Question: Need a help re Blastn against short (~50 nt seq)
0
gravatar for ritujoshi18
5.3 years ago by
Sweden
ritujoshi180 wrote:

Hi Evevryone,

In stand-alone blast, I'm blasting several thousands of ~450nt long sequences against one short ~50nt long motif sequence to find the sequences that has this ~50nt motif. In blastn output I get couple of hits with >90% (and alignment length = 50) So my questions are-

1. Why I'm not getting the hits below 90% identity (like sequences that has 85 or 70% identity to the motif seq)?

2. Is there other efficient way/tool available to find the sequences from the data that has my seq of interest in it?

I'm new to blast and bioinfo in general so  any help/inputs would be highly appreciated!

Thanks very much!

 

blast tool • 1.6k views
ADD COMMENTlink modified 5.3 years ago by Asaf6.1k • written 5.3 years ago by ritujoshi180

For some weird reason, I've seen legacy BLAST's blastall work better on the shorter nucl sequences than blast+ 's blastn. Have you tried blastall?

Also, I think 70% of 50 nucl (30 nucl) might too short of a match for BLAST to consider, and you might wanna tweak parameters a bit.

ADD REPLYlink written 5.3 years ago by RamRS23k

Thanks Ram for your reply. Haven't try the blastall yet but will try that now. And which parameters exactly I will have to change?

Few more Qs-

1. Btw, do you think if its a correct approach to blast long (in my case ~400-600bp) seqs against short read (40- 60 bp) to pull out the sequences from NGS data? 

2. Would I miss the sequences that has mismatches in the string where my short sequence is aligned ?

Again, thanks.

R

ADD REPLYlink modified 5.3 years ago • written 5.3 years ago by ritujoshi180
2
gravatar for Asaf
5.3 years ago by
Asaf6.1k
Israel
Asaf6.1k wrote:

1. You should probably search with a higher E-value threshold, this will give you more answers, you can (or should?) use a small word length (try 7) - this is the minimal perfect matching BLAST requires between the two for the alignment to be found.

2. I think this problem is a classical global alignment problem instead of local alignment (which BLAST does). You can use needle of the EMBOSS package to align the short sequence to the long ones, you should probably use a high gap penalty. The only issue here is that there is no statistics - you have to decide if the alignment returned is satisfactory or not,

ADD COMMENTlink written 5.3 years ago by Asaf6.1k

Thanks very much Asaf!

ADD REPLYlink written 5.3 years ago by ritujoshi180
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