Trinotate generates an empty report, only headers and no data inside
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Entering edit mode
16 days ago
jway • 0

Hello,

I've been running the most recent Dockerized version of Trinotate. I completed steps 2, 3, and 4 as seen in https://github.com/Trinotate/Trinotate/wiki/Software-installation-and-data-required, and while I was running the sequence analyses and database searches, I got confirmations that the results were loaded into the SQLite database (see in trace below).

When I tried to generate the report, however, I got a blank .tsv file with only the headers. I looked through the SQLite file to make sure that my annotations worked, and they are showing up. No error comes up when I generate the report, it's just blank. Is there anything that I'm doing wrong? Any guidance would be greatly appreciated.

Part of the sequence analysis/database search trace

* [Tue Jul  2 18:04:19 2024] Running CMD: diamond blastx -d /data/NEWTRINITYDATADIR/uniprot_sprot -q /data/trinity_out_dir_sortednew.Trinity.fasta -p 32 -k 1 -e 1e-5 -o uniprot_sprot.diamond.blastx.outfmt6 --outfmt 6
diamond v2.0.15.153 (C) Max Planck Society for the Advancement of Science
Documentation, support and updates available at http://www.diamondsearch.org
Please cite: http://dx.doi.org/10.1038/s41592-021-01101-x Nature Methods (2021)

#CPU threads: 32
Scoring parameters: (Matrix=BLOSUM62 Lambda=0.267 K=0.041 Penalties=11/1)
Temporary directory:
#Target sequences to report alignments for: 1
Opening the database...  [0.048s]
Database: /data/NEWTRINITYDATADIR/uniprot_sprot.dmnd (type: Diamond database, sequences: 571609, letters: 206878625)
Block size = 2000000000
Opening the input file...  [0.058s]
Opening the output file...  [0s]
Loading query sequences...  [3.501s]
Masking queries...  [0.687s]
Algorithm: Double-indexed
Building query histograms...  [0.779s]
Allocating buffers...  [0s]
Loading reference sequences...  [0.182s]
Masking reference...  [0.241s]
Initializing temporary storage...  [0s]
Building reference histograms...  [0.295s]
Allocating buffers...  [0s]
Processing query block 1, reference block 1/1, shape 1/2, index chunk 1/4.
Building reference seed array...  [0.167s]
Building query seed array...  [0.381s]
Computing hash join...  [0.133s]
Masking low complexity seeds...  [0.037s]
Searching alignments...  [0.531s]
Processing query block 1, reference block 1/1, shape 1/2, index chunk 2/4.
Building reference seed array...  [0.139s]
Building query seed array...  [0.257s]
Computing hash join...  [0.118s]
Masking low complexity seeds...  [0.038s]
Searching alignments...  [0.438s]
Processing query block 1, reference block 1/1, shape 1/2, index chunk 3/4.
Building reference seed array...  [0.144s]
Building query seed array...  [0.279s]
Computing hash join...  [0.117s]
Masking low complexity seeds...  [0.038s]
Searching alignments...  [0.36s]
Processing query block 1, reference block 1/1, shape 1/2, index chunk 4/4.
Building reference seed array...  [0.127s]
Building query seed array...  [0.304s]
Computing hash join...  [0.116s]
Masking low complexity seeds...  [0.037s]
Searching alignments...  [0.323s]
Processing query block 1, reference block 1/1, shape 2/2, index chunk 1/4.
Building reference seed array...  [0.121s]
Building query seed array...  [0.298s]
Computing hash join...  [0.113s]
Masking low complexity seeds...  [0.038s]
Searching alignments...  [0.334s]
Processing query block 1, reference block 1/1, shape 2/2, index chunk 2/4.
Building reference seed array...  [0.141s]
Building query seed array...  [0.332s]
Computing hash join...  [0.113s]
Masking low complexity seeds...  [0.038s]
Searching alignments...  [0.35s]
Processing query block 1, reference block 1/1, shape 2/2, index chunk 3/4.
Building reference seed array...  [0.145s]
Building query seed array...  [0.348s]
Computing hash join...  [0.114s]
Masking low complexity seeds...  [0.038s]
Searching alignments...  [0.286s]
Processing query block 1, reference block 1/1, shape 2/2, index chunk 4/4.
Building reference seed array...  [0.129s]
Building query seed array...  [0.304s]
Computing hash join...  [0.114s]
Masking low complexity seeds...  [0.038s]
Searching alignments...  [0.283s]
Deallocating buffers...  [0s]
Clearing query masking...  [0.052s]
Computing alignments...  [12.05s]
Deallocating reference...  [0s]
Loading reference sequences...  [0s]
Deallocating buffers...  [0.001s]
Deallocating queries...  [0.001s]
Loading query sequences...  [0s]
Closing the input file...  [0s]
Closing the output file...  [0.012s]
Cleaning up...  [0s]
Total time = 25.781s
Reported 198395 pairwise alignments, 198395 HSPs.
198395 queries aligned.
* [Tue Jul  2 18:04:45 2024] Running CMD: /usr/local/src/Trinotate/Trinotate --db /data/NEWTRINITYDATADIR/TrinotateBoilerplate.sqlite --LOAD_swissprot_blastx uniprot_sprot.diamond.blastx.outfmt6
-LOADING as per --LOAD_swissprot_blastx
CMD: /usr/local/src/Trinotate/util/trinotateSeqLoader/Trinotate_BLAST_loader.pl --sqlite /data/NEWTRINITYDATADIR/TrinotateBoilerplate.sqlite --outfmt6 uniprot_sprot.diamond.blastx.outfmt6 --prog blastx --dbtype Swissprot
CMD: echo "pragma journal_mode=memory;
pragma synchronous=0;
pragma cache_size=4000000;
.mode tabs
.import tmp.blast_bulk_load.3745 BlastDbase" | sqlite3 /data/NEWTRINITYDATADIR/TrinotateBoilerplate.sqlite
memory


