funannotate predict fails with test and with real data: "Not enough gene models ... to train Augustus"

[IanDMedeiros]

Are you using the latest release?
funannotate v1.8.11.

Describe the bug
funannotate test is failing at the predict step with error "Not enough gene models 175 to train Augustus (200 required), exiting". Appears to be identical error to #552. I am also receiving similar errors with real data. End of #552 discussion suggested that the error might be related to GeneMark, but I am having troubled setting up GeneMark-ES so wouldn't the program just run without it?

What command did you issue?
funannotate test -t all --cpus 10

What probably isn't the problem, based on what I have tried so far
Bad Augustus installation. I was getting an Augustus error even earlier in funannotate test, so I replaced the Augustus that was installed by mamba with one (v. 3.3.3) already available on our system.
AUGUSTUS_CONFIG_PATH permissions. Ran chmod 777 $AUGUSTUS_CONFIG_PATH/species and error did not go away.
Multithreading. Tried with --cpus 1 and 10 ... same error.

Logfiles
funannotate-predict.log
`[06/29/22 21:19:42]: /hpc/home/idm7/miniconda3/envs/annotate/bin/funannotate predict -i test.softmasked.fa --protein_evidence protein.evidence.fasta -o annotate --cpus 10 --species Awesome busco

[06/29/22 21:19:42]: OS: CentOS Stream 8, 46 cores, ~ 230 GB RAM. Python: 3.8.12
[06/29/22 21:19:42]: Running funannotate v1.8.11
[06/29/22 21:19:42]: GeneMark path: /hpc/group/bio1/ian/envs/funannotate/gmes_petap
[06/29/22 21:19:42]: Full path to gmes_petap.pl: /hpc/group/bio1/ian/envs/funannotate/gmes_petap/gmes_petap.pl
[06/29/22 21:19:42]: GeneMark appears to be functional? False
[06/29/22 21:19:43]: {'augustus': 1, 'hiq': 2, 'genemark': 0, 'pasa': 6, 'codingquarry': 0, 'snap': 1, 'glimmerhmm': 1, 'proteins': 1, 'transcripts': 1}
[06/29/22 21:19:43]: Skipping CodingQuarry as no --rna_bam passed
[06/29/22 21:19:43]: {'augustus': 'busco', 'snap': 'busco', 'glimmerhmm': 'busco'}
[06/29/22 21:19:43]: Parsed training data, run ab-initio gene predictors as follows:
[06/29/22 21:19:44]: {'augustus': 1, 'hiq': 2, 'genemark': 0, 'pasa': 6, 'codingquarry': 0, 'snap': 1, 'glimmerhmm': 1, 'proteins': 1, 'transcripts': 1}
[06/29/22 21:19:45]: Loading genome assembly and parsing soft-masked repetitive sequences
[06/29/22 21:19:45]: Genome loaded: 6 scaffolds; 3,776,588 bp; 19.75% repeats masked
[06/29/22 21:20:12]: join_mult_hints.pl
[06/29/22 21:20:12]: Running BUSCO to find conserved gene models for training ab-initio predictors
[06/29/22 21:20:12]: /hpc/home/idm7/miniconda3/envs/annotate/bin/python /hpc/home/idm7/miniconda3/envs/annotate/lib/python3.8/site-packages/funannotate/aux_scripts/funannotate-BUSCO2.py -i /hpc/group/bio1/ian/envs/annotate/test-busco_b548c447-5a92-4bca-b6c4-d116e6f7177e/annotate/predict_misc/genome.softmasked.fa -m genome --lineage /hpc/group/bio1/ian/envs/funannotate_db/dikarya -o awesome_busco -c 10 --species anidulans -f --local_augustus /hpc/group/bio1/ian/envs/annotate/test-busco_b548c447-5a92-4bca-b6c4-d116e6f7177e/annotate/predict_misc/ab_initio_parameters/augustus
[06/29/22 21:25:12]: 175 valid BUSCO predictions found, validating protein sequences
[06/29/22 21:26:04]: 175 BUSCO predictions validated
[06/29/22 21:26:04]: Not enough gene models 175 to train Augustus (200 required), exiting
busco.logINFO ****************** Start a BUSCO 2.0 analysis, current time: 06/29/2022 21:20:12 ******************
INFO The lineage dataset is: dikarya_odb9 (eukaryota)
INFO Mode is: genome
INFO Maximum number of regions limited to: 3
INFO To reproduce this run: python /hpc/home/idm7/miniconda3/envs/annotate/lib/python3.8/site-packages/funannotate/aux_scripts/funannotate-BUSCO2.py -i /hpc/group/bio1/ian/envs/annotate/test-busco_b548c447-5a92-4bca-b6c4-d116e6f7177e/annotate/predict_misc/genome.softmasked.fa -o awesome_busco -l /hpc/group/bio1/ian/envs/funannotate_db/dikarya/ -m genome -c 10 -sp anidulans
INFO Check dependencies...
INFO Check input file...
INFO Temp directory is ./tmp/

INFO ****** Phase 1 of 2, initial predictions ******
INFO ****** Step 1/3, current time: 06/29/2022 21:20:12 ******
INFO Create blast database...
INFO [makeblastdb] Building a new DB, current time: 06/29/2022 21:20:12
INFO [makeblastdb] New DB name: /hpc/group/bio1/ian/envs/annotate/test-busco_b548c447-5a92-4bca-b6c4-d116e6f7177e/annotate/predict_misc/busco/tmp/awesome_busco_4188679581
INFO [makeblastdb] New DB title: /hpc/group/bio1/ian/envs/annotate/test-busco_b548c447-5a92-4bca-b6c4-d116e6f7177e/annotate/predict_misc/genome.softmasked.fa
INFO [makeblastdb] Sequence type: Nucleotide
INFO [makeblastdb] Keep Linkouts: T
INFO [makeblastdb] Keep MBits: T
INFO [makeblastdb] Maximum file size: 1000000000B
INFO [makeblastdb] Adding sequences from FASTA; added 6 sequences in 0.0434968 seconds.
INFO Running tblastn, writing output to /hpc/group/bio1/ian/envs/annotate/test-busco_b548c447-5a92-4bca-b6c4-d116e6f7177e/annotate/predict_misc/busco/run_awesome_busco/blast_output/tblastn_awesome_busco.tsv...
INFO ****** Step 2/3, current time: 06/29/2022 21:20:21 ******
INFO Getting coordinates for candidate regions...
INFO Pre-Augustus scaffold extraction...
INFO Running Augustus prediction using anidulans as species:
INFO [augustus] Please find all logs related to Augustus here: /hpc/group/bio1/ian/envs/annotate/test-busco_b548c447-5a92-4bca-b6c4-d116e6f7177e/annotate/predict_misc/busco/run_awesome_busco/augustus_output/augustus.log
INFO 06/29/2022 21:20:21 => 0% of predictions performed (743 to be done)
INFO 06/29/2022 21:20:56 => 10% of predictions performed (75/743 candidate regions)
INFO 06/29/2022 21:21:24 => 20% of predictions performed (149/743 candidate regions)
INFO 06/29/2022 21:22:04 => 30% of predictions performed (223/743 candidate regions)
INFO 06/29/2022 21:22:39 => 40% of predictions performed (298/743 candidate regions)
INFO 06/29/2022 21:23:01 => 50% of predictions performed (372/743 candidate regions)
INFO 06/29/2022 21:23:21 => 60% of predictions performed (446/743 candidate regions)
INFO 06/29/2022 21:23:38 => 70% of predictions performed (521/743 candidate regions)
INFO 06/29/2022 21:23:55 => 80% of predictions performed (596/743 candidate regions)
INFO 06/29/2022 21:24:08 => 90% of predictions performed (669/743 candidate regions)
INFO 06/29/2022 21:24:20 => 100% of predictions performed
INFO Extracting predicted proteins...
INFO ****** Step 3/3, current time: 06/29/2022 21:24:49 ******
INFO Running HMMER to confirm orthology of predicted proteins:
INFO 06/29/2022 21:24:49 => 0% of predictions performed (602 to be done)
INFO [hmmersearch] Parse failed (sequence file /hpc/group/bio1/ian/envs/annotate/test-busco_b548c447-5a92-4bca-b6c4-d116e6f7177e/annotate/predict_misc/busco/run_awesome_busco/augustus_output/extracted_proteins/EOG092600SD.faa.1):
INFO [hmmersearch] Line 2: illegal character %
INFO [hmmersearch] Parse failed (sequence file /hpc/group/bio1/ian/envs/annotate/test-busco_b548c447-5a92-4bca-b6c4-d116e6f7177e/annotate/predict_misc/busco/run_awesome_busco/augustus_output/extracted_proteins/EOG092603EH.faa.1):
INFO [hmmersearch] Line 2: illegal character %
INFO [hmmersearch] Parse failed (sequence file /hpc/group/bio1/ian/envs/annotate/test-busco_b548c447-5a92-4bca-b6c4-d116e6f7177e/annotate/predict_misc/busco/run_awesome_busco/augustus_output/extracted_proteins/EOG092600T4.faa.1):
INFO [hmmersearch] Line 2: illegal character %
INFO [hmmersearch] Parse failed (sequence file /hpc/group/bio1/ian/envs/annotate/test-busco_b548c447-5a92-4bca-b6c4-d116e6f7177e/annotate/predict_misc/busco/run_awesome_busco/augustus_output/extracted_proteins/EOG092600X0.faa.1):
INFO [hmmersearch] Line 2: illegal character %
INFO [hmmersearch] Parse failed (sequence file /hpc/group/bio1/ian/envs/annotate/test-busco_b548c447-5a92-4bca-b6c4-d116e6f7177e/annotate/predict_misc/busco/run_awesome_busco/augustus_output/extracted_proteins/EOG0926009O.faa.1):
INFO [hmmersearch] Line 2: illegal character %
INFO [hmmersearch] Parse failed (sequence file /hpc/group/bio1/ian/envs/annotate/test-busco_b548c447-5a92-4bca-b6c4-d116e6f7177e/annotate/predict_misc/busco/run_awesome_busco/augustus_output/extracted_proteins/EOG092602I6.faa.3):
INFO [hmmersearch] Line 2: illegal character %

<This goes on for many lines, apparently through all the BUSCO loci. Omitting here for space.>

INFO 06/29/2022 21:24:58 => 100% of predictions performed
INFO Results:
INFO C:13.5%[S:13.3%,D:0.2%],F:0.1%,M:86.4%,n:1312
INFO 177 Complete BUSCOs (C)
INFO 175 Complete and single-copy BUSCOs (S)
INFO 2 Complete and duplicated BUSCOs (D)
INFO 1 Fragmented BUSCOs (F)
INFO 1134 Missing BUSCOs (M)
INFO 1312 Total BUSCO groups searched

