Target enrichment from pooled libraries

[gendxdoc]

Does anyone have any experience performing target enrichment from pooled libraries ? We're very interested in performing SureSelect (Agilent) in-solution capture (or array-based capture) with pooled libraries (SOLiD or Illumina). We'd like to pool 16 barcoded libraries and perform a single enrichment. Has anyone tried this ?

We would expect there to be a limit to the number of individuals that can be pooled, while still retaining sufficient sequence complexity after hybridization across all targets.

[wjeck]

We're interested in doing what sounds like a nearly identical procedure at our institution. We're planning on using Illumina's multiplexing (barcoding) kit followed by SureSelect. We'd love to know the expected efficiency and variation in read representation between subsamples.

[elaney_k]

Hi guys,

I've enriched a set of 9 indexed samples using 1 Agilent sureselect assay. I got very even representation of the indexed samples in the final sequenced data (9.4-11.7% representation of each indexed sample in the final pool).
I did have a lot of off-target reads though, on average only 21% of our reads were on-target, however I had relaxed the default repeat masking during array design to allow me to design baits for regions normally masked and am pretty sure it's responsible for most of the off-target reads.

My target was just over 300Kb and I got on average just under 1700 fold enrichment of my target region so I was fairly pleased with the results- not bad for a first go

I think expected on-target reads is ~60% (if you don't mess with the repeat masker!). I'm pretty sure we will increase our on-target reads with our next array design.

Elaine

[gendxdoc]

Elaine -- This is a good result ! We're getting ready to hybridize a pool of 16 indexed libraries with a SureSelect assay targeted at ~1.5Mb. We'll let you know how it goes.
Michael

[wjeck]

This is great news, and I'll look forward to more posts about the effectiveness of the protocol. We'll be ordering our selection kit soon and I'll do my best to post with the quality of our results as well.

[brasj]

Hi guys,

I've enriched a set of 9 indexed samples using 1 Agilent sureselect assay. I got very even representation of the indexed samples in the final sequenced data (9.4-11.7% representation of each indexed sample in the final pool).
I did have a lot of off-target reads though, on average only 21% of our reads were on-target, however I had relaxed the default repeat masking during array design to allow me to design baits for regions normally masked and am pretty sure it's responsible for most of the off-target reads.

My target was just over 300Kb and I got on average just under 1700 fold enrichment of my target region so I was fairly pleased with the results- not bad for a first go

I think expected on-target reads is ~60% (if you don't mess with the repeat masker!). I'm pretty sure we will increase our on-target reads with our next array design.

Elaine

Hi Elaine,

those are indeed good results - and I agree that messing with the repeat masker may be the sole reason for lower on-target reads.

How did you quantify your samples prior to pooling them? Was this bioanalyzer, qpcr, nanodrop....?

Thanks!

[elaney_k]

Thanks guys!

I quantified the samples using a qubit flourimeter (Invitrogen) as it can quantify dsDNA even in the presence of ssDNA e.g. primers. I wouldn't recommend a nanodrop or bioanalyzer for quantification.

Elaine

[brasj]

Thanks Elaine,

I'll be using the qpcr approach - I agree on the nanodrop and bioanalyzer remark. unfortunately we don't have the qubit flourimeter - would make it a lot easier than the qpcr, IMO.

[elaney_k]

Hi guys,

Shameless plug I know but we finally managed to publish the multiplex target enrichment study for anyone that's still interested in it

Multiplex Target Enrichment Using DNA Indexing for Ultra-High Throughput SNP Detection. Kenny EM, Cormican P, Gilks WP, Gates AS, O'Dushlaine CT, Pinto C, Corvin AP, Gill M, Morris DW. DNA Res. 2010 Dec 16 PMID: 21163834

Elaine