BlastDbase loading complete..

* [Tue Jul  2 18:04:47 2024] Running CMD: hmmsearch --cpu 32 --noali --domtblout TrinotatePFAM.out /data/NEWTRINITYDATADIR/Pfam-A.hmm /data/transdecoder_data/trinity_out_dir_sortednew.Trinity.fasta.transdecoder.pep > pfam.log
* [Tue Jul  2 21:42:28 2024] Running CMD: /usr/local/src/Trinotate/Trinotate --db /data/NEWTRINITYDATADIR/TrinotateBoilerplate.sqlite --LOAD_pfam  TrinotatePFAM.out
-LOADING as per --LOAD_pfam
CMD: /usr/local/src/Trinotate/util/trinotateSeqLoader/Trinotate_PFAM_loader.pl --sqlite /data/NEWTRINITYDATADIR/TrinotateBoilerplate.sqlite --pfam TrinotatePFAM.out
CMD: echo "pragma journal_mode=memory;
pragma synchronous=0;
pragma cache_size=4000000;
.mode tabs
.import tmp.pfam_bulk_load.707083 HMMERDbase" | sqlite3 /data/NEWTRINITYDATADIR/TrinotateBoilerplate.sqlite
memory


Loading complete..

* [Tue Jul  2 21:42:42 2024] Running CMD: cmscan -Z 323 --cut_ga --rfam --nohmmonly --tblout infernal.out --fmt 2 --cpu 32 --clanin /data/NEWTRINITYDATADIR/Rfam.clanin /data/NEWTRINITYDATADIR/Rfam.cm /data/trinity_out_dir_sortednew.Trinity.fasta > infernal.log
* [Wed Jul  3 17:00:36 2024] Running CMD: /usr/local/src/Trinotate/Trinotate --db /data/NEWTRINITYDATADIR/TrinotateBoilerplate.sqlite --LOAD_infernal infernal.out
-LOADING as per --LOAD_infernal
CMD: /usr/local/src/Trinotate/util/trinotateSeqLoader/Trinotate_Infernal_loader.pl --sqlite /data/NEWTRINITYDATADIR/TrinotateBoilerplate.sqlite --infernal infernal.out
CMD: echo "pragma journal_mode=memory;
pragma synchronous=0;
pragma cache_size=4000000;
.mode tabs
.import tmp.infernal_bulk_load.3198127 Infernal" | sqlite3 /data/NEWTRINITYDATADIR/TrinotateBoilerplate.sqlite
memory


Loading complete..