INFO ****** Phase 2 of 2, predictions using species specific training ******
INFO ****** Step 1/3, current time: 06/29/2022 21:25:00 ******
INFO Extracting missing and fragmented buscos from the ancestral_variants file...
WARNING The busco id(s) ['EOG0926457R', 'EOG09264XJC', 'EOG0926129I', 'EOG09265F2Y', 'EOG09262N3C', 'EOG092602UY', 'EOG09264R4M', 'EOG09264P3R', 'EOG09264HX6', 'EOG09260OCI', 'EOG09261J0P', 'EOG092610TN', 'EOG09264BOA', 'EOG09261DRB', 'EOG092608L0', 'EOG09261FAX', 'EOG09264X8J', 'EOG09262R8O', 'EOG09262K67', 'EOG09260K5F', 'EOG09263LP3', 'EOG09264S3E', 'EOG09262FJB', 'EOG09260VEY', 'EOG09260TLW', 'EOG092608WI', 'EOG09261OXV', 'EOG09264PE5', 'EOG09261JWS', 'EOG09260NNR', 'EOG09264RJL', 'EOG09262KJA', 'EOG09260VYK', 'EOG092641UM', 'EOG092644N1', 'EOG09262V3O', 'EOG09260XMI', 'EOG09263BDA', 'EOG09264I6B', 'EOG092635ST', 'EOG0926071Q', 'EOG09264PK5', 'EOG09263D2P', 'EOG09260VHV', 'EOG09265RGS', 'EOG092603YJ', 'EOG092621ZV', 'EOG09261801', 'EOG09260ZWU', 'EOG09260PI1', 'EOG092607QZ', 'EOG09262W7C', 'EOG09264V3H', 'EOG09261UWT', 'EOG09264881', 'EOG09263E49', 'EOG09265KQ4', 'EOG09260AZM', 'EOG09264T3S', 'EOG09261TEQ', 'EOG09265BJ3', 'EOG0926522L', 'EOG09262CDO', 'EOG09262H34', 'EOG09264J8E', 'EOG09265FL1', 'EOG0926431P', 'EOG09263M8W', 'EOG09265FTY', 'EOG09262Z2S', 'EOG09264719', 'EOG092625AX', 'EOG09265HEP', 'EOG092618J9', 'EOG09260RVQ', 'EOG09263K45', 'EOG09264R1U', 'EOG09261ICI', 'EOG09263RVR', 'EOG09260WG2', 'EOG09263QUM', 'EOG09264ZWF', 'EOG092646WF', 'EOG09261OLD', 'EOG09263W48', 'EOG092632TF', 'EOG09265552', 'EOG09261D4D', 'EOG09264SET', 'EOG092627XA', 'EOG09262JRP', 'EOG09261P7G', 'EOG09262GNE', 'EOG092636T6', 'EOG092625P1', 'EOG092641M3', 'EOG09262POL', 'EOG09264Z3D', 'EOG09260K29', 'EOG092659DX', 'EOG09264G1I', 'EOG09260289', 'EOG09264C3N', 'EOG09262387', 'EOG09264HU0', 'EOG09264W7W', 'EOG09263WM5', 'EOG092629FB', 'EOG09260KM4', 'EOG092604A0', 'EOG09260FZZ', 'EOG09260GKG', 'EOG09262MJW', 'EOG09260XSR', 'EOG092621S9', 'EOG09261IEV', 'EOG09262TEV', 'EOG092641A6', 'EOG09263DQH', 'EOG09263YBT', 'EOG09263KVG', 'EOG092650VI', 'EOG092653O3', 'EOG09264441', 'EOG0926369X', 'EOG092643IE', 'EOG09261XZ6', 'EOG09264XUV', 'EOG092645OU', 'EOG09261I8J', 'EOG09263WWI', 'EOG09260NAN', 'EOG09260S2Z', 'EOG09264XYD', 'EOG0926484N', 'EOG09263FGN', 'EOG09260ETR', 'EOG0926506U', 'EOG09262KVB', 'EOG092605ZA', 'EOG0926248P', 'EOG092635DF', 'EOG092641K1', 'EOG0926315C', 'EOG092658QH', 'EOG09261JVS', 'EOG0926307V', 'EOG0926587S', 'EOG092604KQ', 'EOG09260J97', 'EOG09262HP3', 'EOG09264OQ8', 'EOG09263L7Y', 'EOG09261I0F', 'EOG09264ZDZ', 'EOG09262CXO', 'EOG09261I1I', 'EOG09261727', 'EOG09262BVA', 'EOG09265QTV', 'EOG092605VL', 'EOG09260KDB', 'EOG092617S2', 'EOG09262YP5', 'EOG0926407T', 'EOG092629RT', 'EOG092605OK', 'EOG09260EPS', 'EOG09265JNA', 'EOG09260DBG', 'EOG09260NZ8', 'EOG092621F2', 'EOG09261IOS', 'EOG0926539T', 'EOG09264W1U', 'EOG09260KNR', 'EOG09263PWF', 'EOG092610VI', 'EOG09264KDO', 'EOG09261G1Y', 'EOG09262IY3', 'EOG09261VD2', 'EOG09263KDI', 'EOG092658SK', 'EOG09265A08', 'EOG09263K05', 'EOG09263QPR', 'EOG092644WX', 'EOG092631ML', 'EOG09260KUC', 'EOG09262M0W', 'EOG092658NW', 'EOG09263XN3', 'EOG0926506Z', 'EOG09263U71', 'EOG09262TUR', 'EOG09265040', 'EOG092655IF', 'EOG09262E7I', 'EOG092641G3', 'EOG09261XNU', 'EOG09260EE7', 'EOG092645QN', 'EOG0926092K', 'EOG09263MR4', 'EOG09264XVU', 'EOG092610KH', 'EOG09261WJ8', 'EOG09261HZD', 'EOG09261SS1', 'EOG09261CQG', 'EOG0926273Q', 'EOG092619L1', 'EOG09265CCT', 'EOG09260KIY', 'EOG09262N5O', 'EOG092604ZZ', 'EOG09260R9L', 'EOG092654KW', 'EOG092615Y4', 'EOG09261CWO', 'EOG09260NXC', 'EOG09265G5K', 'EOG092612XD', 'EOG092605T6', 'EOG09261ZFN', 'EOG092620FM', 'EOG092646C6', 'EOG09264VC6', 'EOG092649VG', 'EOG09260LVD', 'EOG09265PWR', 'EOG09262PPU', 'EOG09262F22', 'EOG092615CC', 'EOG092616YZ', 'EOG09264RQY', 'EOG092616QN', 'EOG0926400M', 'EOG092648O6', 'EOG09264KO7', 'EOG09264NDD', 'EOG09262GWQ', 'EOG0926458I', 'EOG0926115V', 'EOG09265M98', 'EOG09260TVA', 'EOG09261RWJ', 'EOG09264A2D', 'EOG09260UA2', 'EOG092634MM', 'EOG09265IT6', 'EOG09263760', 'EOG092642UD', 'EOG092609O9', 'EOG09265FTN', 'EOG09265EKJ', 'EOG0926534P', 'EOG09263KZJ', 'EOG09261DG0', 'EOG09260NHN', 'EOG09262OX9', 'EOG09261T98', 'EOG09260WCZ', 'EOG09262HKC', 'EOG09263F11', 'EOG09261G92', 'EOG09262U7S', 'EOG09264VZ7', 'EOG092602I6', 'EOG09262E4T', 'EOG09262WQX', 'EOG09265HP0', 'EOG09264SSI', 'EOG09260FMW', 'EOG092612AK', 'EOG092600SD', 'EOG09261ACJ', 'EOG09260ZG2', 'EOG09263Y3L', 'EOG09261NLY', 'EOG092655SO', 'EOG092609RF', 'EOG09263CAC', 'EOG09261ABB', 'EOG09264272', 'EOG092651BA', 'EOG09265L8N', 'EOG09261OSU', 'EOG09262MEK', 'EOG09263UN3', 'EOG09260DP1', 'EOG09261AMX', 'EOG09262UAS', 'EOG09262SI7', 'EOG09263KRO', 'EOG09261TPN', 'EOG09260T4S', 'EOG092610QT', 'EOG09262X7T', 'EOG092629ZN', 'EOG092634B1', 'EOG092620EL', 'EOG0926009O', 'EOG09264G0H', 'EOG09262528', 'EOG09260QNB', 'EOG09261EM7', 'EOG092617RY', 'EOG092646CB', 'EOG09261O4Y', 'EOG09263G4R', 'EOG0926248W', 'EOG09260T28', 'EOG092624KK', 'EOG09263OD3', 'EOG09261Q18', 'EOG092658WY', 'EOG09265GSM', 'EOG09265B95', 'EOG092604I8', 'EOG09264FXB', 'EOG09264ZQC', 'EOG09264PI4', 'EOG09262VPD', 'EOG09262QS5', 'EOG09261ZPW', 'EOG09263ZBF', 'EOG09262YAU', 'EOG09262SMG', 'EOG092608ZS', 'EOG0926229Z', 'EOG09261YRA', 'EOG09263EQZ', 'EOG09260TWS', 'EOG09265OQH', 'EOG09263720', 'EOG092653NM', 'EOG09260AZK', 'EOG09261AH9', 'EOG09265B1X', 'EOG09263817', 'EOG0926112A', 'EOG092601KZ', 'EOG09264X31', 'EOG09264398', 'EOG09261N2L', 'EOG09262LI4', 'EOG0926074Y', 'EOG09260FPA', 'EOG09264MGU', 'EOG092626EQ', 'EOG09264U81', 'EOG09265FCK', 'EOG09260BFE', 'EOG09264CA0', 'EOG092603EH', 'EOG092653VU', 'EOG09262NB1', 'EOG092619MJ', 'EOG09260CKC', 'EOG09261DHR', 'EOG09262TO9', 'EOG092625U6', 'EOG09263MGE', 'EOG09264PDD', 'EOG09263IMF', 'EOG092648K5', 'EOG092602MO', 'EOG09263C55', 'EOG09260EZT', 'EOG09264NC7', 'EOG09262JAT', 'EOG09260E8K', 'EOG0926133I', 'EOG092612CC', 'EOG092600SK', 'EOG092648LP', 'EOG09260VTN', 'EOG092648VW', 'EOG09264O6J', 'EOG0926514P', 'EOG09263W7L', 'EOG09262LYR', 'EOG09265PQX', 'EOG09263QH4', 'EOG09260DXP', 'EOG09260WU6', 'EOG09263NE7', 'EOG09265G9U', 'EOG0926388H', 'EOG0926425H', 'EOG09264HTG', 'EOG09260EAZ', 'EOG0926357F', 'EOG09262JWJ', 'EOG092608RH', 'EOG092629WA', 'EOG092657UN', 'EOG09265PUI', 'EOG0926419M', 'EOG09264JHE', 'EOG09263OQH', 'EOG092638CT', 'EOG09262CBI', 'EOG09262X01', 'EOG092640BS', 'EOG09264DY4', 'EOG09264Y0W', 'EOG092619VG', 'EOG092651FJ', 'EOG09261LPY', 'EOG09261OXD', 'EOG09262ESR', 'EOG0926251E', 'EOG0926310O', 'EOG09264T8I', 'EOG092602FH', 'EOG092607OQ', 'EOG09265NHW', 'EOG09264331', 'EOG09261666', 'EOG09260LRX', 'EOG09260A27', 'EOG09262N10', 'EOG09261B18', 'EOG09260SAH', 'EOG09260ERO', 'EOG09261Y04', 'EOG09261EU7', 'EOG09263EVJ', 'EOG09263MEM', 'EOG09260274', 'EOG09264OYZ', 'EOG09264DT4', 'EOG09263OZR', 'EOG09261W90', 'EOG0926347W', 'EOG09264NEF', 'EOG09264LC7', 'EOG09263FR7', 'EOG09260AQB', 'EOG0926306O', 'EOG09260QVP', 'EOG09261JUE', 'EOG09261I1G', 'EOG09264XOZ', 'EOG09260SIZ', 'EOG09264LBC', 'EOG09262V8E', 'EOG09262GXD', 'EOG09263C4C', 'EOG09260RRC', 'EOG092640WA', 'EOG09263A5D', 'EOG09265313', 'EOG092632WW', 'EOG09263U08', 'EOG09265SHM', 'EOG09260SL3', 'EOG092619GP', 'EOG09263690', 'EOG09263ULA', 'EOG09264RIE', 'EOG09262CMP', 'EOG0926073O', 'EOG09264NJ1', 'EOG09263OAE', 'EOG09263BE5', 'EOG09260RS7', 'EOG09260NY2', 'EOG09261O7R', 'EOG092653YS', 'EOG092657YR', 'EOG09260WUA', 'EOG09262JZW', 'EOG09263LNF', 'EOG09264THP', 'EOG09260Z3X', 'EOG0926115P', 'EOG09261WVT', 'EOG09262E4Q', 'EOG09265I60', 'EOG09262DUV', 'EOG09261C0G', 'EOG09261XNJ', 'EOG092658X5', 'EOG092658CI', 'EOG09263A3Y', 'EOG09263IQ5', 'EOG092654LJ', 'EOG09260KGS', 'EOG09262MXH', 'EOG092611HB', 'EOG09263J6Z', 'EOG09260BRA', 'EOG09264903', 'EOG09262GVX', 'EOG09263R4M', 'EOG09264IIZ', 'EOG09262NNS', 'EOG092606AD', 'EOG09263ZW6', 'EOG09263JTO', 'EOG092651K1', 'EOG09263Q8J', 'EOG09261X9E', 'EOG09262PAY', 'EOG09262CUO', 'EOG09261B3Q', 'EOG09263L9T', 'EOG09260W9L', 'EOG09263X1F', 'EOG09263YFX', 'EOG09260DUR', 'EOG09261DW8', 'EOG092654VM', 'EOG09260NJW', 'EOG09260JTZ', 'EOG09263YFH', 'EOG09260JED', 'EOG092613QA', 'EOG09263KB4', 'EOG09262GLP', 'EOG09265GGX', 'EOG092625OH', 'EOG09265KSE', 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'EOG09261Q5L', 'EOG09260KWP', 'EOG09260RGH', 'EOG092634G9', 'EOG09263RDD', 'EOG09264B74', 'EOG09261H5E', 'EOG09262YQG', 'EOG09260S5R', 'EOG09261UMG', 'EOG09264TQ5', 'EOG092603JK', 'EOG09264F1U', 'EOG09260FL2', 'EOG09261S0S', 'EOG09263DFA', 'EOG0926077L', 'EOG09260LI6', 'EOG09263IT0', 'EOG09260W8D', 'EOG09264IG5', 'EOG09261EMF', 'EOG09262A8G', 'EOG09260Y2Q', 'EOG09265D7J', 'EOG09260Z5E', 'EOG09261031', 'EOG092621YU', 'EOG092630MJ', 'EOG092634M1', 'EOG09263FAK', 'EOG09261N64', 'EOG09260NE0', 'EOG09262X74', 'EOG09260BSW', 'EOG092609YT', 'EOG09263X4B', 'EOG09264CP8', 'EOG092638XA', 'EOG09264OXC', 'EOG092658WS', 'EOG09260S3L', 'EOG09262H50', 'EOG092608AV', 'EOG09263RY2', 'EOG092631QQ', 'EOG09260TDY', 'EOG09262L1P', 'EOG092656RK', 'EOG09263Z41', 'EOG092636Y6', 'EOG09264DMU', 'EOG09261NLR', 'EOG09262X8R', 'EOG09264SUZ', 'EOG092657H8', 'EOG09264OM7', 'EOG0926591L', 'EOG09260FFP', 'EOG09263H5H', 'EOG09264B3O', 'EOG09264JW6', 'EOG09261YV6', 'EOG09262E7W', 'EOG09261A3K', 'EOG092646PE', 'EOG09260B3X', 'EOG09260JPV', 'EOG0926312D', 'EOG09260C2V', 'EOG09264VRO', 'EOG092628LW', 'EOG09264O2D', 'EOG09260LJ8', 'EOG092609JB', 'EOG09262HA6', 'EOG09264DOU', 'EOG09260U6R', 'EOG09261PJZ', 'EOG09260EPQ', 'EOG09261UPM', 'EOG092649QJ', 'EOG09261DJC', 'EOG09260JO5', 'EOG09263GUT', 'EOG09264GMT', 'EOG09264ENO', 'EOG09263WB5', 'EOG09260K4V', 'EOG09261VI3', 'EOG09260K24', 'EOG09260XOG', 'EOG09265BTA', 'EOG092628HC', 'EOG09264DYD', 'EOG09262H1X', 'EOG09264DJ8', 'EOG09264XKX', 'EOG092600W1', 'EOG09262VOC', 'EOG09261LV7', 'EOG092652KR', 'EOG09261VUC', 'EOG09262KZ3', 'EOG092631UM', 'EOG09262IZO', 'EOG09262UB3', 'EOG09262ILV', 'EOG09261MOX', 'EOG09263X22', 'EOG09263FKC', 'EOG09260WGT', 'EOG09264X41', 'EOG09263KEE', 'EOG09264JQ1', 'EOG09260EQD', 'EOG09261V9P', 'EOG092656IY', 'EOG09265CQO', 'EOG09264XTW', 'EOG09260H81', 'EOG09263ZSC', 'EOG092655L0', 'EOG092648XW', 'EOG09264ZDJ', 'EOG09264LH2', 'EOG09263FTE', 'EOG09265A4E', 'EOG092604ML', 'EOG092615IE', 'EOG09262914', 'EOG09263HYJ', 'EOG09262Q8D', 'EOG09263JFQ', 'EOG09264C3V', 'EOG092612LP', 'EOG09260NC6', 'EOG09264E6Z', 'EOG09264V51', 'EOG092643QM', 'EOG09262Z0M', 'EOG09265AT5', 'EOG09264MZ1', 'EOG092606AJ', 'EOG09264829', 'EOG09264SQJ', 'EOG0926131E', 'EOG09260MRU', 'EOG09263274', 'EOG0926423H', 'EOG09262B1U', 'EOG09260VCG', 'EOG09264D7Y', 'EOG09264L6D', 'EOG09264BJC', 'EOG09262MFL', 'EOG09263OWL', 'EOG092645U1', 'EOG09260NWN', 'EOG0926025H', 'EOG09262DC1', 'EOG09262K8C', 'EOG092611G7', 'EOG09260LBU', 'EOG09262JZK', 'EOG092648U2', 'EOG09264OBO', 'EOG09263EBB', 'EOG09262IZ6', 'EOG09264G1F', 'EOG09260XQV', 'EOG09262QRH', 'EOG09264A8D', 'EOG09261B6Y', 'EOG09265DDU', 'EOG09265GXF', 'EOG09260SJV', 'EOG09265E8A', 'EOG09265H9T', 'EOG09261M78', 'EOG09265JA7', 'EOG0926137U', 'EOG09261FM4', 'EOG09261QXC', 'EOG09264UJF', 'EOG09264XT5', 'EOG09261MMR', 'EOG09262PMC', 'EOG09262HQM', 'EOG09263JZO', 'EOG09265ANI', 'EOG09261QR8', 'EOG09263XZN', 'EOG09260EOI', 'EOG09262N0U', 'EOG09262PIH', 'EOG092652ZZ', 'EOG09262A6N', 'EOG092642I5', 'EOG09260VTA', 'EOG09260E2O', 'EOG092600T9', 'EOG09265K60', 'EOG09263LR1', 'EOG092614E6', 'EOG09262M2J', 'EOG0926140Q', 'EOG092621CK', 'EOG092619EA', 'EOG09264LKR', 'EOG092621CP', 'EOG09262M7B', 'EOG09260779', 'EOG09262N47', 'EOG09265RGI', 'EOG09260XL0', 'EOG09261ZJR', 'EOG09261IEH', 'EOG09265C25', 'EOG09264RBX', 'EOG09263EDC', 'EOG092629U5', 'EOG09265822', 'EOG09265BBJ', 'EOG09261404', 'EOG09263RW3', 'EOG09260375', 'EOG09261LEU', 'EOG09262341', 'EOG092631IR', 'EOG092618M2', 'EOG09264U78', 'EOG09263CG4', 'EOG092645LS', 'EOG09262VVF', 'EOG09261XAF', 'EOG09261N20', 'EOG09260UXC', 'EOG09265HPJ', 'EOG09261V2P', 'EOG09262516', 'EOG09260LS9', 'EOG09260KCB', 'EOG09260V5Q', 'EOG09260FKU', 'EOG09264OML', 'EOG09262V9N', 'EOG09265CN0', 'EOG09260HPO', 'EOG092644DY', 'EOG09260XH5', 'EOG09262F7P', 'EOG09263A64', 'EOG0926505R', 'EOG09264RJ7', 'EOG09261QR5', 'EOG09263ZBJ', 'EOG09261PUF', 'EOG09262SR7', 'EOG09260RNZ', 'EOG09260WYQ', 'EOG09260BYL', 'EOG092644X6', 'EOG09263G3M', 'EOG0926510L', 'EOG092606WZ', 'EOG092638EN', 'EOG0926489S', 'EOG09264JK1', 'EOG092608T8', 'EOG09264RR2', 'EOG09264O9F', 'EOG092600X0', 'EOG09264EGS', 'EOG092634ZL', 'EOG09264ABT', 'EOG09264FYY', 'EOG09260M87', 'EOG09264B2P', 'EOG092630YS', 'EOG09264HN5', 'EOG092644TW', 'EOG09262G8Y', 'EOG09263MNN', 'EOG09263BGW', 'EOG09261ONU', 'EOG09264L06', 'EOG09261KHB', 'EOG092603D0', 'EOG09262J7K', 'EOG09261M4S', 'EOG09262WSH', 'EOG09263Z8I', 'EOG09265EOF', 'EOG092604MJ', 'EOG09261V03', 'EOG09260HSP', 'EOG092608WU', 'EOG09260NHB', 'EOG092638RC', 'EOG0926158Y', 'EOG09260GF5', 'EOG09263OCO', 'EOG09260AEC', 'EOG092610LQ', 'EOG092603SM', 'EOG09260DFH', 'EOG092624UF', 'EOG09265I7S', 'EOG092614DJ', 'EOG09263CG2', 'EOG09263I7I', 'EOG09260N53', 'EOG09264BWL', 'EOG09260J8F', 'EOG09264T1U', 'EOG09265KF9', 'EOG09264KTU', 'EOG092613R2', 'EOG09264TJN', 'EOG0926354S', 'EOG0926420U', 'EOG09261ZXZ', 'EOG092654O3', 'EOG09265FGA', 'EOG09260MBW', 'EOG09264CST', 'EOG092619NK', 'EOG09263WZ2', 'EOG09264UD3', 'EOG092650I8', 'EOG09261FH7', 'EOG09261225', 'EOG09264KK7', 'EOG09264YKY', 'EOG09262P1W', 'EOG09262C5Z', 'EOG09262KUJ', 'EOG09264L3W', 'EOG09264G04', 'EOG09260WUS', 'EOG09264XPY', 'EOG09264FVQ', 'EOG09260DUW', 'EOG092653LT', 'EOG09265LEG', 'EOG092656CM', 'EOG09264Z1B', 'EOG09263TQ5', 'EOG092658ZO', 'EOG09260OM6', 'EOG092635SS', 'EOG09261KRX', 'EOG092647CM', 'EOG09265BG5', 'EOG09264IKZ', 'EOG09261UOJ', 'EOG09263UWJ', 'EOG09260HMA', 'EOG09262Q6S', 'EOG09260APA', 'EOG09264MN3', 'EOG09265591', 'EOG09265ER6', 'EOG09262I0R', 'EOG09260931', 'EOG092633QB', 'EOG09261RFF', 'EOG092603KJ', 'EOG09262BHE', 'EOG09262IP2', 'EOG09264DIM', 'EOG09262E98', 'EOG092649VA', 'EOG09264YHS', 'EOG09260PJ9', 'EOG092628SP', 'EOG09264K2W', 'EOG09264IV9', 'EOG09261W1K', 'EOG09260JDM', 'EOG09260H6E', 'EOG092613UB', 'EOG09264P74', 'EOG09261V87', 'EOG092624JL', 'EOG09262O0R', 'EOG09262PZ9', 'EOG09264GQZ', 'EOG09261HB6', 'EOG09264IOS', 'EOG09262MOO', 'EOG09261CM0', 'EOG09265K4K', 'EOG09265AL8', 'EOG09261EY9', 'EOG092651HW', 'EOG09260B65', 'EOG092629WJ', 'EOG092628FW', 'EOG09260OZU', 'EOG09261OKK', 'EOG092604S1', 'EOG092631MU', 'EOG09264USX', 'EOG09260TPT', 'EOG09261G4Z', 'EOG09261RWU', 'EOG09262LQT', 'EOG092605VU'] were not found in the ancestral_variants file
INFO Running tblastn, writing output to /hpc/group/bio1/ian/envs/annotate/test-busco_b548c447-5a92-4bca-b6c4-d116e6f7177e/annotate/predict_misc/busco/run_awesome_busco/blast_output/tblastn_awesome_busco_missing_and_frag_rerun.tsv...
INFO [tblastn] Warning: [tblastn] Query is Empty!
INFO Getting coordinates for candidate regions...
INFO ****** Step 2/3, current time: 06/29/2022 21:25:01 ******
INFO Training Augustus using Single-Copy Complete BUSCOs:
INFO 06/29/2022 21:25:01 => Converting predicted genes to short genbank files...
INFO 06/29/2022 21:25:07 => All files converted to short genbank files, now running the training scripts...
INFO Pre-Augustus scaffold extraction...
INFO Re-running Augustus with the new metaparameters, number of target BUSCOs: 1135
INFO 06/29/2022 21:25:09 => 0% of predictions performed (0 to be done)
INFO 06/29/2022 21:25:09 => 100% of predictions performed
INFO Extracting predicted proteins...
INFO ****** Step 3/3, current time: 06/29/2022 21:25:09 ******
INFO Running HMMER to confirm orthology of predicted proteins:
INFO 06/29/2022 21:25:09 => 0% of predictions performed (0 to be done)
INFO 06/29/2022 21:25:09 => 100% of predictions performed
INFO Results:
INFO C:13.5%[S:13.3%,D:0.2%],F:0.1%,M:86.4%,n:1312
INFO 177 Complete BUSCOs (C)
INFO 175 Complete and single-copy BUSCOs (S)
INFO 2 Complete and duplicated BUSCOs (D)
INFO 1 Fragmented BUSCOs (F)
INFO 1134 Missing BUSCOs (M)
INFO 1312 Total BUSCO groups searched

