Low exomic yeld after capture.

[durandg]

Dear all,

After having used the Targetseq Exome Capture kit for years, LifeTech provided us with a new version of the kit called Ion TargetSeq™ Exome Enrichment kit for the exome capture of the libraries. In my first Solid 5500XL run using it, i only got circa 20-25% of exomic sequences, instead of the 75% of "read on target" that i usually get with the former kit.

Would anyone have an idea of what could be the cause? Did you already used this kit and what results did you get? Is there any tricky change in the way you do perform the capture with this new version and that would not be mentionned in the procotol?

My samples were FFPE; i'm wondering if the formalin and/or paraffin could interfer with a step of the library or the capture and could dramatically reduce its yeld...

Thank you,
durandg.

[JPC]

If you have 25% mapping and 75% off target then either your hyb failed of your post hyb washing wasn't efficient. I wouldn't suspect FFPE at this stage if the only issue with your data is the off target %.

Why did LT give you an Ion kit for your 5500, are they dropping the original version?

[Zaag]

How about duplicate levels?

[durandg]

Our FAS told me that this "new version" of the capture kit has redesigned probes and more probes hat before but she wasn't able to tell me whether it is discontinued or not.

Investigation ongoing...

[BurgundiaPR]

Hi everybody,

I re-open this old post because we are experimenting the same issue, using Agilent QXT capture.

The % of on-target reads is about 25%. The off-target reads do not seem to have a particular pattern, nor the regions where they map - they uniformely map on the entire genome.

Have you any idea of what can explain this result ?

Thank you by advance,
BPR

[Joe CHEN]

low capture efficiency

Does anyone find out the reason about the low capture efficiency? I still have the same problem. Different style of libraries,the same probe, same protocol. style 1 library on target 60%,style 2 library on target 30%