Command to generate report, no errors appeared

root@4c6df2aa90d4:/data# $TRINOTATE_HOME/Trinotate --db /data/NEWTRINITYDATADIR/TrinotateBoilerplate.sqlite --report > myTrinotateattempt3.tsv
-REPORT being generated.
CMD: /usr/local/src/Trinotate/util/Trinotate_report_writer.pl --sqlite /data/NEWTRINITYDATADIR/TrinotateBoilerplate.sqlite -E 1e-5 --pfam_cutoff DNC

Opened up some of the entries in the SQLite file to make sure that everything was annotated correctly

root@4c6df2aa90d4:/data# sqlite3 /data/NEWTRINITYDATADIR/TrinotateBoilerplate.sqlite
SQLite version 3.31.1 2020-01-27 19:55:54
Enter ".help" for usage hints.
sqlite> .tables
BlastDbase           ORF                  UniprotIndex
Diff_expression      PFAMreference        eggNOGIndex
EggnogMapper         RNAMMERdata          go
ExprClusterAnalyses  Replicates           go_slim
ExprClusters         Samples              go_slim_mapping
Expression           SignalP              pfam2go
HMMERDbase           TaxonomyIndex        tmhmm
Infernal             Transcript
sqlite> SELECT * FROM EggnogMapper LIMIT 10;
TRINITY_DN0_c0_g1_i1.p2|760192.Halhy_5172|2.41e-32|132.0|COG3210@1|root,COG3210@2|Bacteria,4PMJA@976|Bacteroidetes|976|Bacteroidetes|U|domain, Protein|-|-|-|-|-|-|-|-|-|-|-|-|CHU_C
TRINITY_DN0_c0_g1_i10.p2|335543.Sfum_2553|5.35e-07|57.0|COG4966@1|root,COG4966@2|Bacteria,1Q8WE@1224|Proteobacteria,439J3@68525|delta/epsilon subdivisions,2X4VK@28221|Deltaproteobacteria,2MSHQ@213462|Syntrophobacterales|28221|Deltaproteobacteria|NU|Pfam:N_methyl_2|-|-|-|-|-|-|-|-|-|-|-|-|N_methyl
TRINITY_DN0_c0_g1_i10.p1|243090.RB11769|4.25e-32|134.0|COG2911@1|root,COG2911@2|Bacteria|2|Bacteria|S|protein secretion|-|-|-|ko:K20276|ko02024,map02024|-|-|-|ko00000,ko00001|-|-|-|CHU_C,Calx-beta,F5_F8_type_C,He_PIG,SLH
TRINITY_DN0_c0_g1_i10.p6|1232437.KL662002_gene4685|7.65e-09|58.5|COG4967@1|root,COG4967@2|Bacteria,1NI49@1224|Proteobacteria,42X6W@68525|delta/epsilon subdivisions,2WSY4@28221|Deltaproteobacteria,2MMCT@213118|Desulfobacterales|28221|Deltaproteobacteria|NU|Prokaryotic N-terminal methylation motif|-|-|-|ko:K02458,ko:K02671|ko03070,ko05111,map03070,map05111|M00331|-|-|ko00000,ko00001,ko00002,ko02035,ko02044|3.A.15|-|-|N_methyl
TRINITY_DN0_c0_g1_i10.p4|1232437.KL662002_gene4687|1.31e-12|70.5|COG4970@1|root,COG4970@2|Bacteria,1N85U@1224|Proteobacteria,42WBU@68525|delta/epsilon subdivisions,2WSG1@28221|Deltaproteobacteria,2MMBS@213118|Desulfobacterales|28221|Deltaproteobacteria|NU|Prokaryotic N-terminal methylation motif|-|-|-|ko:K08084|-|-|-|-|ko00000,ko02044|3.A.15.2|-|-|GspH,N_methyl