INFO BUSCO analysis done with WARNING(s). Total running time: 300.09778451919556 seconds
INFO Results written in /hpc/group/bio1/ian/envs/annotate/test-busco_b548c447-5a92-4bca-b6c4-d116e6f7177e/annotate/predict_misc/busco/run_awesome_busco/

INFO ****************** Start a BUSCO 2.0 analysis, current time: 06/29/2022 21:25:30 ******************
INFO The lineage dataset is: dikarya_odb9 (eukaryota)
INFO Mode is: proteins
INFO To reproduce this run: python /hpc/home/idm7/miniconda3/envs/annotate/lib/python3.8/site-packages/funannotate/aux_scripts/funannotate-BUSCO2.py -i /hpc/group/bio1/ian/envs/annotate/test-busco_b548c447-5a92-4bca-b6c4-d116e6f7177e/annotate/predict_misc/busco_augustus.proteins.fasta -o awesome_busco -l /hpc/group/bio1/ian/envs/funannotate_db/dikarya/ -m proteins -c 10 -sp anidulans
INFO Check dependencies...
INFO Check input file...
INFO Temp directory is ./tmp/
INFO Running HMMER on the proteins:
INFO 06/29/2022 21:25:30 => 0% of predictions performed (1312 to be done)
INFO 06/29/2022 21:25:32 => 10% of predictions performed (134/1312 candidate proteins)
INFO 06/29/2022 21:25:33 => 20% of predictions performed (263/1312 candidate proteins)
INFO 06/29/2022 21:25:35 => 30% of predictions performed (396/1312 candidate proteins)
INFO 06/29/2022 21:25:38 => 40% of predictions performed (525/1312 candidate proteins)
INFO 06/29/2022 21:25:41 => 50% of predictions performed (659/1312 candidate proteins)
INFO 06/29/2022 21:25:44 => 60% of predictions performed (791/1312 candidate proteins)
INFO 06/29/2022 21:25:47 => 70% of predictions performed (922/1312 candidate proteins)
INFO 06/29/2022 21:25:52 => 80% of predictions performed (1054/1312 candidate proteins)
INFO 06/29/2022 21:25:56 => 90% of predictions performed (1181/1312 candidate proteins)
INFO 06/29/2022 21:26:00 => 100% of predictions performed
INFO Results:
INFO C:13.3%[S:13.3%,D:0.0%],F:0.0%,M:86.7%,n:1312
INFO 175 Complete BUSCOs (C)
INFO 175 Complete and single-copy BUSCOs (S)
INFO 0 Complete and duplicated BUSCOs (D)
INFO 0 Fragmented BUSCOs (F)
INFO 1137 Missing BUSCOs (M)
INFO 1312 Total BUSCO groups searched

INFO BUSCO analysis done. Total running time: 33.35695195198059 seconds
INFO Results written in /hpc/group/bio1/ian/envs/annotate/test-busco_b548c447-5a92-4bca-b6c4-d116e6f7177e/annotate/predict_misc/busco_proteins/run_awesome_busco/

`

OS/Install Information

Installed using mamba on computing cluster running Red Hat Enterprise Linux 8.

