--- author: Sam White toc-title: Contents toc-depth: 5 toc-location: left layout: post title: Genome Annotation - Pgenerosa_v074 Using GenSAS date: '2019-07-10 09:26' tags: - GenSAS - geoduck - Panopea generosa - v074 - Pgenerosa_v074 - Panopea-generosa-vv0.74.a3 - v074.a3 - annotation categories: - 2019 - Geoduck Genome Sequencing --- In our various attempts to get the _Panopea generosa_ genome annotated in such a manner that we're comfortable with (the previous annotation attempts we're lacking any annotations in almost all of the largest scaffolds, which didn't seem right), Steven stumbled across [GenSAS](https://www.gensas.org/gensas), a web/GUI-based genome annotation program, so we gave it a shot. This version of the genome annotation will be referred to as: - Panopea-generosa-vv0.74.a3 I uploaded the following to the GenSAS website to potentially use as "evidence files": #### Transcriptome FastA files (links to notebook entries): - [singular transcriptome](https://robertslab.github.io/sams-notebook/posts/2018/2018-09-04-transcriptome-assembly-geoduck-rnaseq-data/) - [ctenidia](https://robertslab.github.io/sams-notebook/posts/2019/2019-04-09-Transcriptome-Assembly---Geoduck-Tissue-specific-Assembly-Ctenidia-with-HiSeq-and-NovaSeq-Data-on-Mox/) - [gonad](https://robertslab.github.io/sams-notebook/posts/2019/2019-04-09-Transcriptome-Assembly---Geoduck-Tissue-specific-Assembly-Gonad-HiSeq-and-NovaSeq-Data-on-Mox/) - [heart](https://robertslab.github.io/sams-notebook/posts/2019/2019-02-15-Transcriptome-Assembly---Geoduck-Tissue-Specific-Assembly-Heart/) - [EPI99 (larvae)](https://robertslab.github.io/sams-notebook/posts/2019/2019-04-09-Transcriptome-Assembly---Geoduck-Tissue-specific-Assembly-Larvae-Day5-EPI99-with-HiSeq-and-NovaSeq-Data-on-Mox/) - [EPI115 (juvenile)](https://robertslab.github.io/sams-notebook/posts/2019/2019-04-09-Transcriptome-Assembly---Geoduck-Tissue-specific-Assembly-Juvenile-Super-Low-OA-EPI115-with-HiSeq-Data-on-Mox/) - [EPI116 (juvenile)](https://robertslab.github.io/sams-notebook/posts/2019/2019-04-09-Transcriptome-Assembly---Geoduck-Tissue-specific-Assembly-Juvenile-Super-Low-OA-EPI116-with-HiSeq-Data-on-Mox/) - [EPI123 (juvenile)](https://robertslab.github.io/sams-notebook/posts/2019/2019-04-09-Transcriptome-Assembly---Geoduck-Tissue-specific-Assembly-Juvenile-Ambient-OA-EPI123-with-HiSeq-Data-on-Mox/) - [EPI124 (juvenile)](https://robertslab.github.io/sams-notebook/posts/2019/2019-04-09-Transcriptome-Assembly---Geoduck-Tissue-specific-Assembly-Juvenile-Ambient-OA-EPI124-with-HiSeq-and-NovaSeq-Data-on-Mox/) #### TransDecoder protein FastA files (links to notebook entries) - [singular proteome](https://robertslab.github.io/sams-notebook/posts/2018/2018-11-21-Annotation---Geoduck-Transcritpome-with-TransDecoder/) - [ctenidia](https://robertslab.github.io/sams-notebook/posts/2019/2019-06-27-Transcriptome-Annotation---Geoduck-Ctenidia-with-Transdecoder-on-Mox/) - [EPI99 (larvae)](https://robertslab.github.io/sams-notebook/posts/2019/2019-06-27-Transcriptome-Annotation---Geoduck-Larvae-Day5-EPI99-with-Transdecoder-on-Mox/) - [EPI115 (juvenile)](https://robertslab.github.io/sams-notebook/posts/2019/2019-06-27-Transcriptome-Annotation---Geoduck-Juvenile-Super-Low-OA-EPI115-with-Transdecoder-on-Mox/) - [EPI116 (juvenile)](https://robertslab.github.io/sams-notebook/posts/2019/2019-06-27-Transcriptome-Annotation---Geoduck-Juvenile-Super-Low-OA-EPI116-with-Transdecoder-on-Mox/) - [EPI123 (juvenile)](https://robertslab.github.io/sams-notebook/posts/2019/2019-06-27-Transcriptome-Annotation---Geoduck-Juvenile-Ambient-OA-EPI123-with-Transdecoder-on-Mox/) - [EPI124 (juvenile)](https://robertslab.github.io/sams-notebook/posts/2019/2019-06-27-Transcriptome-Annotation---Geoduck-Juvenile-Ambient-OA-EPI124-with-Transdecoder-on-Mox/) - [gonad](https://robertslab.github.io/sams-notebook/posts/2019/2019-06-27-Transcriptome-Annotation---Geoduck-Gonad-with-Transdecoder-on-Mox/) - [heart](https://robertslab.github.io/sams-notebook/posts/2019/2019-03-18-Transcriptome-Annotation---Geoduck-Heart-with-Transdecoder-on-Mox/) #### Repeats