TRINITY_DN0_c0_g1_i11.p6|234267.Acid_5017|4.69e-77|237.0|COG3383@1|root,COG3383@2|Bacteria,3Y2T6@57723|Acidobacteria|2|Bacteria|C|PFAM NADH ubiquinone oxidoreductase, subunit G, iron-sulphur binding|hoxU|-|1.17.1.10,1.6.5.3|ko:K05299,ko:K05588|ko00190,ko00680,ko00720,ko01100,ko01120,ko01200,map00190,map00680,map00720,map01100,map01120,map01200|M00377|R00134,R11945|RC00061,RC02796|ko00000,ko00001,ko00002,ko01000|-|-|iJN678.hoxU|Fer2_4,Fer4,Fer4_10,Fer4_6,Fer4_7,Fer4_9,Molybdop_Fe4S4,NADH-G_4Fe-4S_3
TRINITY_DN0_c0_g1_i11.p4|926569.ANT_12520|2.77e-185|531.0|COG1894@1|root,COG1894@2|Bacteria,2G5K9@200795|Chloroflexi|200795|Chloroflexi|C|PFAM Respiratory-chain NADH dehydrogenase domain, 51 kDa subunit|hoxF|-|1.6.5.3|ko:K00335,ko:K05587|ko00190,ko01100,map00190,map01100|M00144|R11945|RC00061|ko00000,ko00001,ko00002,ko01000|3.D.1|-|-|2Fe-2S_thioredx,Complex1_51K,NADH_4Fe-4S,SLBB
TRINITY_DN0_c0_g1_i11.p3|926569.ANT_12550|8.23e-203|582.0|COG3259@1|root,COG3259@2|Bacteria,2G68W@200795|Chloroflexi|200795|Chloroflexi|C|PFAM nickel-dependent hydrogenase, large subunit|-|-|1.12.1.2,1.8.98.5|ko:K00436,ko:K14126|ko00680,map00680|-|R00019,R00700,R11943|RC00011|ko00000,ko00001,ko01000|-|-|-|NiFeSe_Hases
TRINITY_DN0_c0_g1_i11.p1|439235.Dalk_4404|1.61e-193|644.0|COG3419@1|root,COG3506@1|root,COG3419@2|Bacteria,COG3506@2|Bacteria,1NUAV@1224|Proteobacteria,42NJ7@68525|delta/epsilon subdivisions,2WKJV@28221|Deltaproteobacteria,2MHMF@213118|Desulfobacterales|28221|Deltaproteobacteria|NU|Tfp pilus assembly protein tip-associated adhesin|pilY1|-|-|ko:K02674|-|-|-|-|ko00000,ko02035,ko02044|-|-|-|Neisseria_PilC,PA14
TRINITY_DN0_c0_g1_i2.p4|1232437.KL662002_gene4687|6.28e-15|76.6|COG4970@1|root,COG4970@2|Bacteria,1N85U@1224|Proteobacteria,42WBU@68525|delta/epsilon subdivisions,2WSG1@28221|Deltaproteobacteria,2MMBS@213118|Desulfobacterales|28221|Deltaproteobacteria|NU|Prokaryotic N-terminal methylation motif|-|-|-|ko:K08084|-|-|-|-|ko00000,ko02044|3.A.15.2|-|-|GspH,N_methyl
sqlite> SELECT * FROM HMMERDbase LIMIT 10;
TRINITY_DN239_c0_g1_i8.p3|PF10417.14|1-cysPrx_C|NULL|165.0|204.0|1.0|41.0|2.1e-16|4.8e-16|63.5|62.3
TRINITY_DN196518_c0_g1_i1.p1|PF10417.14|1-cysPrx_C|NULL|173.0|211.0|1.0|40.0|1.0e-15|1.7e-15|61.2|60.5
TRINITY_DN12898_c0_g1_i6.p1|PF10417.14|1-cysPrx_C|NULL|55.0|91.0|1.0|38.0|1.1e-15|2.2e-15|61.1|60.2
TRINITY_DN75604_c0_g1_i1.p1|PF10417.14|1-cysPrx_C|NULL|170.0|209.0|1.0|41.0|1.7e-15|3.4e-15|60.5|59.6
TRINITY_DN12898_c0_g1_i2.p1|PF10417.14|1-cysPrx_C|NULL|79.0|115.0|1.0|38.0|1.8e-15|3.2e-15|60.5|59.7