`-------------------------------------------------------
Checking dependencies for 1.8.11

You are running Python v 3.8.12. Now checking python packages...
biopython: 1.77
goatools: 1.2.3
matplotlib: 3.4.3
natsort: 8.1.0
numpy: 1.23.0
pandas: 1.4.3
psutil: 5.9.1
requests: 2.28.1
scikit-learn: 1.1.1
scipy: 1.8.1
seaborn: 0.11.2
All 11 python packages installed

You are running Perl v b'5.026002'. Now checking perl modules...
Carp: 1.38
Clone: 0.42
DBD::SQLite: 1.64
DBD::mysql: 4.046
DBI: 1.642
DB_File: 1.855
Data::Dumper: 2.173
File::Basename: 2.85
File::Which: 1.23
Getopt::Long: 2.5
Hash::Merge: 0.300
JSON: 4.02
LWP::UserAgent: 6.39
Logger::Simple: 2.0
POSIX: 1.76
Parallel::ForkManager: 2.02
Pod::Usage: 1.69
Scalar::Util::Numeric: 0.40
Storable: 3.15
Text::Soundex: 3.05
Thread::Queue: 3.12
Tie::File: 1.02
URI::Escape: 3.31
YAML: 1.29
local::lib: 2.000024
threads: 2.15
threads::shared: 1.56
All 27 Perl modules installed

Checking Environmental Variables...
$FUNANNOTATE_DB=/hpc/group/bio1/ian/envs/funannotate_db
$PASAHOME=/hpc/home/idm7/miniconda3/envs/annotate/opt/pasa-2.5.2
$TRINITY_HOME=/hpc/home/idm7/miniconda3/envs/annotate/opt/trinity-2.8.5
$EVM_HOME=/hpc/home/idm7/miniconda3/envs/annotate/opt/evidencemodeler-1.1.1
$AUGUSTUS_CONFIG_PATH=/hpc/home/idm7/miniconda3/envs/annotate/config/
$GENEMARK_PATH=/hpc/group/bio1/ian/envs/funannotate/gmes_petap
All 6 environmental variables are set

Checking external dependencies...
PASA: 2.5.2
CodingQuarry: 2.0
Trinity: 2.8.5
augustus: 3.3.3
bamtools: bamtools 2.5.1
bedtools: bedtools v2.30.0
blat: BLAT v35
diamond: 2.0.15
ete3: 3.1.2
exonerate: exonerate 2.4.0
fasta: no way to determine
glimmerhmm: 3.0.4
gmap: 2021-08-25
hisat2: 2.2.1
hmmscan: HMMER 3.3.2 (Nov 2020)
hmmsearch: HMMER 3.3.2 (Nov 2020)
java: 11.0.1-internal
kallisto: 0.46.1
mafft: v7.505 (2022/Apr/10)
makeblastdb: makeblastdb 2.2.31+
minimap2: 2.24-r1122
pigz: pigz 2.7
proteinortho: 6.1.0
pslCDnaFilter: no way to determine
salmon: salmon 0.14.1
samtools: samtools 1.12
snap: 2006-07-28
stringtie: 2.2.1
tRNAscan-SE: 2.0.9 (July 2021)
tantan: tantan 39
tbl2asn: no way to determine, likely 25.X
tblastn: tblastn 2.2.31+
trimal: trimAl v1.4.rev15 build[2013-12-17]
trimmomatic: 0.39
ERROR: emapper.py not installed
ERROR: gmes_petap.pl not installed
ERROR: signalp not installed`

[nextgenusfs]

Seems like related to these errors, which suggest that BUSCO did not parse the output properly perhaps?

INFO [hmmersearch] Parse failed (sequence file /hpc/group/bio1/ian/envs/annotate/test-busco_b548c447-5a92-4bca-b6c4-d116e6f7177e/annotate/predict_misc/busco/run_awesome_busco/augustus_output/extracted_proteins/EOG092603EH.faa.1):
INFO [hmmersearch] Line 2: illegal character %

This seems like the error you get when you use Augustus 3.4.

So it seems like the Augustus config path ENV variable is still pointing to the conda install $AUGUSTUS_CONFIG_PATH=/hpc/home/idm7/miniconda3/envs/annotate/config/, so how did you "replace" augustus? You should probably uninstall it, ie I think you can use conda remove but need to use --force to only delete that package. After you remove the conda augustus, you can manually add back the location of your system AUGUSTUS_CONFIG_PATH variable. I'd first try to fix that and then rerun funannotate setup because it copies over Augustus parameters files from that location. Maybe its possible that the parameters files for this version from mamba are incompatible with your v3.3.3?

But perhaps the other thing to try would be to downgrade hmmer to the previous versions as maybe that is reason for the failure? I've never seen this be the problem before - seems like most likely augustus (as it nearly always is augustus issues). The other one for awhile was tblastn and multithreaded, but seems like you have older version so that shouldn't be an issue.

[IanDMedeiros]

Thanks for the quick reply—I think the piece I was missing was re-running funannotate setup with the new augustus. (/hpc/home/idm7/miniconda3/envs/annotate/config/ is actually the config directory associated with the new augustus.) With that correction, funannotate predict successfully completed in the test (it is still throwing an error that there are not enough gene models to train augustus, but that is now coming at the very end, after funannotate predict completes). I am now trying the pipeline with actual data.

[editing my comment, I now see that HMMER is there as hmmscan and hmmsearch]

######################################################### Running funannotate clean` unit testing: minimap2 mediated assembly duplications
Downloading: https://osf.io/8pjbe/download?version=1 Bytes: 252076
[DOWNLOAD PROGRESS OMITTED FOR SPACE]
6 input contigs, 6 larger than 500 bp, N50 is 427,039 bp
Checking duplication of 6 contigs

scaffold_73 appears duplicated: 100% identity over 100% of the contig. contig length: 15153
scaffold_91 appears duplicated: 100% identity over 100% of the contig. contig length: 8858
scaffold_27 appears duplicated: 100% identity over 100% of the contig. contig length: 427039

6 input contigs; 6 larger than 500 bp; 3 duplicated; 3 written to file
CMD: funannotate clean -i test.clean.fa -o test.exhaustive.fa --exhaustive
#########################################################
#########################################################
SUCCESS: funannotate clean test complete.
#########################################################

#########################################################
Running funannotate mask unit testing: RepeatModeler --> RepeatMasker
Downloading: https://osf.io/hbryz/download?version=1 Bytes: 375687
[DOWNLOAD PROGRESS OMITTED FOR SPACE]
������������������[Jun 30 01:06 AM]: OS: CentOS Stream 8, 46 cores, ~ 230 GB RAM. Python: 3.8.12
[Jun 30 01:06 AM]: Running funanotate v1.8.11
[Jun 30 01:06 AM]: Soft-masking simple repeats with tantan
[Jun 30 01:06 AM]: Repeat soft-masking finished:
Masked genome: /hpc/group/bio1/ian/envs/test-mask_e3d485dd-d34b-4a2b-b684-f7b62bc53bba/test.masked.fa
num scaffolds: 2
assembly size: 1,216,048 bp
masked repeats: 50,965 bp (4.19%)

---

CMD: funannotate mask -i test.fa -o test.masked.fa --cpus 16
#########################################################
#########################################################
SUCCESS: funannotate mask test complete.
#########################################################

#########################################################
Running funannotate predict unit testing
Downloading: https://osf.io/te2pf/download?version=1 Bytes: 1489808
[DOWNLOAD PROGRESS OMITTED FOR SPACE]
�������������������-------------------------------------------------------
[Jun 30 01:06 AM]: OS: CentOS Stream 8, 46 cores, ~ 230 GB RAM. Python: 3.8.12
[Jun 30 01:06 AM]: Running funannotate v1.8.11
[Jun 30 01:06 AM]: Skipping CodingQuarry as no --rna_bam passed
[Jun 30 01:06 AM]: Parsed training data, run ab-initio gene predictors as follows:
�[4mProgram Training-Method�[0m
augustus pretrained
glimmerhmm busco
snap busco
[Jun 30 01:06 AM]: Loading genome assembly and parsing soft-masked repetitive sequences
[Jun 30 01:06 AM]: Genome loaded: 6 scaffolds; 3,776,588 bp; 19.75% repeats masked
[Jun 30 01:06 AM]: Mapping 1,065 proteins to genome using diamond and exonerate
[Jun 30 01:06 AM]: Found 1,505 preliminary alignments with diamond in 0:00:01 --> generated FASTA files for exonerate in 0:00:00
[PROGRESS UPDATES OMITTED FOR SPACE]
Progress: 99.93%
[Jun 30 01:07 AM]: Exonerate finished in 0:00:19: found 1,270 alignments
[Jun 30 01:07 AM]: Running BUSCO to find conserved gene models for training ab-initio predictors
[Jun 30 01:11 AM]: 175 valid BUSCO predictions found, validating protein sequences
[Jun 30 01:12 AM]: 175 BUSCO predictions validated
[Jun 30 01:12 AM]: Running Augustus gene prediction using saccharomyces parameters
[PROGRESS UPDATES OMITTED FOR SPACE]
Progress: 90.91%
[Jun 30 01:13 AM]: 1,485 predictions from Augustus
[Jun 30 01:13 AM]: Pulling out high quality Augustus predictions
[Jun 30 01:13 AM]: Found 371 high quality predictions from Augustus (>90% exon evidence)
[Jun 30 01:13 AM]: Running SNAP gene prediction, using training data: annotate/predict_misc/busco.final.gff3
[Jun 30 01:14 AM]: 1,512 predictions from SNAP
[Jun 30 01:14 AM]: Running GlimmerHMM gene prediction, using training data: annotate/predict_misc/busco.final.gff3
[Jun 30 01:15 AM]: 1,597 predictions from GlimmerHMM
[Jun 30 01:15 AM]: Summary of gene models passed to EVM (weights):
[Jun 30 01:15 AM]: EVM: partitioning input to ~ 35 genes per partition using min 1500 bp interval
[PROGRESS UPDATES OMITTED FOR SPACE]
Progress: 97.96%
[Jun 30 01:18 AM]: Converting to GFF3 and collecting all EVM results
�[4mSource Weight Count�[0m
Augustus 1 1325
Augustus HiQ 2 372
GlimmerHMM 1 1597
snap 1 1512
Total - 4806
[Jun 30 01:18 AM]: 1,654 total gene models from EVM
[Jun 30 01:18 AM]: Generating protein fasta files from 1,654 EVM models
[Jun 30 01:18 AM]: now filtering out bad gene models (< 50 aa in length, transposable elements, etc).
[Jun 30 01:18 AM]: Found 128 gene models to remove: 0 too short; 0 span gaps; 128 transposable elements
[Jun 30 01:18 AM]: 1,526 gene models remaining
[Jun 30 01:18 AM]: Predicting tRNAs
[Jun 30 01:18 AM]: 112 tRNAscan models are valid (non-overlapping)
[Jun 30 01:18 AM]: Generating GenBank tbl annotation file
[Jun 30 01:18 AM]: Collecting final annotation files for 1,638 total gene models
[Jun 30 01:18 AM]: Converting to final Genbank format
[Jun 30 01:18 AM]: Funannotate predict is finished, output files are in the annotate/predict_results folder
[Jun 30 01:18 AM]: Your next step might be functional annotation, suggested commands:

Run InterProScan (manual install):
funannotate iprscan -i annotate -c 16

Run antiSMASH (optional):
funannotate remote -i annotate -m antismash -e youremail@server.edu

Annotate Genome:
funannotate annotate -i annotate --cpus 16 --sbt yourSBTfile.txt

[Jun 30 01:18 AM]: Training parameters file saved: annotate/predict_results/saccharomyces.parameters.json
[Jun 30 01:18 AM]: Add species parameters to database:

funannotate species -s saccharomyces -a annotate/predict_results/saccharomyces.parameters.json

---

[Jun 30 01:19 AM]: OS: CentOS Stream 8, 46 cores, ~ 230 GB RAM. Python: 3.8.12
[Jun 30 01:19 AM]: Running funannotate v1.8.11
[Jun 30 01:19 AM]: Skipping CodingQuarry as no --rna_bam passed
[Jun 30 01:19 AM]: Parsed training data, run ab-initio gene predictors as follows:
�[4mProgram Training-Method�[0m
augustus busco
glimmerhmm busco
snap busco
[Jun 30 01:19 AM]: Loading genome assembly and parsing soft-masked repetitive sequences
[Jun 30 01:19 AM]: Genome loaded: 6 scaffolds; 3,776,588 bp; 19.75% repeats masked
[Jun 30 01:19 AM]: Mapping 1,065 proteins to genome using diamond and exonerate
[Jun 30 01:19 AM]: Found 1,505 preliminary alignments with diamond in 0:00:01 --> generated FASTA files for exonerate in 0:00:00
[PROGRESS UPDATES OMITTED FOR SPACE]
Progress: 99.93%
[Jun 30 01:19 AM]: Exonerate finished in 0:00:19: found 1,270 alignments
[Jun 30 01:19 AM]: Running BUSCO to find conserved gene models for training ab-initio predictors
[Jun 30 01:24 AM]: 175 valid BUSCO predictions found, validating protein sequences
[Jun 30 01:24 AM]: 175 BUSCO predictions validated
[Jun 30 01:24 AM]: Not enough gene models 175 to train Augustus (200 required), exiting
CMD: funannotate predict -i test.softmasked.fa --protein_evidence protein.evidence.fasta -o annotate --augustus_species saccharomyces --cpus 16 --species Awesome testicus
#########################################################
#########################################################
SUCCESS: funannotate predict test complete.
#########################################################

#########################################################
Running funannotate predict BUSCO-mediated training unit testing
CMD: funannotate predict -i test.softmasked.fa --protein_evidence protein.evidence.fasta -o annotate --cpus 16 --species Awesome busco
#########################################################
#########################################################
Traceback (most recent call last):
File "/hpc/home/idm7/miniconda3/envs/annotate/bin/funannotate", line 10, in
sys.exit(main())
File "/hpc/home/idm7/miniconda3/envs/annotate/lib/python3.8/site-packages/funannotate/funannotate.py", line 716, in main
mod.main(arguments)
File "/hpc/home/idm7/miniconda3/envs/annotate/lib/python3.8/site-packages/funannotate/test.py", line 407, in main
runBuscoTest(args)
File "/hpc/home/idm7/miniconda3/envs/annotate/lib/python3.8/site-packages/funannotate/test.py", line 200, in runBuscoTest
assert 1500 <= countGFFgenes(os.path.join(
File "/hpc/home/idm7/miniconda3/envs/annotate/lib/python3.8/site-packages/funannotate/test.py", line 45, in countGFFgenes
with open(input, 'r') as f:
FileNotFoundError: [Errno 2] No such file or directory: 'test-busco_e3d485dd-d34b-4a2b-b684-f7b62bc53bba/annotate/predict_results/Awesome_busco.gff3'
`

[IanDMedeiros]

No, still failing on real data...

