw_reads.4.raw_reads.19.N3 && LAmerge -v L1.4.19 raw_reads.4.raw_reads.19.C0.S raw_reads.4.raw_reads.19.N0.S raw_reads.4.raw_reads.19.C1.S raw_reads.4.raw_reads.19.N1.S raw_reads.4.raw_reads.19.C2.S raw_reads.4.raw_reads.19.N2.S raw_reads.4.raw_reads.19.C3.S raw_reads.4.raw_reads.19.N3.S + +Once these jobs have run to completion, you can try restarting the pipeline. + + +