Exome Sequencing with nimblegen SeqCap EZ v3

[airtime]

Hi,

we working with a GAllx from Illumina and have good experience with nimblegen SeqCap EZ v2 (on target).
We compare the nimblegen SeqCap EZ v3 and Illumina TrueSeq exome kits and the results shocked me .
For nimblegen we have nice mapping rate and low duplication level, but on target it is only 30 %.
For illumina mapping is well but duplicate level is high (20 - 30 %). On target we can reach 76 %.

How it could be that the on target of nimblen v3 kit is so bad? We have good results in the pre steps and everything looks similar to nimblegen v2.

About hints or sharing experience about the nimblegen v3 kit I would be very lucky.

[josdegraaf]

What do you call on target?

[airtime]

Hi,

there are design files (.bed) with region information. And that reads mapped to the genome, including a region from the design file too, are on target.

[josdegraaf]

I know but some people take +- 100 or 200 bases as on target and some don't. Anyway 30% is very low, should be more towards >95% bases covered. We also used V3 and had +- 98%. Illumina performs slightly less with +- 92%.
How deep did you sequence?

[airtime]

Hi,

we used this desing file "SeqCap_EZ_Exome_v3_capture.bed" it include +- 100bp.
how deep means how many reads we get out or how many mapped?

[airtime]

Hi,

what you mentioned that 95% of bases are called means the uniformity.
But the issue is that we mapped >90% of 100 000 000 reads to the genome and then we calculate the 30% on target amount (after duplication removement).