---

[Jun 30 01:32 AM]: OS: CentOS Stream 8, 12 cores, ~ 33 GB RAM. Python: 3.8.12
[Jun 30 01:32 AM]: Running funannotate v1.8.11
[Jun 30 01:32 AM]: Skipping CodingQuarry as no --rna_bam passed
[Jun 30 01:32 AM]: Parsed training data, run ab-initio gene predictors as follows:
�[4mProgram Training-Method�[0m
augustus busco
glimmerhmm busco
snap busco
[Jun 30 01:32 AM]: Loading genome assembly and parsing soft-masked repetitive sequences
[Jun 30 01:32 AM]: Genome loaded: 74 scaffolds; 26,734,419 bp; 2.37% repeats masked
[Jun 30 01:32 AM]: Mapping 554,696 proteins to genome using diamond and exonerate
[Jun 30 01:35 AM]: Found 293,585 preliminary alignments with diamond in 0:01:35 --> generated FASTA files for exonerate in 0:00:58
[PROGRESS OMITTED FOR SPACE]
[Jun 30 02:34 AM]: Exonerate finished in 0:59:08: found 1,406 alignments
[Jun 30 02:35 AM]: Running BUSCO to find conserved gene models for training ab-initio predictors
[Jun 30 02:45 AM]: 0 valid BUSCO predictions found, validating protein sequences
Traceback (most recent call last):
File "/hpc/home/idm7/miniconda3/envs/annotate/bin/funannotate", line 10, in
sys.exit(main())
File "/hpc/home/idm7/miniconda3/envs/annotate/lib/python3.8/site-packages/funannotate/funannotate.py", line 716, in main
mod.main(arguments)
File "/hpc/home/idm7/miniconda3/envs/annotate/lib/python3.8/site-packages/funannotate/predict.py", line 1354, in main
buscoProtComplete = lib.getCompleteBuscos(buscoProtOutput,
File "/hpc/home/idm7/miniconda3/envs/annotate/lib/python3.8/site-packages/funannotate/library.py", line 5370, in getCompleteBuscos
with open(input, 'r') as infile:
FileNotFoundError: [Errno 2] No such file or directory: 'annotated/predict_misc/busco_proteins/run_eurotiomycetes_af_10-2_af_10-2/full_table_eurotiomycetes_af_10-2_af_10-2.tsv'

[nextgenusfs]

The test is still somewhat failing for the same reason -- a successful test should produce > 200 models from BUSCO. So I think it is still related to augustus.

FYI you can use --no-progress to suppress that annoying progress meter on HPC.

Here is my successful test:

$ ./funannotate_dev/funannotate-docker test -t predict --cpus 4
#########################################################
Running `funannotate predict` unit testing
CMD: funannotate predict -i test.softmasked.fa --protein_evidence protein.evidence.fasta -o annotate --augustus_species saccharomyces --cpus 4 --species Awesome testicus
#########################################################
-------------------------------------------------------
[Jun 30 09:01 AM]: OS: Debian GNU/Linux 10, 4 cores, ~ 8 GB RAM. Python: 3.8.13
[Jun 30 09:01 AM]: Running funannotate v1.8.12
[Jun 30 09:01 AM]: GeneMark not found and $GENEMARK_PATH environmental variable missing. Will skip GeneMark ab-initio prediction.
[Jun 30 09:01 AM]: Skipping CodingQuarry as no --rna_bam passed
[Jun 30 09:01 AM]: Parsed training data, run ab-initio gene predictors as follows:
  Program      Training-Method
  augustus     pretrained     
  glimmerhmm   busco          
  snap         busco          
[Jun 30 09:01 AM]: Loading genome assembly and parsing soft-masked repetitive sequences
[Jun 30 09:01 AM]: Genome loaded: 6 scaffolds; 3,776,588 bp; 19.75% repeats masked
[Jun 30 09:01 AM]: Mapping 1,065 proteins to genome using diamond and exonerate
[Jun 30 09:01 AM]: Found 1,505 preliminary alignments with diamond in 0:00:02 --> generated FASTA files for exonerate in 0:00:00
[Jun 30 09:02 AM]: Exonerate finished in 0:00:34: found 1,270 alignments
[Jun 30 09:02 AM]: Running BUSCO to find conserved gene models for training ab-initio predictors
[Jun 30 09:14 AM]: 373 valid BUSCO predictions found, validating protein sequences
[Jun 30 09:15 AM]: 370 BUSCO predictions validated
[Jun 30 09:15 AM]: Running Augustus gene prediction using saccharomyces parameters
[Jun 30 09:18 AM]: 1,485 predictions from Augustus
[Jun 30 09:18 AM]: Pulling out high quality Augustus predictions
[Jun 30 09:18 AM]: Found 371 high quality predictions from Augustus (>90% exon evidence)
[Jun 30 09:18 AM]: Running SNAP gene prediction, using training data: annotate/predict_misc/busco.final.gff3
[Jun 30 09:20 AM]: 1,362 predictions from SNAP
[Jun 30 09:20 AM]: Running GlimmerHMM gene prediction, using training data: annotate/predict_misc/busco.final.gff3
[Jun 30 09:23 AM]: 1,769 predictions from GlimmerHMM
[Jun 30 09:23 AM]: Summary of gene models passed to EVM (weights):
  Source         Weight   Count
  Augustus       1        1325 
  Augustus HiQ   2        372  
  GlimmerHMM     1        1769 
  snap           1        1362 
  Total          -        4828 
[Jun 30 09:23 AM]: EVM: partitioning input to ~ 35 genes per partition using min 1500 bp interval
[Jun 30 09:34 AM]: Converting to GFF3 and collecting all EVM results
[Jun 30 09:34 AM]: 1,695 total gene models from EVM
[Jun 30 09:34 AM]: Generating protein fasta files from 1,695 EVM models
[Jun 30 09:34 AM]: now filtering out bad gene models (< 50 aa in length, transposable elements, etc).
[Jun 30 09:34 AM]: Found 137 gene models to remove: 0 too short; 0 span gaps; 137 transposable elements
[Jun 30 09:34 AM]: 1,558 gene models remaining
[Jun 30 09:34 AM]: Predicting tRNAs
[Jun 30 09:35 AM]: 112 tRNAscan models are valid (non-overlapping)
[Jun 30 09:35 AM]: Generating GenBank tbl annotation file
[Jun 30 09:35 AM]: Collecting final annotation files for 1,670 total gene models
[Jun 30 09:35 AM]: Converting to final Genbank format
[Jun 30 09:35 AM]: Funannotate predict is finished, output files are in the annotate/predict_results folder
[Jun 30 09:35 AM]: Your next step might be functional annotation, suggested commands:
-------------------------------------------------------
Run InterProScan (manual install): 
funannotate iprscan -i annotate -c 4

Run antiSMASH (optional): 
funannotate remote -i annotate -m antismash -e youremail@server.edu

Annotate Genome: 
funannotate annotate -i annotate --cpus 4 --sbt yourSBTfile.txt
-------------------------------------------------------
                
[Jun 30 09:35 AM]: Training parameters file saved: annotate/predict_results/saccharomyces.parameters.json
[Jun 30 09:35 AM]: Add species parameters to database:

  funannotate species -s saccharomyces -a annotate/predict_results/saccharomyces.parameters.json

#########################################################
SUCCESS: `funannotate predict` test complete.
#########################################################

And then the busco.log:

INFO	****************** Start a BUSCO 2.0 analysis, current time: 06/30/2022 09:02:33 ******************
INFO	The lineage dataset is: dikarya_odb9 (eukaryota)
INFO	Mode is: genome
INFO	Maximum number of regions limited to: 3
INFO	To reproduce this run: python /venv/lib/python3.8/site-packages/funannotate/aux_scripts/funannotate-BUSCO2.py -i /Users/jon/test-predict_6004e1e9-1eda-4469-a5b0-c06c104a1135/annotate/predict_misc/genome.softmasked.fa -o saccharomyces -l /opt/databases/dikarya/ -m genome -c 4 -sp anidulans
INFO	Check dependencies...
INFO	Check input file...
INFO	Temp directory is ./tmp/

INFO	****** Phase 1 of 2, initial predictions ******
INFO	****** Step 1/3, current time: 06/30/2022 09:02:33 ******
INFO	Create blast database...
INFO	[makeblastdb]	Building a new DB, current time: 06/30/2022 09:02:33
INFO	[makeblastdb]	New DB name:   /Users/jon/test-predict_6004e1e9-1eda-4469-a5b0-c06c104a1135/annotate/predict_misc/busco/tmp/saccharomyces_3039243825
INFO	[makeblastdb]	New DB title:  /Users/jon/test-predict_6004e1e9-1eda-4469-a5b0-c06c104a1135/annotate/predict_misc/genome.softmasked.fa
INFO	[makeblastdb]	Sequence type: Nucleotide
INFO	[makeblastdb]	Keep Linkouts: T
INFO	[makeblastdb]	Keep MBits: T
INFO	[makeblastdb]	Maximum file size: 1000000000B
INFO	[makeblastdb]	Adding sequences from FASTA; added 6 sequences in 0.0503011 seconds.
INFO	Running tblastn, writing output to /Users/jon/test-predict_6004e1e9-1eda-4469-a5b0-c06c104a1135/annotate/predict_misc/busco/run_saccharomyces/blast_output/tblastn_saccharomyces.tsv...
INFO	****** Step 2/3, current time: 06/30/2022 09:02:42 ******
INFO	Getting coordinates for candidate regions...
INFO	Pre-Augustus scaffold extraction...
INFO	Running Augustus prediction using anidulans as species:
INFO	[augustus] Please find all logs related to Augustus here: /Users/jon/test-predict_6004e1e9-1eda-4469-a5b0-c06c104a1135/annotate/predict_misc/busco/run_saccharomyces/augustus_output/augustus.log
INFO	06/30/2022 09:02:42 =>	0% of predictions performed (743 to be done)
INFO	06/30/2022 09:04:21 =>	10% of predictions performed (75/743 candidate regions)
INFO	06/30/2022 09:05:44 =>	20% of predictions performed (149/743 candidate regions)
INFO	06/30/2022 09:07:25 =>	30% of predictions performed (223/743 candidate regions)
INFO	06/30/2022 09:08:50 =>	40% of predictions performed (298/743 candidate regions)
INFO	06/30/2022 09:09:50 =>	50% of predictions performed (372/743 candidate regions)
INFO	06/30/2022 09:10:44 =>	60% of predictions performed (446/743 candidate regions)
INFO	06/30/2022 09:11:28 =>	70% of predictions performed (521/743 candidate regions)
INFO	06/30/2022 09:12:15 =>	80% of predictions performed (595/743 candidate regions)
INFO	06/30/2022 09:12:51 =>	90% of predictions performed (669/743 candidate regions)
INFO	06/30/2022 09:13:22 =>	100% of predictions performed
INFO	Extracting predicted proteins...
INFO	****** Step 3/3, current time: 06/30/2022 09:13:42 ******
INFO	Running HMMER to confirm orthology of predicted proteins:
INFO	06/30/2022 09:13:42 =>	0% of predictions performed (686 to be done)
INFO	06/30/2022 09:13:43 =>	10% of predictions performed (69/686 candidate proteins)
INFO	06/30/2022 09:13:44 =>	20% of predictions performed (139/686 candidate proteins)
INFO	06/30/2022 09:13:45 =>	30% of predictions performed (208/686 candidate proteins)
INFO	06/30/2022 09:13:46 =>	40% of predictions performed (277/686 candidate proteins)
INFO	06/30/2022 09:13:48 =>	50% of predictions performed (346/686 candidate proteins)
INFO	06/30/2022 09:13:49 =>	60% of predictions performed (412/686 candidate proteins)
INFO	06/30/2022 09:13:51 =>	70% of predictions performed (481/686 candidate proteins)
INFO	06/30/2022 09:13:53 =>	80% of predictions performed (549/686 candidate proteins)
INFO	06/30/2022 09:13:55 =>	90% of predictions performed (619/686 candidate proteins)
INFO	06/30/2022 09:13:57 =>	100% of predictions performed
INFO	Results:
INFO	C:28.9%[S:28.4%,D:0.5%],F:0.8%,M:70.3%,n:1312
INFO	380 Complete BUSCOs (C)
INFO	373 Complete and single-copy BUSCOs (S)
INFO	7 Complete and duplicated BUSCOs (D)
INFO	10 Fragmented BUSCOs (F)
INFO	922 Missing BUSCOs (M)
INFO	1312 Total BUSCO groups searched

