Standalone blast output from paired end reads

[sp24]

Hi Everyone,

I have a question, which is probably simple. I'm new to this area though so I need a little help.

I have paired end reads. I have taken the R1 files (not the R2, I hear I have to reverse complement those) and converted them to Fasta, and made a blast database. I used tblastn because I have an amino acid query and the paired reads are nucleotides. The output is huge, and I have no idea how to even interpret it. I have a tabular output.

I'd just like to know if anyone has done a similar project and what to do with the blast outputs. Someone suggested to use Cap3 sequence assembler. The problem for me is working with the blast output and figuring out how to deal with it.

Thank you!

[rhinoceros]

Hi Everyone,
The output is huge, and I have no idea how to even interpret it. I have a tabular output.

It lists all your hits. What's there to interpret? You can slim it down easily with simple unix commands, e.g. assuming you used default '-outfmt 6', the 4th column lists the alignment lengths. To only keep hits that produced ≥ 100 bp alignments, do:

awk -F'\t' 'int($4)>=100' result.tsv > result_slimmed_down.tsv

Want to see only the unique sequences that produced these hits?

cat result_slimmed_down.tsv | cut -f 2 | sort -u > unique_hit_contig_ids.txt

etc.


p.s. I think you should delete your db, go read a few articles relative to paired-end reads (pay special attention to M&M), process your data properly, and only then build the db and run your blasts..

[sp24]

Thanks for your input. By interpret I just meant that once I'm done blasting, that's not the end of the process. I need to figure out a way to actually get the sequences out, maybe assemble them using cap3.

I'm not sure if I'll be able to use that first command since I'm only using paired end reads, which are not that long to begin with.

I did use that second command on my blast output and got the names, which will be useful because I can use the blastdbcmd command to extract the hits.

Right now I'm just experimenting, I'm sure I will have to delete my database and start over anyway. I just wanted to have something to work with and practice blasting, and getting sequences out, and then actually doing something with those sequences. I'm not sure what M&M is though.