Files - [RepeatModeler library](https://robertslab.github.io/sams-notebook/posts/2019/2019-06-26-RepeatModeler---Pgenerosa_v074-for-MAKER-Annotation-on-Emu/) --- # RESULTS This took way longer than I was expecting! This took nearly an entire month (the majority of that time was running Augustus _ab initio_ gene prediction, which took ~3 weeks): ![GenSAS project summary processes and runtimes screencap](https://github.com/RobertsLab/sams-notebook/blob/master/images/screencaps/20190710_gensas_pgen-074_runtimes.png?raw=true) --- Output folder: - [20190710_Pgenerosa_v074_gensas_annotation/](https://gannet.fish.washington.edu/Atumefaciens/20190710_Pgenerosa_v074_gensas_annotation/) Merged GFF (SwissProt IDs in Column 9 - from BLASTp and DIAMOND): - [Panopea-generosa-vv0.74.a3-merged-2019-09-24-9-20-04.gff3](https://gannet.fish.washington.edu/Atumefaciens/20190710_Pgenerosa_v074_gensas_annotation/Panopea-generosa-vv0.74.a3-merged-2019-09-24-9-20-04.gff3) Feature counts: ```shell awk 'NR>3 { print $3 }' Panopea-generosa-vv0.74.a3-merged-2019-09-03-6-14-33.gff3 | sort | uniq -c 192022 CDS 192022 exon 45748 gene 45748 mRNA ``` BUSCO assessment: - 68.4% complete BUSCOs present in predicted genes Individual feature GFFs were made with the following shell commands: ```shell features_array=(CDS exon gene mRNA) for feature in ${features_array[@]} do output="Panopea-generosa-vv0.74.a3.${feature}.gff3" input="Panopea-generosa-vv0.74.a3-merged-2019-09-24-9-20-04.gff3" head -n 3 Panopea-generosa-vv0.74.a3-merged-2019-09-24-9-20-04.gff3 \ >> ${output} awk -v feature="$feature" '$3 == feature {print}' ${input} \ >> ${output} done ``` - [Panopea-generosa-vv0.74.a3.CDS.gff3](https://gannet.fish.washington.edu/Atumefaciens/20190710_Pgenerosa_v074_gensas_annotation/Panopea-generosa-vv0.74.a3.CDS.gff3) - [Panopea-generosa-vv0.74.a3.exon.gff3](https://gannet.fish.washington.edu/Atumefaciens/20190710_Pgenerosa_v074_gensas_annotation/Panopea-generosa-vv0.74.a3.exon.gff3) - [Panopea-generosa-vv0.74.a3.gene.gff3](https://gannet.fish.washington.edu/Atumefaciens/20190710_Pgenerosa_v074_gensas_annotation/Panopea-generosa-vv0.74.a3.gene.gff3) - [Panopea-generosa-vv0.74.a3.mRNA.gff3](https://gannet.fish.washington.edu/Atumefaciens/20190710_Pgenerosa_v074_gensas_annotation/Panopea-generosa-vv0.74.a3.mRNA.gff3) SwissProt functional annotations (tab-delimited text): - BLASTp - [Panopea-generosa-vv0.74.a3.5d66e5e537f02-blast_functional.tab](https://gannet.fish.washington.edu/Atumefaciens/20190710_Pgenerosa_v074_gensas_annotation/Panopea-generosa-vv0.74.a3.5d66e5e537f02-blast_functional.tab) - DIAMOND - [Panopea-generosa-vv0.74.a3.5d66e5f03c48d-diamond_functional.tab](https://gannet.fish.washington.edu/Atumefaciens/20190710_Pgenerosa_v074_gensas_annotation/) Pfam annotations (tab-delimited text): - [Panopea-generosa-vv0.74.a3.5d65aaa449919-pfam.tab](https://gannet.fish.washington.edu/Atumefaciens/20190710_Pgenerosa_v074_gensas_annotation/Panopea-generosa-vv0.74.a3.5d65aaa449919-pfam.tab) Grabbed the top 10 most abundant Pfam Accessions to see how things looked: ```shell awk '{print $2}' Panopea-generosa-vv0.74.a3.5d65aaa449919-pfam.tab | sort | uniq -c | sort -nr | head ``` | Feature Count | Pfam Accession | Pfam | |---------------|----------------|------------------------------------------------------| | 1062 | PF00078.22 | Reverse transcriptase (RNA-dependent DNA polymerase) | | 370 | PF00665.21 | Integrase | | 353 | PF13358.1 | DDE superfamily endonuclease | | 264 | PF03372.18 | Endonuclease/Exonuclease/phosphatase family | | 213 | PF14529.1 | Endonuclease-reverse transcriptase | | 208 | PF00643.19 | B-box zinc finger | | 199 | PF07690.11 | Major Facilitator Superfamily | | 158 | PF00001.16 | 7 transmembrane receptor (rhodopsin family) | | 148 | PF12796.2 | Ankyrin repeat | | 136 | PF00096.21 | Zinc finger | A couple of interesting things that I notice from this table: 1. The four of the top five most abundant are involved in DNA transposition. 