INFO	****** Phase 2 of 2, predictions using species specific training ******
INFO	****** Step 1/3, current time: 06/30/2022 09:13:58 ******
INFO	Extracting missing and fragmented buscos from the ancestral_variants file...
WARNING	The busco id(s) ['EOG092647L7', 'EOG09264L0C', 'EOG09262X7T', 'EOG09264RR2', 'EOG09264RIE', 'EOG09265SHM', 'EOG09262TO9', 'EOG0926448Q', 'EOG092641G3', 'EOG09262O0R', 'EOG092610VI', 'EOG09261V2P', 'EOG0926400M', 'EOG09261DG0', 'EOG09264881', 'EOG0926457R', 'EOG09264331', 'EOG09260LRX', 'EOG092643IE', 'EOG09260JNY', 'EOG09260Z5E', 'EOG0926458I', 'EOG092657UN', 'EOG09264Z3D', 'EOG092629RT', 'EOG092612XD', 'EOG09261QR8', 'EOG09265FTY', 'EOG09260JO5', 'EOG09262V8E', 'EOG092630YS', 'EOG092644ZU', 'EOG092621CK', 'EOG09262FE3', 'EOG092624X0', 'EOG09261NLY', 'EOG09260WCZ', 'EOG09260WGT', 'EOG09262KUJ', 'EOG09262N0U', 'EOG09262SI7', 'EOG092645OU', 'EOG092632TF', 'EOG09261JCQ', 'EOG09264XTW', 'EOG09261XNJ', 'EOG09264XT5', 'EOG09263IT0', 'EOG09264U78', 'EOG09262NB1', 'EOG092610TN', 'EOG09264ABT', 'EOG092610ZY', 'EOG09263Q8J', 'EOG09264FXB', 'EOG09261W2O', 'EOG09263G4R', 'EOG0926510L', 'EOG09264OYZ', 'EOG092640WA', 'EOG092602FH', 'EOG09262UTQ', 'EOG092608WI', 'EOG09260EQD', 'EOG09263WM5', 'EOG09263FAK', 'EOG092605ZA', 'EOG09263FKC', 'EOG09263YBT', 'EOG09263R4M', 'EOG092659DX', 'EOG09261QXC', 'EOG092605VL', 'EOG09263ZSC', 'EOG09262WXK', 'EOG092644DY', 'EOG09260LVD', 'EOG092608AV', 'EOG09261727', 'EOG09261OXD', 'EOG09260EE7', 'EOG092611HB', 'EOG09262N3C', 'EOG09264DIM', 'EOG092604MJ', 'EOG09264HOY', 'EOG09264XPY', 'EOG092626HU', 'EOG092650I8', 'EOG0926315C', 'EOG09260DXP', 'EOG09263BG5', 'EOG092616QN', 'EOG09264OBA', 'EOG09264BIX', 'EOG092644N1', 'EOG09264DT4', 'EOG09262N47', 'EOG09263CLY', 'EOG09265I7S', 'EOG09262W7C', 'EOG092654LJ', 'EOG09260K29', 'EOG09264LH2', 'EOG09265822', 'EOG09263BE5', 'EOG09261OKK', 'EOG09260NE0', 'EOG09262D0D', 'EOG09262SWJ', 'EOG09261P7G', 'EOG0926306O', 'EOG0926140Q', 'EOG09261H5E', 'EOG092615IE', 'EOG092650VI', 'EOG09260BFE', 'EOG09261VI3', 'EOG092635DF', 'EOG09264SUZ', 'EOG09260VYK', 'EOG09264L06', 'EOG09264904', 'EOG09261S7X', 'EOG092631MU', 'EOG09261I0F', 'EOG09260LJ8', 'EOG09262MXH', 'EOG09263RW3', 'EOG09260HPO', 'EOG092658ZO', 'EOG092610KH', 'EOG09263BDA', 'EOG09260OZU', 'EOG09263760', 'EOG09263KVG', 'EOG092615SM', 'EOG092629WA', 'EOG092652Y6', 'EOG09263Z8I', 'EOG0926115P', 'EOG09264398', 'EOG09262J7K', 'EOG09263TQ5', 'EOG09260W8D', 'EOG09260J97', 'EOG092600W1', 'EOG092604ZZ', 'EOG09262D4G', 'EOG09261UPM', 'EOG09264873', 'EOG09261LV7', 'EOG092605T6', 'EOG09260NAN', 'EOG09263EQZ', 'EOG09261V87', 'EOG09260EAZ', 'EOG09262528', 'EOG09260VEY', 'EOG09260MRU', 'EOG09260RRC', 'EOG09260APA', 'EOG092653VU', 'EOG09262YP5', 'EOG09261KRX', 'EOG092628SP', 'EOG09262YQG', 'EOG09263KDI', 'EOG092634ZL', 'EOG09264HU0', 'EOG0926357F', 'EOG09261TEQ', 'EOG09260AQB', 'EOG09265040', 'EOG092629ZN', 'EOG092605KN', 'EOG09262K8C', 'EOG09260OLB', 'EOG09261660', 'EOG092628HC', 'EOG09264G7L', 'EOG0926248P', 'EOG0926112A', 'EOG09260QNB', 'EOG09264ZDZ', 'EOG09262KVB', 'EOG09263SZM', 'EOG092656RK', 'EOG09263RY2', 'EOG0926158Y', 'EOG092654XA', 'EOG0926436T', 'EOG09264W71', 'EOG092619MJ', 'EOG09260TWS', 'EOG09260BYL', 'EOG09262GVX', 'EOG09264PE5', 'EOG09261CXQ', 'EOG09265GXF', 'EOG09260E2O', 'EOG09261476', 'EOG092649VG', 'EOG09262QTY', 'EOG092645U1', 'EOG092652ZZ', 'EOG09260JTZ', 'EOG092625AX', 'EOG09263CUQ', 'EOG09261Y04', 'EOG09263D2P', 'EOG0926049A', 'EOG09265G5K', 'EOG09264EGS', 'EOG09265BG5', 'EOG092619GP', 'EOG09264J8E', 'EOG09260RVQ', 'EOG09262GWQ', 'EOG09261801', 'EOG092658SK', 'EOG092644X6', 'EOG09264JQ1', 'EOG09265HEP', 'EOG092604KQ', 'EOG09260XQV', 'EOG09262ZZ8', 'EOG09265DWT', 'EOG09264U81', 'EOG09260BSW', 'EOG09260OCI', 'EOG09264W1U', 'EOG09262C5Z', 'EOG09262B1U', 'EOG092654VM', 'EOG09262MJW', 'EOG09261404', 'EOG09261I1I', 'EOG09261JR0', 'EOG0926025H', 'EOG092648LP', 'EOG09264R4M', 'EOG09264KTU', 'EOG09264T1U', 'EOG09264O2D', 'EOG09263EDC', 'EOG09264GMT', 'EOG092629FB', 'EOG092643Y5', 'EOG09264I6B', 'EOG092600SK', 'EOG09265KNL', 'EOG09264RJ7', 'EOG09264CA0', 'EOG09262645', 'EOG092617S2', 'EOG09260V5Q', 'EOG09260Z3X', 'EOG09262UB3', 'EOG092603SM', 'EOG0926195C', 'EOG09261QR5', 'EOG0926307V', 'EOG092609O9', 'EOG09264OM7', 'EOG092654KW', 'EOG092634B5', 'EOG092658WY', 'EOG09262POL', 'EOG0926229Z', 'EOG09260274', 'EOG09264UJF', 'EOG09264L3W', 'EOG09260KM4', 'EOG09260JPV', 'EOG0926514P', 'EOG09260XH5', 'EOG09261RWU', 'EOG0926079Q', 'EOG092647CM', 'EOG09265552', 'EOG09262SR7', 'EOG092619RJ', 'EOG092652TN', 'EOG09263A3Y', 'EOG09260H6E', 'EOG092602OP', 'EOG09260RS7', 'EOG09260RGH', 'EOG09263LNF', 'EOG092645LS', 'EOG09263C55', 'EOG09262CBI', 'EOG09263FR7', 'EOG092620IA', 'EOG092608AE', 'EOG09260SJV', 'EOG092600SD', 'EOG09264OXC', 'EOG09260W52', 'EOG09264USX', 'EOG092605VU', 'EOG09264XYD', 'EOG09260DUR', 'EOG092604A0', 'EOG092649QJ', 'EOG092605FC', 'EOG09265DRM', 'EOG09261OXV', 'EOG092659OC', 'EOG092644WX', 'EOG09262U7S', 'EOG092618M2', 'EOG09264P74', 'EOG09262GNE', 'EOG09265F2Y', 'EOG09264LC7', 'EOG09263ULA', 'EOG09261PUF', 'EOG092638XA', 'EOG09264IDN', 'EOG09261HB6', 'EOG09265K4K', 'EOG092608ZS', 'EOG09260KUC', 'EOG092615CC', 'EOG09265QTV', 'EOG0926248W', 'EOG09262WHU', 'EOG0926213Q', 'EOG09264TQ5', 'EOG09260EPS', 'EOG09262GLP', 'EOG092646PE', 'EOG092624RX', 'EOG0926073O', 'EOG09264GQZ', 'EOG092638RC', 'EOG092634MM', 'EOG09261NW2', 'EOG09262R8O', 'EOG09264OML', 'EOG0926506Z', 'EOG09264G1I', 'EOG09260FL2', 'EOG09265CN0', 'EOG092654QZ', 'EOG09261DW8', 'EOG09260XSR', 'EOG0926133I', 'EOG09265G9U', 'EOG09260FZZ', 'EOG092645L9', 'EOG09262Z2S', 'EOG09264ZQC', 'EOG09262OX9', 'EOG09263QUM', 'EOG092619NK', 'EOG09264CP8', 'EOG09260RRN', 'EOG09260GR8', 'EOG09262VOC', 'EOG09264FVQ', 'EOG09260DUW', 'EOG09264BOA', 'EOG09262E4T', 'EOG09265BE5', 'EOG09261OSU', 'EOG0926049S', 'EOG09263UN3', 'EOG092603D0', 'EOG09264WF4', 'EOG092631IR', 'EOG09262BVA', 'EOG092631QQ', 'EOG09263I7I', 'EOG09264C3V', 'EOG09264ZDJ', 'EOG09260NZ8', 'EOG092653LT', 'EOG09260K24', 'EOG09262WSH', 'EOG09264C3N', 'EOG09263EVJ', 'EOG09260PI1', 'EOG0926131E', 'EOG092653YS', 'EOG092656JA', 'EOG09262VVF', 'EOG09260A98', 'EOG09261WVT', 'EOG09263720', 'EOG09260CKC', 'EOG092629WJ', 'EOG09261127', 'EOG092602MO', 'EOG09261UOJ', 'EOG09264W46', 'EOG09264NJ1', 'EOG092610QT', 'EOG0926273Q', 'EOG09265FCK', 'EOG09265ER6', 'EOG09260GKG', 'EOG092608T8', 'EOG09261FM4', 'EOG09262A6N', 'EOG09263CGP', 'EOG092621CP', 'EOG09260779', 'EOG09265B95', 'EOG09261NLR', 'EOG09265RGI', 'EOG09261ZXZ', 'EOG09261K9J', 'EOG09263MR4', 'EOG09263E87', 'EOG09260WG2', 'EOG09262G8Y', 'EOG09260FMW', 'EOG09263G3M', 'EOG09260PJ9', 'EOG092657YR', 'EOG092646WF', 'EOG09264DY4', 'EOG09263HED', 'EOG09260931', 'EOG092636T6', 'EOG09260U6R', 'EOG09263MGE', 'EOG092619L1', 'EOG09260N2T', 'EOG09264IG5', 'EOG09264O9F', 'EOG09265H9T', 'EOG09260TDY', 'EOG09262X74', 'EOG0926213Z', 'EOG09261I8J', 'EOG09263GSR', 'EOG09262KJA', 'EOG09264O6J', 'EOG09262P1W', 'EOG09261N20', 'EOG09262QS5', 'EOG092603EH', 'EOG09260Y2Q', 'EOG09260XL0', 'EOG09263KZJ', 'EOG09264A2D', 'EOG09263K05', 'EOG092604S1', 'EOG09265M98', 'EOG09265PWR', 'EOG09260EOI', 'EOG09265LEG', 'EOG09264272', 'EOG09261G1Y', 'EOG09261666', 'EOG09264T0J', 'EOG09261IEV', 'EOG092605QM', 'EOG09261Q18', 'EOG092606WZ', 'EOG09261JVS', 'EOG09265CCT', 'EOG09261EY9', 'EOG09262QJW', 'EOG09260UXC', 'EOG0926077L', 'EOG09265KQ4', 'EOG09264AWW', 'EOG092600T9', 'EOG0926539T', 'EOG09262K67', 'EOG09263CG4', 'EOG09263DQH', 'EOG09265D7J', 'EOG09262BCZ', 'EOG092614UB', 'EOG09261EM7', 'EOG09264OQ8', 'EOG0926423H', 'EOG09264NEF', 'EOG092620CR', 'EOG092630MJ', 'EOG092651HW', 'EOG09262SMG', 'EOG09264E6Z', 'EOG092641M3', 'EOG09262A8G', 'EOG092601KZ', 'EOG092642I5', 'EOG09265DDU', 'EOG09263JTO', 'EOG09263OZR', 'EOG09262XRU', 'EOG0926534P', 'EOG09260DFH', 'EOG09260VCG', 'EOG09260VTN', 'EOG092618J9', 'EOG09264RJL', 'EOG09262IY3', 'EOG09264S3E', 'EOG09264V2U', 'EOG09262NNS', 'EOG09260E8K', 'EOG09261MOX', 'EOG09260M87', 'EOG09261CM0', 'EOG09262BHE', 'EOG09261W90', 'EOG09260WUA', 'EOG09260S2Z', 'EOG09264TJN', 'EOG092646CB', 'EOG09263RDD', 'EOG09264RBX', 'EOG09263OCO', 'EOG09263F11', 'EOG092648VW', 'EOG09264VRO', 'EOG09261ACJ', 'EOG09261OLD', 'EOG09264ENO', 'EOG09262PPU', 'EOG092612LP', 'EOG09263Z41', 'EOG09263X22', 'EOG09261N64', 'EOG09261OIA', 'EOG09262VPD', 'EOG09262LQT', 'EOG092609YT', 'EOG092627F1', 'EOG09260FFP', 'EOG092602I6', 'EOG0926477X', 'EOG09261JWS', 'EOG09265GYD', 'EOG09264XUV', 'EOG092655L0', 'EOG092608L0', 'EOG0926419M', 'EOG09264PD5', 'EOG09260LI6', 'EOG09261IEH', 'EOG09261UWT', 'EOG09262CXO', 'EOG09262LI4', 'EOG09265KPR', 'EOG09264T3S', 'EOG092628FW', 'EOG092639H5', 'EOG09261FH7', 'EOG09262Q6S', 'EOG0926505R', 'EOG09264B3O', 'EOG092653SU', 'EOG09260S5R', 'EOG0926004Z', 'EOG092603KJ', 'EOG09263OWL', 'EOG09264DJ8', 'EOG09264T8I', 'EOG09264SQJ', 'EOG09262TEV', 'EOG092653NM', 'EOG09260VTA', 'EOG0926092K', 'EOG09262X01', 'EOG092649XV', 'EOG09262V3O', 'EOG092635YY', 'EOG09261YLQ', 'EOG09262FJB', 'EOG0926499W', 'EOG09260JJW', 'EOG092607OQ', 'EOG09260ERO', 'EOG09262V9N', 'EOG09260B3X', 'EOG09265I60', 'EOG09264VC6', 'EOG09261FAX', 'EOG09262XMN', 'EOG09264R0D', 'EOG092638CT', 'EOG09265EKJ', 'EOG09261SS1', 'EOG092602UY', 'EOG092616YZ', 'EOG09263RVR', 'EOG09261ICI', 'EOG09263UWJ', 'EOG09262N10', 'EOG09261ZJR', 'EOG09260KCB', 'EOG09263J3H', 'EOG09263FGN', 'EOG09264ZWF', 'EOG09264W7W', 'EOG09262UAS', 'EOG0926310O', 'EOG09261LPY', 'EOG0926071Q', 'EOG09262E3Q', 'EOG09262DPL', 'EOG09260AZK', 'EOG09264KDO', 'EOG092617RY', 'EOG09264HN5', 'EOG09260QVP', 'EOG09260375', 'EOG09264X31', 'EOG09264G04', 'EOG092609JB', 'EOG092606AJ', 'EOG092606AD', 'EOG09260NNR', 'EOG09261W1K', 'EOG092613UB', 'EOG09264I14', 'EOG09260H81', 'EOG09261XNU', 'EOG09260NWN', 'EOG09264IQ7', 'EOG09264CP4', 'EOG09265RGS', 'EOG09260GIX', 'EOG092658X5', 'EOG0926251E', 'EOG092621GA', 'EOG09263LR1', 'EOG09263E9V', 'EOG09262KZ3', 'EOG09262E7W', 'EOG092600NM', 'EOG09260RNZ', 'EOG09264IV9', 'EOG09262PMC', 'EOG09260R9L', 'EOG092631UM', 'EOG09260NC6', 'EOG09264P4J', 'EOG09260KGS', 'EOG09261B3Q', 'EOG09264ZXA', 'EOG09265B1X', 'EOG09262F22', 'EOG09263OAE', 'EOG09261V9P', 'EOG09261Q5L', 'EOG09264MGU', 'EOG09264KK7', 'EOG092611G7', 'EOG092644TW', 'EOG09261D4D', 'EOG09265PQX', 'EOG09263X1F', 'EOG09260KDB', 'EOG09263ZW6', 'EOG09263690', 'EOG09260NHB', 'EOG09261J0P', 'EOG09262JZK', 'EOG09263L9T', 'EOG092656CM', 'EOG09263GIG', 'EOG09262516', 'EOG09263IMF', 'EOG09265FTN', 'EOG09262WQX', 'EOG09265K60', 'EOG09264SSI', 'EOG09263OD3', 'EOG09265GGX', 'EOG09264D7Y', 'EOG09260T4S', 'EOG09260WUS', 'EOG09261KHB', 'EOG09261EMF', 'EOG092617AN', 'EOG09265PUI', 'EOG09263K45', 'EOG0926489S', 'EOG09262HP3', 'EOG09263ZBJ', 'EOG092619VG', 'EOG09261T98', 'EOG09260NXC', 'EOG09261ZFN', 'EOG09260MBW', 'EOG09265313', 'EOG09265NHW', 'EOG09260289', 'EOG092624KK', 'EOG09263S2P', 'EOG09264719', 'EOG092624UF', 'EOG09261DRB', 'EOG09261MMR', 'EOG09261VD2', 'EOG09265JA7', 'EOG092613R2', 'EOG09262914', 'EOG09260KNI', 'EOG0926369X', 'EOG09265A08', 'EOG09264A8D', 'EOG09260J8F', 'EOG09260KNR', 'EOG09262TUR', 'EOG09264NC7', 'EOG09264XKX', 'EOG092621ZV', 'EOG092600S9', 'EOG09263CAC', 'EOG09262JRP', 'EOG09260NHN', 'EOG09260B65', 'EOG092643NE', 'EOG09262KXK', 'EOG09265AT5', 'EOG0926431P', 'EOG092620E4', 'EOG092605OK', 'EOG09260JDM', 'EOG092652KR', 'EOG09260LS9', 'EOG09260UA2', 'EOG092641A6', 'EOG09261RWJ', 'EOG09264VZ7', 'EOG09260WU6', 'EOG092641UM', 'EOG0926354S', 'EOG09263M8W', 'EOG09263PWF', 'EOG092658NW', 'EOG092612MY', 'EOG092632WW', 'EOG0926390Q', 'EOG09263QPR', 'EOG09265HP0', 'EOG09263OQH', 'EOG092628LW', 'EOG09263U08', 'EOG092612CC', 'EOG09263AZP', 'EOG092620U5', 'EOG09265OQH', 'EOG09261I1G', 'EOG09260SAH', 'EOG092643QM', 'EOG09263KRO', 'EOG09263817', 'EOG09263MEM', 'EOG09265BJ3', 'EOG09264BWL', 'EOG09263WB5', 'EOG09263X0V', 'EOG09262QRH', 'EOG09264YEG', 'EOG09264PDD', 'EOG092651BA', 'EOG092608WU', 'EOG09264MN3', 'EOG092643JW', 'EOG09262N5O', 'EOG092646VF', 'EOG09261YRA', 'EOG092633QB', 'EOG09261IOS', 'EOG09260C2V', 'EOG09263U71', 'EOG09261225', 'EOG092617RN', 'EOG09261AH9', 'EOG09263SFX', 'EOG0926129I', 'EOG09264JHE', 'EOG09262A65', 'EOG092619EA', 'EOG092648O6', 'EOG092643VB', 'EOG09260DP1', 'EOG09264YKY', 'EOG09263EBB', 'EOG092641K1', 'EOG0926388H', 'EOG09260GF5', 'EOG09263HD8', 'EOG09262PZ9', 'EOG092634G9', 'EOG09265E8A', 'EOG092644O2', 'EOG09264PK5', 'EOG092652YI', 'EOG09264FYY', 'EOG09262387', 'EOG09265IT6', 'EOG09262X8R', 'EOG09264JK1', 'EOG09262M0W', 'EOG092645G0', 'EOG092648K5', 'EOG09264V30', 'EOG09262MFL', 'EOG092657H8', 'EOG09260SRF', 'EOG09265BTA', 'EOG09260TPT', 'EOG09264JW6', 'EOG09264K2W', 'EOG092629U5', 'EOG09262E98', 'EOG09265ANI', 'EOG0926142Y', 'EOG092645QN', 'EOG09262ILV', 'EOG09261B6Y', 'EOG09261ZPW', 'EOG09264SET', 'EOG092646C6', 'EOG09265JNA', 'EOG092636Y6', 'EOG092631ML', 'EOG09263W7L', 'EOG092627XA', 'EOG09261A3K', 'EOG092638EN', 'EOG09264RW6', 'EOG0926009O', 'EOG092651FJ', 'EOG09260HMA', 'EOG092648XW', 'EOG092646EZ', 'EOG092655SO', 'EOG09261YV6', 'EOG092634B1', 'EOG09264IIZ', 'EOG09260075', 'EOG092653KS', 'EOG09263MNN', 'EOG09263Y3L', 'EOG09260S3L', 'EOG09262PAY', 'EOG09261VUC', 'EOG09262M7B', 'EOG09262H1X', 'EOG09262YAU', 'EOG09262DC1', 'EOG09264HX6', 'EOG09261G92', 'EOG09263GUT', 'EOG0926506U', 'EOG09261RFF', 'EOG092624SJ', 'EOG092621F2', 'EOG09260N53', 'EOG09263J6Z', 'EOG092656IY', 'EOG09261PJZ', 'EOG09265JVH', 'EOG09260DBG', 'EOG09262341', 'EOG09263E5F', 'EOG09261V03', 'EOG09264XVU', 'EOG09263KB4', 'EOG09261HQU', 'EOG09262JWJ', 'EOG0926074Y', 'EOG092635ST', 'EOG0926115V', 'EOG09265EOF', 'EOG092635SS', 'EOG092614DJ', 'EOG09264DOU', 'EOG092600T4', 'EOG092626EQ', 'EOG09264LKR', 'EOG09262PIH', 'EOG09260ETR', 'EOG09264KIV', 'EOG09260OE9', 'EOG09261WJ8', 'EOG09265C25', 'EOG09264YIJ', 'EOG09263WZ2', 'EOG09262M2J', 'EOG09263RF8', 'EOG092603YJ', 'EOG092610LQ', 'EOG09261FAB', 'EOG09260KWP', 'EOG09262IP2', 'EOG0926137U', 'EOG09264F1U', 'EOG092648Q0', 'EOG09261G4Z', 'EOG09261B18', 'EOG09261N2L', 'EOG09260HS3', 'EOG092649VA', 'EOG0926591L', 'EOG09264XJC', 'EOG09264B2P', 'EOG09264DMU', 'EOG09264NNY', 'EOG092653O3', 'EOG092608RH', 'EOG092640BS', 'EOG09263JW5', 'EOG09262CDO', 'EOG092655M5', 'EOG09260EPQ', 'EOG09264CND', 'EOG09261F73', 'EOG09264LJU', 'EOG09263A5D', 'EOG09263DFA', 'EOG0926312D', 'EOG09260FKU', 'EOG09263FTE', 'EOG0926347W', 'EOG09264441', 'EOG09262CUO', 'EOG09261MPU', 'EOG0926587S'] were not found in the ancestral_variants file
INFO	Running tblastn, writing output to /Users/jon/test-predict_6004e1e9-1eda-4469-a5b0-c06c104a1135/annotate/predict_misc/busco/run_saccharomyces/blast_output/tblastn_saccharomyces_missing_and_frag_rerun.tsv...
INFO	[tblastn]	Warning: [tblastn] Query is Empty!
INFO	Getting coordinates for candidate regions...
INFO	****** Step 2/3, current time: 06/30/2022 09:13:58 ******
INFO	Training Augustus using Single-Copy Complete BUSCOs:
INFO	06/30/2022 09:13:58 =>	Converting predicted genes to short genbank files...
INFO	06/30/2022 09:14:04 =>	All files converted to short genbank files, now running the training scripts...
INFO	Pre-Augustus scaffold extraction...
INFO	Re-running Augustus with the new metaparameters, number of target BUSCOs: 932
INFO	06/30/2022 09:14:21 =>	0% of predictions performed (0 to be done)
INFO	06/30/2022 09:14:21 =>	100% of predictions performed
INFO	Extracting predicted proteins...
INFO	****** Step 3/3, current time: 06/30/2022 09:14:21 ******
INFO	Running HMMER to confirm orthology of predicted proteins:
INFO	06/30/2022 09:14:21 =>	0% of predictions performed (0 to be done)
INFO	06/30/2022 09:14:21 =>	100% of predictions performed
INFO	Results:
INFO	C:28.9%[S:28.4%,D:0.5%],F:0.8%,M:70.3%,n:1312
INFO	380 Complete BUSCOs (C)
INFO	373 Complete and single-copy BUSCOs (S)
INFO	7 Complete and duplicated BUSCOs (D)
INFO	10 Fragmented BUSCOs (F)
INFO	922 Missing BUSCOs (M)
INFO	1312 Total BUSCO groups searched

