Jun 12, 2024
Transcriptome annotation
- 1Universidade Federal do Sul da Bahia
Protocol Citation: Rafael Rodrigues Ferrari, Thiago Mafra Batista 2024. Transcriptome annotation. protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvok92bl4o/v1
Manuscript citation:
Ferrari RR, Batista TM, Zhou QS, Hilário HO, Orr MC, Luo A, Zhu CD. The Whole Genome of Colletes collaris (Hymenoptera: Colletidae): An Important Step in Comparative Genomics of Cellophane Bees. Genome Biol Evol. 2023 May 5;15(5):evad062. doi: 10.1093/gbe/evad062. PMID: 37075227; PMCID: PMC10159585.
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 12, 2024
Last Modified: June 12, 2024
Protocol Integer ID: 101716
Keywords: transcriptome annotation, functional annotation, Trinotate
Funders Acknowledgement:
Suzano Papel e Celulose S/A
Grant ID: 23746.009802/2021-88
Veracel Celulose
Grant ID: 23746.001092/2022-30
Abstract
This protocol provides detailed, step-by-step instructions for students and researchers to annotate transcriptomes. In this tutorial, we will follow the Trinity -> TransDecoder -> Trinotate pipeline, using the SwissProt and Pfam databases for functional annotation of protein-coding transcripts.
FINDIG CODING REGIONS WITHIN TRANSCRIPTS
FINDIG CODING REGIONS WITHIN TRANSCRIPTS
****TransDecoder (https://github.com/TransDecoder/TransDecoder/wiki)****
***Extracting the long open reading frames (ORFs)***
**Prepare a .pbs file to run the analysis remotely on Sagarana**
/home/fafinha/bin/TransDecoder-TransDecoder-v5.5.0/TransDecoder.LongOrfs -t /home/fafinha/collaris/Trinity_run/assembly/Trinity.fasta
***Including homology searches as ORF retention criteria***
**BlastP search**
*Prepare a .pbs file to run the analysis remotely on Sagarana*
blastp -query /home/fafinha/collaris/TransDecoder_run/2_homology_searches/blastp/Trinity.fasta.transdecoder_dir/longest_orfs.pep \
-db /home/fafinha/collaris/TransDecoder_run/uniprot_sprot.fasta -max_target_seqs 1 -outfmt 6 -evalue 1e-5 -num_threads 64 \
-out /home/fafinha/collaris/TransDecoder_run/2_homology_searches/blastp/blastp_output.fmt6
**Pfam search**
*Download the Pfam database (Pfam-A.hmm)*
*Decompress the file
$gzip -d Pfam-A.hmm.gz
*Index the database*
$/programs/hmmer-3.3.2/bin/hmmpress Pfam-A.hmm
*Prepare a .pbs file to run the analysis remotely on Sagarana*
/home/fafinha/anaconda3/bin/hmmscan --cpu 64 --domtblout /home/fafinha/collaris/TransDecoder_run/2_homology_searches/pfam/pfam.domtblout \
/home/fafinha/bin/pfam/Pfam-A.hmm /home/fafinha/collaris/TransDecoder_run/2_homology_searches/blastp/Trinity.fasta.transdecoder_dir/longest_orfs.pep
***Predicting the likely coding regions***
#Run the 'TransDecoder.Predict' script in the same directory where the 'Trinity.fasta.transdecoder_dir' folder is located
**Without homology**
$/home/fafinha/bin/TransDecoder-TransDecoder-v5.5.0/TransDecoder.Predict -t /home/fafinha/collaris/Trinity_run/assembly/Trinity.fasta
**With homology**
*BlastP*
$/home/fafinha/bin/TransDecoder-TransDecoder-v5.5.0/TransDecoder.Predict -t /home/fafinha/collaris/Trinity_run/assembly/Trinity.fasta \
--retain_blastp_hits /home/fafinha/collaris/TransDecoder_run/run2/homology/blast/blastp_output.fmt6
*Pfam*
$/home/fafinha/bin/TransDecoder-TransDecoder-v5.5.0/TransDecoder.Predict -t /home/fafinha/collaris/Trinity_run/assembly/Trinity.fasta \
--retain_pfam_hits /home/fafinha/collaris/TransDecoder_run/run2/homology/pfam/pfam.domtblout
*BlastP + Pfam*
$/home/fafinha/bin/TransDecoder-TransDecoder-v5.5.0/TransDecoder.Predict -t /home/fafinha/collaris/Trinity_run/assembly/Trinity.fasta \
--retain_blastp_hits /home/fafinha/collaris/TransDecoder_run/run2/homology/blast/blastp_output.fmt6 \
--retain_pfam_hits /home/fafinha/collaris/TransDecoder_run/run2/homology/pfam/pfam.domtblout
FUNCTIONAL ANNOTATION
FUNCTIONAL ANNOTATION
#Perform 'FINDIG CODING REGIONS WITHIN TRANSCRIPTS' first