2. A rhodopsin protein family appears in the Top 10 most abundant Pfams?! Proteins in this family are involved in light detection... InterProScan annotations (tab-delimited text): - [Panopea-generosa-vv0.74.a3.5d65aaa22961e-interproscan.tab](https://gannet.fish.washington.edu/Atumefaciens/20190710_Pgenerosa_v074_gensas_annotation/Panopea-generosa-vv0.74.a3.5d65aaa22961e-interproscan.tab) - Contains gene ontology (GO) terms Project Summary file (TEXT): - [t_5d25021b0d20b-5d6843e21278a-publish-project-summary.txt](https://gannet.fish.washington.edu/Atumefaciens/20190710_Pgenerosa_v074_gensas_annotation/t_5d25021b0d20b-5d6843e21278a-publish-project-summary.txt) ``` ================================= Project Summary --------------------------------- # Project Information Project Name : Pgenerosa_v074 Create Date : 2019-07-09 14:07:39 # Project Properties Genus : Panopea Species : generosa Project Type : invertebrate Prefix : PGEN_ Common Name : Pacific geoduck Genetic Code : Standard Code # Input FASTA Filename : Pgenerosa_v074.fa Filesize : 913.68 MB Number of Sequence : 18 ================================= Job Information --------------------------------- # Official Gene Set >PASA Refinement - version : 2.3.3 - Transcripts FASTA file : Trinity.fasta # The source Job of the refinement job >Augustus-01 - version : 3.3.1 - Species : fly - Report genes on : both - Allowed gene structure : partial - cDNA (transcripts) sequences : Trinity.fasta - Protein sequences : 20180827_trinity_geoduck.fasta.transdecoder.fa # The consensus mask Job >Masked Repeat Consensus # The source jobs for consensus mask job >RepeatMasker >RepeatModeler # Family copy number summary Family Copy Numbers DNA 675 DNA/Academ 1327 DNA/Crypton 344 DNA/Ginger 130 DNA/Kolobok-T2 141 DNA/Maverick 942 DNA/MuLE-MuDR 201 DNA/MuLE-NOF? 142 DNA/P 167 DNA/PIF-Harbinger 227 DNA/RC 587 DNA/Sola 508 DNA/TcMar-Fot1 117 DNA/TcMar-Mariner 6734 DNA/TcMar-Tc1 3718 DNA/hAT-Tip100 516 DNA/hAT-hAT5 1037 Type:DNA 17513 LINE 883 LINE/CR1 5204 LINE/CR1-Zenon 14653 LINE/I 980 LINE/I-Nimb 1119 LINE/L1 4031 LINE/L1-Tx1 6620 LINE/L2 8879 LINE/L2-Hydra 113 LINE/Penelope 1026 LINE/RTE-X 21214 Type:LINE 64722 SINE/tRNA-Core-L2 41152 Type:SINE 41152 LTR/Caulimovirus 140 LTR/DIRS 448 LTR/Gypsy 1031 LTR/Ngaro 343 LTR/Pao 82 Type:LTR 2044 Type:EVERYTHING_TE 125431 Type:Simple_repeat 19235 Type:Unknown 1465471 # The functional Jobs on the OGS >InterProScan - version : 5.29-68.0 >Pfam - version : 1.6 - E-value Sequence : 1 - E-value Domain : 10 >SignalP - version : 4.1 - Organism group : euk - Method : best - D-cutoff for SignalP-noTM networks : 0.45 - D-cutoff for SignalP-TM networks : 0.50 - Minimal predicted signal peptide length : 10 - Truncate to sequence length : 70 >BLAST protein vs protein (blastp)_SP01 - version : 2.7.1 - Protein Data Set : SwissProt - Maximum HSP Distinace : 30000 - Output type : tab - Matrix : BLOSUM62 - Expect : 1e-8 - Word Size : 3 - Gap Open : 11 - Gap Extend : 1 >DIAMOND Functional_SP01 - version : 0.9.22 - Protein Data Set : SwissProt >BLAST protein vs protein (blastp) - version : 2.7.1 - Protein Data Set : 20180827_trinity_geoduck.fasta.transdecoder.fa - Maximum HSP Distinace : 30000 - Output type : tab - Matrix : BLOSUM62 - Expect : 1e-8 - Word Size : 3 - Gap Open : 11 - Gap Extend : 1 >DIAMOND Functional - version : 0.9.22 - Protein Data Set : 20180827_trinity_geoduck.fasta.transdecoder.fa ``` Overall, this annotation is much more believable than the previous MAKER annotations, due to the fact that GenSAS actually predicts genes to exist on all the scaffolds (unlike MAKER)! As such, this will likely become the canonical _P.generosa_ genome going forward. With that being said, we still should manually curate this when we have the time to see how well the predictions line up with the evidence.