INFO	BUSCO analysis done with WARNING(s). Total running time: 711.759425163269 seconds
INFO	Results written in /Users/jon/test-predict_6004e1e9-1eda-4469-a5b0-c06c104a1135/annotate/predict_misc/busco/run_saccharomyces/


INFO	****************** Start a BUSCO 2.0 analysis, current time: 06/30/2022 09:14:41 ******************
INFO	The lineage dataset is: dikarya_odb9 (eukaryota)
INFO	Mode is: proteins
INFO	To reproduce this run: python /venv/lib/python3.8/site-packages/funannotate/aux_scripts/funannotate-BUSCO2.py -i /Users/jon/test-predict_6004e1e9-1eda-4469-a5b0-c06c104a1135/annotate/predict_misc/busco_augustus.proteins.fasta -o saccharomyces -l /opt/databases/dikarya/ -m proteins -c 4 -sp anidulans
INFO	Check dependencies...
INFO	Check input file...
INFO	Temp directory is ./tmp/
INFO	Running HMMER on the proteins:
INFO	06/30/2022 09:14:42 =>	0% of predictions performed (1312 to be done)
INFO	06/30/2022 09:14:44 =>	10% of predictions performed (133/1312 candidate proteins)
INFO	06/30/2022 09:14:47 =>	20% of predictions performed (264/1312 candidate proteins)
INFO	06/30/2022 09:14:49 =>	30% of predictions performed (396/1312 candidate proteins)
INFO	06/30/2022 09:14:53 =>	40% of predictions performed (525/1312 candidate proteins)
INFO	06/30/2022 09:14:57 =>	50% of predictions performed (658/1312 candidate proteins)
INFO	06/30/2022 09:15:01 =>	60% of predictions performed (788/1312 candidate proteins)
INFO	06/30/2022 09:15:06 =>	70% of predictions performed (921/1312 candidate proteins)
INFO	06/30/2022 09:15:11 =>	80% of predictions performed (1050/1312 candidate proteins)
INFO	06/30/2022 09:15:17 =>	90% of predictions performed (1182/1312 candidate proteins)
INFO	06/30/2022 09:15:22 =>	100% of predictions performed
INFO	Results:
INFO	C:28.3%[S:28.2%,D:0.1%],F:0.0%,M:71.7%,n:1312
INFO	371 Complete BUSCOs (C)
INFO	370 Complete and single-copy BUSCOs (S)
INFO	1 Complete and duplicated BUSCOs (D)
INFO	0 Fragmented BUSCOs (F)
INFO	941 Missing BUSCOs (M)
INFO	1312 Total BUSCO groups searched