****Trinotate (https://github.com/Trinotate/Trinotate.github.io/blob/master/index.asciidoc) (on kiko)****
***Generate databases***
$/home/thiagomafra/instaladores/Trinotate-Trinotate-v3.2.2/admin/Build_Trinotate_Boilerplate_SQLite_db.pl Trinotate
****Blastn****
***Prepare a .pbs file to run the analysis remotely on Sagarana***
/programs/ncbi-blast-2.10.1+/bin/blastp -query /home/fafinha/collaris/Trinotate_run/1st_step/Trinity_reduced.fasta.transdecoder.pep -db \ /home/fafinha/collaris/Trinotate_run/1st_step/uniprot_sprot.fasta -num_threads 64 -outfmt 6 -evalue 1e-6 \
-out /home/fafinha/collaris/Trinotate_run/2nd_step/blastp.tab
***Keep best hits only***
$cat blastp.tab | sort -k1,1 -k12,12nr -k11,11n | sort -k1,1 -u > blastp_besthits.tab
****Blastx****
***Prepare a .pbs file to run the analysis remotely on Sagarana***
/programs/ncbi-blast-2.10.1+/bin/blastx -query /home/fafinha/collaris/Trinotate_run/1st_step/Trinity_reduced.fasta \ -db /home/fafinha/collaris/Trinotate_run/1st_step/uniprot_sprot.fasta -num_threads 64 -outfmt 6 -evalue 1e-6 \
-out /home/fafinha/collaris/Trinotate_run/2nd_step/blastx.tab
***Keep best hits only***
$cat blastx.tab | sort -k1,1 -k12,12nr -k11,11n | sort -k1,1 -u > blastx_besthits.tab
****TMHMM (on kiko)****
$/home/thiagomafra/instaladores/tmhmm-2.0c/bin/tmhmm --short < /home/thiagomafra/collaris/trinotate_run/Trinity_reduced.fasta.transdecoder.pep \
> /home/thiagomafra/collaris/trinotate_run/fafinha/run2/tmhmm.out
****HMMER (on kiko)****
$/home/thiagomafra/instaladores/hmmer-3.1b2-linux-intel-x86_64/binaries/hmmscan --cpu 64 \
--domtblout /home/thiagomafra/collaris/trinotate_run/fafinha/run2/TrinotatePFAM.out /home/thiagomafra/collaris/trinotate_run/fafinha/run2/Pfam-A.hmm \
/home/thiagomafra/collaris/trinotate_run/Trinity_reduced.fasta.transdecoder.pep > /home/thiagomafra/collaris/trinotate_run/fafinha/run2/pfam.log
****SignalP (on kiko)****
$/home/thiagomafra/instaladores/signalp-4.1/signalp -f short -n /home/thiagomafra/collaris/trinotate_run/fafinha/run2/signalp.out \
/home/thiagomafra/collaris/trinotate_run/Trinity_reduced.fasta.transdecoder.pep
****RNAmmer (on kiko)****
$/home/thiagomafra/instaladores/Trinotate-Trinotate-v3.2.2/util/rnammer_support/RnammerTranscriptome.pl --transcriptome /home/thiagomafra/collaris/trinotate_run/Trinity_reduced.fasta --path_to_rnammer /home/thiagomafra/instaladores/rnammer/rnammer
***Generating a .gene_trans_map***
$/home/thiagomafra/instaladores/trinityrnaseq-v2.10.0/util/support_scripts/get_Trinity_gene_to_trans_map.pl \
/home/thiagomafra/collaris/trinotate_run/Trinity_reduced.fasta > Trinity.fasta.gene_trans_map
****Populating the .sqlite file****
***Loading transcripts and coding regions***
$/home/thiagomafra/instaladores/Trinotate-Trinotate-v3.2.2/Trinotate Trinotate.sqlite init --gene_trans_map ./Trinity.fasta.gene_trans_map \
--transcript_fasta /home/thiagomafra/collaris/trinotate_run/Trinity_reduced.fasta \
--transdecoder_pep /home/thiagomafra/collaris/trinotate_run/Trinity_reduced.fasta.transdecoder.pep
***Loading BLAST homologies***
$/home/thiagomafra/instaladores/Trinotate-Trinotate-v3.2.2/Trinotate Trinotate.sqlite LOAD_swissprot_blastp blastp_besthits.tab
$/home/thiagomafra/instaladores/Trinotate-Trinotate-v3.2.2/Trinotate Trinotate.sqlite LOAD_swissprot_blastx blastx_besthits.tab
***Loading Pfam protein domains***
$/home/thiagomafra/instaladores/Trinotate-Trinotate-v3.2.2/Trinotate Trinotate.sqlite LOAD_pfam TrinotatePFAM.out
***Loading transmembrane domains***
$/home/thiagomafra/instaladores/Trinotate-Trinotate-v3.2.2/Trinotate Trinotate.sqlite LOAD_tmhmm tmhmm.out
***Loading signal peptide predictions***
$/home/thiagomafra/instaladores/Trinotate-Trinotate-v3.2.2/Trinotate Trinotate.sqlite LOAD_signalp signalp.out
***Loading rRNA gene predictions***
$/home/thiagomafra/instaladores/Trinotate-Trinotate-v3.2.2/Trinotate Trinotate.sqlite LOAD_rnammer Trinity_reduced.fasta.rnammer.gff
****Generate an output of Trinotate annotation report****
$/home/thiagomafra/instaladores/Trinotate-Trinotate-v3.2.2/Trinotate Trinotate.sqlite report > trinotate_annotation_report.xls