FAQ 2: Did i used the right command?

I have tried everything as per your suggestions using the following command:
perl GKas.pl -query_seq="/home/Packages/GKas/mart_export_4.txt" -gff="Mus_musculus.GRCm38.68_tr.gtf" -hit_seq="/home/Packages/GKas/scaffolds.fasta" -spe=6 -blast2seq=/usr/local/bin -blat=/usr/local/bin -codeml=/home/Packages/paml4.6/bin -blat_done=T -detail="/home/Packages/GKas/detail.txt" -kaks_file="/home/Packages/GKas/kaks.txt"

The GFF issues seem to have been solved. With the above command I have found the following results:
blat_rel.query which is around 30 MB is fiel size
problem_loc around 79 bytes
detail.txt 0 bytes
rub, rst and rst1 each with 0 bytes
new_blatout around 8MB
I dont have any ka ks file or the details of the alignment

I dont know what is wrong now, can you suggest what I am doing wrong or what I should do??

  • you need to change the command to:

perl GKas.pl -query_seq="/home/Packages/GKas/mart_export_4.txt" -gff="Mus_musculus.GRCm38.68_tr.gtf" -hit_seq="/home/Packages/GKas/scaffolds.fasta" -spe=6 -blast2seq=/usr/local/bin/bl2seq -blat=/usr/local/bin/blat -codeml=/home/Packages/paml4.6/bin/codeml -blat_done=T -detail="/home/Packages/GKas/detail.txt" -kaks_file="/home/Packages/GKas/kaks.txt"

  • since you use the "-blast2seq=/usr/local/bin/", then in the line 438:

438: system "$blast2seq -p blastn -i tmpdna1.fa -j tmpdna2.fa -D 1 -o tmp.fas.txt";
it will be system "/usr/local/bin/ -p blastn -i tmpdna1.fa -j tmpdna2.fa -D 1 -o tmp.fas.txt",
obviously, it will not run bl2seq.
so, i still insist to change the command, if the PATH is OK, you can ignore the option like -blast2seq, -blat and -codeml.


  • I notice that u used the gene ID as "ENSMUSG00000020333|ENSMUST00000000145" and "scaffold980|size443694", this may will cause problems, i suggest use "ENSMUSG00000020333" and "scaffold980" if they are unique.

And, since you used the option "-blat_done=T", so, it will skip running blat , and you didn't specify the privious blat rel, so it will use the file defined at line
25: my $blat_out = 'blat_rel.query';
that's why you still get the blat_rel.query and new_blatout .