INFO	BUSCO analysis done. Total running time: 42.64242601394653 seconds
INFO	Results written in /Users/jon/test-predict_6004e1e9-1eda-4469-a5b0-c06c104a1135/annotate/predict_misc/busco_proteins/run_saccharomyces/

[nextgenusfs]

If it helps, I just pushed an update where the dependencies, versions, and full paths are printed to the logfile, ie:

[06/30/22 10:18:12]: /venv/bin/funannotate predict -i test.softmasked.fa --protein_evidence protein.evidence.fasta -o annotate --augustus_species saccharomyces --cpus 4 --species Awesome testicus

[06/30/22 10:18:12]: OS: Debian GNU/Linux 10, 4 cores, ~ 8 GB RAM. Python: 3.8.13
[06/30/22 10:18:12]: Running funannotate v1.8.12
[06/30/22 10:18:12]: GeneMark not found and $GENEMARK_PATH environmental variable missing. Will skip GeneMark ab-initio prediction.
[06/30/22 10:18:12]: exonerate version=exonerate 2.4.0 path=/venv/bin/exonerate
[06/30/22 10:18:12]: diamond version=2.0.15 path=/venv/bin/diamond
[06/30/22 10:18:12]: tbl2asn version=no way to determine, likely 25.X path=/venv/bin/tbl2asn
[06/30/22 10:18:12]: bedtools version=bedtools v2.30.0 path=/venv/bin/bedtools
[06/30/22 10:18:12]: augustus version=3.3.2 path=/usr/bin/augustus
[06/30/22 10:18:12]: etraining version=NA path=/usr/bin/etraining
[06/30/22 10:18:12]: tRNAscan-SE version=2.0.9 (July 2021) path=/venv/bin/tRNAscan-SE
[06/30/22 10:18:12]: bam2hints version=NA path=/usr/bin/bam2hints
[06/30/22 10:18:12]: minimap2 version=2.24-r1122 path=/venv/bin/minimap2
[06/30/22 10:18:12]: $AUGUSTUS_CONFIG_PATH=/usr/share/augustus/config
[06/30/22 10:18:13]: {'augustus': 1, 'hiq': 2, 'genemark': 0, 'pasa': 6, 'codingquarry': 0, 'snap': 1, 'glimmerhmm': 1, 'proteins': 1, 'transcripts': 1}
[06/30/22 10:18:13]: Skipping CodingQuarry as no --rna_bam passed

You can upgrade with pip, ie:

python -m pip install git+https://github.com/nextgenusfs/funannotate.git

Ultimately I think this is a problem with augustus, not entirely sure what is the issue. It is outputting data like its Augustus 3.4 which is incompatible with the internal BUSCO script in funannotate, so I've not seen any version < 3.4 fail like this before.

[IanDMedeiros]

I tried upgrading as you suggested and got a new error:

#########################################################
Running funannotate clean unit testing: minimap2 mediated assembly duplications
Downloading: https://osf.io/8pjbe/download?version=1 Bytes: 252076
8192 [3.25%]��������������16384 [6.50%]���������������24576 [9.75%]���������������32768 [13.00%]����������������40960 [16.25%]����������������49152 [19.50%]����������������57344 [22.75%]����������������65536 [26.00%]����������������73728 [29.25%]����������������81920 [32.50%]����������������90112 [35.75%]����������������98304 [39.00%]����������������106496 [42.25%]�����������������114688 [45.50%]�����������������122880 [48.75%]�����������������131072 [52.00%]�����������������139264 [55.25%]�����������������147456 [58.50%]�����������������155648 [61.75%]�����������������163840 [65.00%]�����������������172032 [68.25%]�����������������180224 [71.50%]�����������������188416 [74.75%]�����������������196608 [78.00%]�����������������204800 [81.25%]�����������������212992 [84.50%]�����������������221184 [87.74%]�����������������229376 [90.99%]�����������������237568 [94.24%]�����������������245760 [97.49%]�����������������252076 [100.00%]������������������Traceback (most recent call last):
File "/hpc/home/idm7/miniconda3/envs/funannotate/bin/funannotate", line 8, in
sys.exit(main())
File "/hpc/home/idm7/miniconda3/envs/funannotate/lib/python3.8/site-packages/funannotate/funannotate.py", line 716, in main
mod.main(arguments)
File "/hpc/home/idm7/miniconda3/envs/funannotate/lib/python3.8/site-packages/funannotate/clean.py", line 194, in main
CheckDependencies(programs)
File "/hpc/home/idm7/miniconda3/envs/funannotate/lib/python3.8/site-packages/funannotate/library.py", line 910, in CheckDependencies
log.debug('{} version={} path={}'.format(f[0], f[2], f[1]))
NameError: name 'log' is not defined
CMD: funannotate clean -i test.clean.fa -o test.exhaustive.fa --exhaustive
#########################################################
#########################################################
Traceback (most recent call last):
File "/hpc/home/idm7/miniconda3/envs/funannotate/bin/funannotate", line 8, in
sys.exit(main())
File "/hpc/home/idm7/miniconda3/envs/funannotate/lib/python3.8/site-packages/funannotate/funannotate.py", line 716, in main
mod.main(arguments)
File "/hpc/home/idm7/miniconda3/envs/funannotate/lib/python3.8/site-packages/funannotate/test.py", line 401, in main
runCleanTest(args)
File "/hpc/home/idm7/miniconda3/envs/funannotate/lib/python3.8/site-packages/funannotate/test.py", line 130, in runCleanTest
assert countfasta(os.path.join(tmpdir, 'test.exhaustive.fa')) == 3
File "/hpc/home/idm7/miniconda3/envs/funannotate/lib/python3.8/site-packages/funannotate/test.py", line 36, in countfasta
with open(input, 'r') as f:
FileNotFoundError: [Errno 2] No such file or directory: 'test-clean_5ed48339-b445-4f05-92d7-20b9e40d7c14/test.exhaustive.fa'

[nextgenusfs]

Oh, that's my fault, I'll fix.

Can you just run funannotate test -t predict --cpus N so it just runs the predict test.

[nextgenusfs]

latest commit should fix this error, thanks for reporting.

[IanDMedeiros]

funannotate test -t predict --cpus 10

The test completes, but still only finds 175 BUSCO loci

#########################################################
Running funannotate predict unit testing
Downloading: https://osf.io/te2pf/download?version=1 Bytes: 1489808
[Jul 02 02:46 PM]: OS: CentOS Stream 8, 46 cores, ~ 230 GB RAM. Python: 3.8.12
[Jul 02 02:46 PM]: Running funannotate v1.8.12
[Jul 02 02:46 PM]: GeneMark not found and $GENEMARK_PATH environmental variable missing. Will skip GeneMark ab-initio prediction.
[Jul 02 02:46 PM]: Skipping CodingQuarry as no --rna_bam passed
[Jul 02 02:46 PM]: Parsed training data, run ab-initio gene predictors as follows:
�[4mProgram Training-Method�[0m
augustus pretrained
glimmerhmm busco
snap busco
[Jul 02 02:46 PM]: Loading genome assembly and parsing soft-masked repetitive sequences
[Jul 02 02:46 PM]: Genome loaded: 6 scaffolds; 3,776,588 bp; 19.75% repeats masked
[Jul 02 02:46 PM]: Mapping 1,065 proteins to genome using diamond and exonerate
[Jul 02 02:46 PM]: Found 1,505 preliminary alignments with diamond in 0:00:01 --> generated FASTA files for exonerate in 0:00:00
[Jul 02 02:46 PM]: Exonerate finished in 0:00:21: found 1,270 alignments
[Jul 02 02:46 PM]: Running BUSCO to find conserved gene models for training ab-initio predictors
[Jul 02 02:51 PM]: 175 valid BUSCO predictions found, validating protein sequences
[Jul 02 02:52 PM]: 175 BUSCO predictions validated
[Jul 02 02:52 PM]: Running Augustus gene prediction using saccharomyces parameters
[Jul 02 02:53 PM]: 1,485 predictions from Augustus
[Jul 02 02:53 PM]: Pulling out high quality Augustus predictions
[Jul 02 02:53 PM]: Found 371 high quality predictions from Augustus (>90% exon evidence)
[Jul 02 02:53 PM]: Running SNAP gene prediction, using training data: annotate/predict_misc/busco.final.gff3
[Jul 02 02:54 PM]: 1,519 predictions from SNAP
[Jul 02 02:54 PM]: Running GlimmerHMM gene prediction, using training data: annotate/predict_misc/busco.final.gff3
[Jul 02 02:55 PM]: 1,586 predictions from GlimmerHMM
[Jul 02 02:55 PM]: Summary of gene models passed to EVM (weights):
[Jul 02 02:55 PM]: EVM: partitioning input to ~ 35 genes per partition using min 1500 bp interval
[Jul 02 02:57 PM]: Converting to GFF3 and collecting all EVM results
�[4mSource Weight Count�[0m
Augustus 1 1325
Augustus HiQ 2 372
GlimmerHMM 1 1586
snap 1 1519
Total - 4802
[Jul 02 02:57 PM]: 1,683 total gene models from EVM
[Jul 02 02:57 PM]: Generating protein fasta files from 1,683 EVM models
[Jul 02 02:57 PM]: now filtering out bad gene models (< 50 aa in length, transposable elements, etc).
[Jul 02 02:57 PM]: Found 131 gene models to remove: 0 too short; 0 span gaps; 131 transposable elements
[Jul 02 02:57 PM]: 1,552 gene models remaining
[Jul 02 02:57 PM]: Predicting tRNAs
[Jul 02 02:57 PM]: 112 tRNAscan models are valid (non-overlapping)
[Jul 02 02:57 PM]: Generating GenBank tbl annotation file
[Jul 02 02:57 PM]: Collecting final annotation files for 1,664 total gene models
[Jul 02 02:57 PM]: Converting to final Genbank format
[Jul 02 02:57 PM]: Funannotate predict is finished, output files are in the annotate/predict_results folder
[Jul 02 02:57 PM]: Your next step might be functional annotation, suggested commands:

Run InterProScan (manual install):
funannotate iprscan -i annotate -c 10

Run antiSMASH (optional):
funannotate remote -i annotate -m antismash -e youremail@server.edu

Annotate Genome:
funannotate annotate -i annotate --cpus 10 --sbt yourSBTfile.txt

[Jul 02 02:57 PM]: Training parameters file saved: annotate/predict_results/saccharomyces.parameters.json
[Jul 02 02:57 PM]: Add species parameters to database:

funannotate species -s saccharomyces -a annotate/predict_results/saccharomyces.parameters.json

CMD: funannotate predict -i test.softmasked.fa --protein_evidence protein.evidence.fasta -o annotate --augustus_species saccharomyces --cpus 10 --species Awesome testicus
#########################################################
#########################################################
SUCCESS: funannotate predict test complete.
#########################################################

The error you commented on earlier,

INFO [hmmersearch] Parse failed (sequence file /hpc/group/bio1/ian/envs/annotate/test-busco_b548c447-5a92-4bca-b6c4-d116e6f7177e/annotate/predict_misc/busco/run_awesome_busco/augustus_output/extracted_proteins/EOG092603EH.faa.1):
INFO [hmmersearch] Line 2: illegal character %

seems to be happening becuase Augustus is appending extra stuff to some of the protein sequences. See for example

">g1[CP022974.1:280548-281798]
MVSSLPKESQAELQLFQNEINAANPSDFLQFSANYFNKRLEQQRAFLKAREPEFKAKNIVLFPEPEESFSRPQSAQSQSRSRSSVMFKSPFVNEDPHSNVFKSGFNLDPHEQDTHQQAQEEQQHTREKTSTPPLPMHFNAQRRTSVSGETLQPNNFDDWTPDHYKEKSEQQLQRLEKSIRNNFLFNKLDSDSKRLVINCLEEKSVPKGATIIKQGDQGDYFYVVEKGTVDFYVNDNKVNSSGPGSSFGELALMYNSPRAATVVATSDCLLWALDRLTFRKILLGSSFKKRLMYDDLLKSMPVLKSLTTYDRAKLADALDTKIYQPGETIIREGDQGENFYLIEYGAVDVSKKGQGVINKLKDHDYFGEVALLNDLPRQATVTATKRTKVATLGKSGFQRLLGPAVDVLKLNDPTRHEvidence%CDSCDS5'UTR3'UTRhintincompatibleRM:"

[nextgenusfs]

And in the logfile it lists Augustus v3.3.3?

So try a different version of Augustus. It needs to be less than v3.4. I thought 3.3.3 was fine but apparently not on your system. Most if not all of the versions on bioconda lately are not compiled properly and won't work with BUSCO. I don't know what changed in bioconda that it stopped working.

[IanDMedeiros]

I am working to set up an alternative version of Augustus now.

The error I am seeing with my existing Augustus 3.3.3 compiled without bioconda seems to be in how the /predicted_genes files (eg., EOG09260A98.out.1) are being converted to /extracted_proteins files (e.g., EOG09260A98.faa.1). Do you happen to know if this is performed by Augustus or one of its associated scripts? If so, I will post an issue in the Augustus github repository.

EDIT: Ok, looks like it is BUSCO that extracts the sequences.

[nextgenusfs]

Yeah BUSCO extracts but Augustus format changed in 3.4. I haven't had chance to update that script. I want to remove the internal BUSCO and use the conda most recent package, however there are too many dependency conflicts and I cannot get a conda environment to build properly. So I haven't changed it yet. The Augustus proteinprofile issue though will continue to be an issue.

[IanDMedeiros]

I still have no idea what is the problem with my Augustus 3.3.3, but I have solved the immediate issue by modifying funannotate-BUSCO2.py. I inserted two lines into the function _extract to account for the format of the predicted_genes .out files:

1606   elif line.startswith('# Evidence'):
1607        check = 0

Attaching the full edited function as well: _extract.py.txt

Thanks for your help thinking through this!