Question: Precise Ngs Alignment
3
gravatar for Leszek
7.1 years ago by
Leszek4.0k
IIMCB, Poland
Leszek4.0k wrote:

I need to precisely align NGS data. The problem is, in global alignment I loose plenty of reads (20%), while local alignment fails to align merely 5% of reads. I don't care that much about 3'-end, but 5'-end has to be aligned with 1bp precision. Iterative hard-clipping of 3'-ends helps, but it would be perfect to do it in one run. Do you know of any program that can precisely align 5'-end and allows for soft-clipping of 3'-end at the same time?

next-gen alignment • 2.1k views
ADD COMMENTlink written 7.1 years ago by Leszek4.0k
1

I see BWA does the soft clipping of the 3' end based on quality (-q option). and also MOSAIK v2.0 and Bowtie 2 supports soft clipping.

ADD REPLYlink written 7.1 years ago by Rm7.9k

bowtie2 does global or local alignment. in local mode, both ends may be clipped and I need entire 5'-end. Concerning BWA, indeed it does soft-clipping of 3-end based on quality down to 35bp ("-q INT quality threshold for read trimming down to 35bp [0]"), but I have noticed some reads are able to align after trimming to 25, 21 or even 16bp. I have never used MOSAIK, but will give it a try:)

ADD REPLYlink written 7.0 years ago by Leszek4.0k

BTW did you tried pre- trimming the reads to specific length and aligning it...Generally I follow pre- trimming the reads after looking at the FastQC report.

ADD REPLYlink written 7.0 years ago by Rm7.9k

http://wiki.bioinformatics.ucdavis.edu/index.php/Subsequence.pl

ADD REPLYlink written 7.0 years ago by Rm7.9k

I did, but the point is some reads align uniqeuly @ 41bp, and other @ 31bp, and other @ 21bp. So I would need to do iterative 3'-trimming of unaligned reads. Novoalign does it for me automatically:)

ADD REPLYlink written 7.0 years ago by Leszek4.0k
3
gravatar for Sean Davis
7.0 years ago by
Sean Davis25k
National Institutes of Health, Bethesda, MD
Sean Davis25k wrote:

Novoalign has a miRNA mode that does something similar to what you are asking, though I haven't used it in quite some time.

ADD COMMENTlink written 7.0 years ago by Sean Davis25k

+1 Novoalign is slower than bowtie2 or bwa, but align much more reads and algs look much better for me

ADD REPLYlink written 7.0 years ago by Leszek4.0k
2
gravatar for JC
7.1 years ago by
JC8.0k
Mexico
JC8.0k wrote:

I don't know an optimal solution for this, once I had a similar case where some reads need trimming with different sizes, I use Blat to align to the reference and then use some Perl scripts to convert the PSLX to a valid SAM format, inserting soft-masking ends and indels flags in the CIGAR when it was required. The mapping worked terrific but it was extremely slow. Do you want to take a look? http://github.com/caballero/RNAseq-Pi/ the files megablat.pl and pslx2sam.pl under bin/

ADD COMMENTlink written 7.1 years ago by JC8.0k

thanks JC, I was thinking about BLAT. I think it could handle current data (4x6mln reads), but we expect a lot more in the following months, so I prefer to find efficient solution for that.

ADD REPLYlink written 7.0 years ago by Leszek4.0k
1

yes, I agree, now I only use Blat in extreme cases when Bowtie/BWA cannot be used.

ADD REPLYlink written 7.0 years ago by JC8.0k

gmap is faster and probably still as sensitive as blat.

ADD REPLYlink written 7.0 years ago by Sean Davis25k
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