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Old 03-18-2010, 04:01 PM   #1
Location: baltimore

Join Date: Oct 2009
Posts: 89
Default insert size

Dear group:
I need some help in understanding insert size concept.

I have a targeted exome sequencing data using paired-end approach with 76 bp. I have lots of duplicates in the sam file. I should use rmdup with insert size correctly mentioned.

i was told by technician that insert size is between 150-300 bp.

When I see 9th tag which is inferred insert size in sam file, I have lots of numbers that range from 0 to 100,000.

Since the experiment is done with an insert size 150-300 bp, and BWA inferred insert size has lots of ranges, what number should I use in using rmdup. Heng Li recommends that we should use correct insert size always. If I have range from 150-300 (technician) and SAM file inferred sizes are spanning across wide ranges, Which insert size should I select to remove duplicates and call SNPs.

OR should I make sets of reads that fall into certain ranges and call SNPs in each bin.

Also what is inferred insert size '0' mean and what is 345,039 mean.

adrian is offline   Reply With Quote
Old 03-18-2010, 05:55 PM   #2
Senior Member
Location: Boston area

Join Date: Nov 2007
Posts: 747

Many if not all of the very large insert sizes are probably due to falsely aligned reads to repeats (LINEs, SINEs, Satellite, etc); take a few of them and look to see whether both reads actually align to non-repeat DNA.

Also, take a sample from your SAM file (ideally random) & look at the size distribution -- you may see a long tail of weird sizes, but you'll probably see most of the counts in a distribution around what the technician said. It's a worthwhile check on the library in any case & easy to generate the data table with a little bit of perl (or even UNIX shell commands).
krobison is offline   Reply With Quote

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