low sureselect on-target reads

[hawaii454-0]

Hello,
I am using Agilent's Sureselect human all exon v4 kit to make libraries for Illumina sequencing.

Experiment details:
Using version 1.3 protocol
Covaris S2 used for shearing
Incubations are carried out on a tube heat block
Ampure XP beads are used for sample clean ups
MJ Research dyad PCR blocks are used for the amplification steps
RNase free eppendorf tubes with filter tips are used throughout
Agilent 2100 bioanalyser instrument used

In the literature, it says that the expected on target reads is between 70-80%, but after analyses we are only getting around 45-60% for most of our libraries. A great number of our reads are even as low as 28% on target. I have attached a document with all the Agilent traces for one library prep which gave 28% bases on target after final analyses.

Can anyone advise on why this may be occurring?

For example, could there be a minimum amount of library needed after each step? I know that 500ng is needed before hybridisation but what about other steps? The reason I'm asking is that some of the Agilent traces show lower peaks than others and I was wondering if this affects the final quantity.

Thanks in advance for your help,
Anna.

[josdegraaf]

You seem to have rather low yields after the post capture PCR, did you quantify this also in a different way or only on the BA?
Using older sureselect versions we obtained much more final library yield if the standard protocol was followed but recently we switched to v4 and also had very low post capture yields. I am waiting for the analysis so cant comment of this leads to less on-target reads.
After talking to techsupp I was told that this is seen at many more sites and they sended me a updated version of the protocol (1.4.1) including a on-bead post capture PCR.

[hawaii454-0]

Hello,
I quantify the final library after PCR by qubit fluorometry and then checking this on a qPCR. Do you do this the same way?

I haven't started using the v4 protocol with on-bead PCR yet.

Is there anything you can suggest to increase yields? Does your lab get higher % on target reads as the norm?

Thanks.

[chongm]

Hi I'm using the V5+ UTR protocol and I'm finding on average 54% reads on target +-100 bp, whereas in their datasheet, they show ~80% on target. Just based on my experience with enrichment kits in general these kits rarely perform to the reported specifications, so I feel like our % on target may not be an anomaly. Did you ever get higher % on target? If you did, what was your issue?

MC