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Old 06-01-2011, 06:47 AM   #1
Location: Paris

Join Date: Jun 2011
Posts: 15
Default Coverage of reads not homogenous (sureselect target enrichement)

Hi all,

I have successfully run a targeted enrichment with SureSelect (Agilent protocol) . And now, I try to analyze the data. I need your help for the interpretation of sequencing results.

The problem is about the coverage of reads which is not homogenous for a lot of regions.

For example, I took the region of exon 34 of NOTCH1 gene. In the middle of the exon, I can see a small region with a very low density of reads. I found this region in the tumoral and germline sample.
I can immediately eliminate the hypothesis of a deletion because we had already sequenced this gene and we had found only one mutation but not deletion. In other words, this gene is a control to check the results.

Other hypothesis is the alignment… so, if you think that is the problem, what would be the criteria to modify? (alignment software : BFAST, condition : 4 mismatches).

Other possibility, the hybridization between the baits (manufactured after the design) and the DNA were not enough specific ? maybe, there was a problem during the synthesis of baits…

What is your opinion about this result?
Have you already met this kind of problem?

Thank you for your help...

Here are parameters of the sequencing :
- sequencing protocol : paired-end
-Bait length : 120bp
-Bait Tiling Frequency : 1X
- Sequencer : Illumina GA II
- length reads = 2*150pb
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