waldeyr/Tsor2

Tsor2_pipeline

Tsor2 annotation workflow

Dependencies

• NCBI-Blast 2.8+

• Signalp 5

• Python3.x libraries:

** pandas

** Biopython

** re

** os

RefSeq Alignment

Database building:

makeblastdb -in refseq_protein.fasta -out refseq_blast/RefSeq_Release_200 -dbtype prot -max_file_sz 2GB -parse_seqids -title RefSeq_Release_200 -logfile log.txt

Blast running (Caution: it will take about 2 weeks to run):

nohup blastx -db refseq_blast/RefSeq_Release_200 -num_threads 48 -evalue 10E-5 -query Tsor2_Trinity_Unigene.fasta -outfmt "6 qseqid qgi qacc qlen qstart qend qseq nident pident evalue positive ppos gaps qframe sseqid" -out Tsor2_RefSeq_blast.result &

Insert headline on the result file (caution: execute it only once)

echo -e "contig_id\tqgi\tqacc\tqlen\tqstart\tqend\tqseq\tnident\tpident\tevalue\tpositive\tppos\tgaps\tqframe\tsseqid" | cat - Tsor2_refseq_blast.result > /tmp/out && mv /tmp/out Tsor2_refseq_blast.result

RefSeq parse and annotation

Before run it, check if the Tsor2_refseq_blast.result has a headline with the command head Tsor2_refseq_blast.result

Caution: recommended 32 GB RAM

python3 Step01_refseq_parse_and_annotation.py

Uniprot Alignment

Database building:

makeblastdb -in uniprot_sprot.fasta -out uniprot_sprot/uniprot_sprot -dbtype prot -max_file_sz 2GB -parse_seqids -title uniprot_sprot -logfile log.txt

Blast running (Caution: it will take about 2 days to run):

nohup blastx -db uniprot_sprot/uniprot_sprot -num_threads 48 -evalue 10E-5 -query Tsor2_Trinity_Unigene.fasta -outfmt "6 qseqid qgi qacc qlen qstart qend qseq nident pident evalue positive ppos gaps qframe sseqid" -out Tsor2_uniprot_blast.result &

Insert headline on the result file (caution: execute it only once)

echo -e "contig_id\tqgi\tqacc\tqlen\tqstart\tqend\tqseq\tnident\tpident\tevalue\tpositive\tppos\tgaps\tqframe\tsseqid" | cat - Tsor2_uniprot_blast.result > /tmp/out && mv /tmp/out Tsor2_uniprot_blast.result

TrEMBL Alignment (only for those contigs that were not annotated by any other means; see the script "Step04_build_input_for_trembl_running.py")

Database building:

makeblastdb -in uniprot_trembl.fasta -out uniprot_trembl/uniprot_trembl -dbtype prot -max_file_sz 2GB -parse_seqids -title uniprot_trembl -logfile log.txt

Blast running (Caution: it will take about 2 days to run):

nohup blastx -db uniprot_trembl/uniprot_trembl -num_threads 36 -evalue 1E-5 -query Tsor2_input_for_TrEMBL.fasta -outfmt "6 qseqid qgi qacc qlen qstart qend qseq nident pident evalue positive ppos gaps qframe sseqid" -out Tsor2_trembl_blast.result &

Insert headline on the result file (caution: execute it only once)

echo -e "contig_id\tqgi\tqacc\tqlen\tqstart\tqend\tqseq\tnident\tpident\tevalue\tpositive\tppos\tgaps\tqframe\tsseqid" | cat - Tsor2_trembl_blast.result > /tmp/out && mv /tmp/out Tsor2_trembl_blast.result

SIGNALP

signalp -fasta Tsor2_Translated_Proteins.fasta -org euk -format short -prefix Tsor2_signalp -verbose -gff3