TRINITY_DN12898_c0_g1_i3.p1|PF10417.14|1-cysPrx_C|NULL|217.0|253.0|1.0|38.0|5.7e-15|1.0e-14|58.9|58.1
TRINITY_DN3190_c0_g1_i3.p2|PF10417.14|1-cysPrx_C|NULL|169.0|201.0|1.0|34.0|2.7e-14|5.3e-14|56.7|55.8
TRINITY_DN371771_c0_g1_i1.p1|PF10417.14|1-cysPrx_C|NULL|73.0|104.0|1.0|33.0|5.3e-14|8.1e-14|55.8|55.2
TRINITY_DN3190_c0_g1_i1.p3|PF10417.14|1-cysPrx_C|NULL|169.0|201.0|1.0|34.0|1.0e-13|2.0e-13|54.9|53.9
TRINITY_DN3190_c0_g1_i6.p3|PF10417.14|1-cysPrx_C|NULL|169.0|201.0|1.0|34.0|1.0e-13|2.0e-13|54.9|53.9
sqlite> SELECT * FROM PFAMreference LIMIT 10;
PF10417.14|1-cysPrx_C|C-terminal domain of 1-Cys peroxiredoxin|21.1|21.1|21.1|21.1|21.0|21.0
PF21734.2|10_blade|10-bladed beta propeller domain|27.0|27.0|28.0|28.3|25.4|22.6
PF21578.2|117-like_vir|Virus, 117-like|27.0|27.0|28.6|27.1|26.3|26.0
PF12574.13|120_Rick_ant|120 KDa Rickettsia surface antigen|25.0|25.0|39.6|64.6|23.6|21.2
PF09847.14|12TM_1|Membrane protein of 12 TMs|33.2|33.2|33.3|33.3|31.7|33.0
PF00244.25|14-3-3|14-3-3 protein|33.2|33.2|33.2|33.2|33.1|33.1
PF16998.10|17kDa_Anti_2|17 kDa outer membrane surface antigen|26.6|26.6|26.6|26.6|26.5|26.5
PF00389.35|2-Hacid_dh|D-isomer specific 2-hydroxyacid dehydrogenase, catalytic domain|24.6|24.6|24.6|24.6|24.5|24.5
PF02826.24|2-Hacid_dh_C|D-isomer specific 2-hydroxyacid dehydrogenase, NAD binding domain|25.1|25.1|25.1|25.1|25.0|25.0
PF00198.28|2-oxoacid_dh|2-oxoacid dehydrogenases acyltransferase (catalytic domain)|23.0|23.0|23.0|23.0|22.9|22.9
sqlite> .exit
trinotate metatranscriptome docker • 243 views
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Entering edit mode
10 days ago

Jway,

Try first re-run the init command prior to exporting results

Trinotate --db <sqlite.db> --init --gene_trans_map <file> --transcript_fasta <file> --transdecoder_pep <file>

If the checkpoint file __trinotate_run_checkpts/*__init.ok was written you may need to remove

Then

Trinotate --db <sqlite.db> --report --incl_pep > Trinotate.xls

As gene_trans_map is a mandatory file, you can create one as follows example:

grep "^>" assembly.fasta | awk '{gsub(/_i[0-9]$|>/,""); print}' > genes

grep "^>" assembly.fasta | awk '{gsub(/>/,""); print}' > trans

paste -d"\t" genes trans > gen_tr_map

the gen_tr_map file will contain transcript and gene list strings separated by a delimiter

Ex:

transcript string: NODE_1_length_21868_cov_160.403166_g0_i0

gene string: NODE_1_length_21868_cov_160.403166_g0

A similar issue was posted here:

https://github.com/Trinotate/Trinotate.github.io/issues/70

Regards

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