need help about blast2

Question

[Kurban]

Hello everyone, there is a warning after I ran blast2:

mrkurban@mrkurban-Lenovo:~/Desktop$ blast2 -p tblastn -i quir.fa -j gene.fa -e 1e-4 > newoutputfile.txt
[blast2] WARNING: comp867: Could not calculate ungapped Karlin-Altschul parameters due to an invalid query sequence or its translation. Please verify the query sequence(s) and/or filtering options
mrkurban@mrkurban-Lenovo:~/Desktop$

I do not know where did I go wrong?

[Cytosine]

I think the warning here is clear enough that you can look up what went wrong yourself.

Did you verify the query?

[Kurban]

Hi @Cytosine

My two sequences file form are as follows:

file1.fasta

>comp80_c0_seq1 len=592 path=[0:0-591]
TTTTTTTTTTTTAAAGTAAATGCTGCATTTTATTAAAACACAGTATCCAAGTTGTAAAAT
TTAACAAAAATATAAATTACGACCTAGGTCTTTCCTTCTTTTCTTTATACAATGCTAGTA
ATGATACATTAGCCACTTTTACGACCTTGAATCTAACGCCAGGAATGTCACCTACGGCAT
GACCTTTACGACCGAAACCTGCAACTAAAACTTCGTCGTTTTCTTCAATGTGATTGAGAC
AACCGTCCCTCGGTACGAACGCAGTGATTTTCTTTCCGTTTTTAATCAACTGCACTCTCA
CGCACTTTCGGATAGCGGAGTTGGGCTGTTTGGCTTCAACTCCTACTTTTTCCAAAACGA
TTCCTTTCGCGTGCGAAGCACCGCCAAAAGGATTGGCCTTCCACCTAGTGCCCAAATGGG
CCTTTTTGTAGTCTTTATCTGCCCATCTTTGATCGCGACGGTGATTCACATGCTTCCTGG
CTGTTCTAAGACCGCGGGGTTTACCCATGTTGACGGCTGCACAACTTCGGCAAAAAAGAA
ATTTTCGTACCAACCTGAGAAATAACTGTAACCGAGTAATACCAACCGTATT
.....

file2.fasta

>comp904_c0_seq6 len=134 path=[6441:0-4 4536:5-133]
AAATAAAACGACGTACTCATAATACTAAGAGATTTTCAAATTTACAAAAAAATGCTGCCT
GGACTACGAATTTTGTCTCGATCGATCATCGATTTCTTTTTTAAATTATAAATACTTAAA
GCAAAAAAAAAAAA
.....

Among this two files, one of them is query seq, another one is db seq. Are they not standard fasta files? How should I further verify them?

Answer 1

[Siva]

I see at least one problem. You are using tblastn to compare two nucleotide sequences. tblastn takes a protein sequence as query and searches against translated nucleotide sequences. Try blastn instead.

Also, blast2 is an outdated tool. NCBI recommends to use the recent BLAST+ application.

ftp://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/LATEST/

If you have to compare only few sequences, you can also use the NCBI BLAST online to compare two nucleotide sequences:

http://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSearch&PROG_DEF=blastn&BLAST_PROG_DEF=megaBlast&BLAST_SPEC=blast2seq

[Kurban]

Thanks @Siva.

I have transcriptome data set. and by using BLAST+ I got what I want.