Target enrichment

[cub103]

Hi- I wonder if anybody has used the Nimblegen Sequence Capture and sequenced the library using Illumina Solexa Sequencer? How it worked? It will be great if you have a protocol. Thanks.

[NextGenSeq]

See

http://www.abrf.org/ResearchGroups/DNASequencing/EPosters/APerera_DSRG-Study-22March2010.pdf

[bioinfosm]

This Clin Chem paper uses nimblegen capture followed by Illumina sequencing

http://www.clinchem.org/cgi/content/abstract/clinchem.2010.145441

[cub103]

Very useful information. Thanks!

[HGENETIC]

We're in the process of setting this system in our lab in a 96 well format, going to take a few weeks to get it optimised but if you're interested I could send you details on our progress?

[cub103]

We're in the process of setting this system in our lab in a 96 well format, going to take a few weeks to get it optimised but if you're interested I could send you details on our progress?

Yes, please! Would be so appreciated if you would share this.

[shangyukui]

very useful information

[NextGenSeq]

Supposedly new solution phase sequence capture kits from Roche (Nimblegen) will be released in the near future. Their current solution phase capture kits require a purchase of ~100 assays but the new ones supposedly will not.

[ec338]

Hi- I wonder if anybody has used the Nimblegen Sequence Capture and sequenced the library using Illumina Solexa Sequencer? How it worked? It will be great if you have a protocol. Thanks.

Hello,

Yes, we had no problem using Nimblegen Seqcap whole exome capture (in solution) with Solexa sequencing (Nimblegen guidelines attachedhttp://www.nimblegen.com/products/li...me_SR_v1p2.pdf). We are currently waiting for results from the combination "Nimblegem array-based sequence capture (custom resequencing)" +"Solexa sequencing" (in-house protocols available if this works).

FUTHER QUESTION: our next aim is to try Nimblegen + multiplex Solexa sequencing - did anybody ever try to capture on a Nimblegen array and sequence on GAIIx several captured samples in multiplex?? Any protocols for indexing aso? Any other option(s) for cost-effective small scale capture/resequencing projects (e.g. 2 Mb for 2 patients = when Agilent Sureselect Target enrichment systems would not fit).

Thanks,

Estelle

[csquared]

We've done a lot of capture/Illumina sequencing. A couple quick comments on our observations:

The Nimblegen methods are reasonably good but we use in-house protocols with very consistent results and find the protocols more convenient. One disclaimer is we have only done the head-to-head a couple times so it may not be a 100% fair statement to say in-house is "better" than Illumina. Generally speaking, the standard Illumina lib prep methods will work fine with the Nimblegen arrays with minimal modification.

Multiplexing works well. We routinley do 4 samples per array capture and depending on the capture region size, multiplex two or three arrays per sequencing lane. Again, methods are pretty straightforward. We didn't need to reinvent the wheel. Just made sure all the libraries were high quality and the hybridizations, washes, and elutions were performed as optimally as possible.

[cutcopy11]

Is there a reliable target enrichment system designed for SOLiD???

[ec338]

We've done a lot of capture/Illumina sequencing. A couple quick comments on our observations:

The Nimblegen methods are reasonably good but we use in-house protocols with very consistent results and find the protocols more convenient. One disclaimer is we have only done the head-to-head a couple times so it may not be a 100% fair statement to say in-house is "better" than Illumina. Generally speaking, the standard Illumina lib prep methods will work fine with the Nimblegen arrays with minimal modification.

Multiplexing works well. We routinley do 4 samples per array capture and depending on the capture region size, multiplex two or three arrays per sequencing lane. Again, methods are pretty straightforward. We didn't need to reinvent the wheel. Just made sure all the libraries were high quality and the hybridizations, washes, and elutions were performed as optimally as possible.

Hello,
Very encouraging comment about multiplexing, thank you! Are you using 385K or 2.1M arrays? Would it be possible to share your in-house protocol / slight modifications from library prep to multiplex capture and sequencing? Many thanks, Estelle.

[upenn_ngs]

and solution-based hybridization? has anyone had success placing several individuals into a single capture?

[csquared]

Yes to the multiplexed solution phase question. However, in limited trials, the results were better to divide the input library into multiple, individual, lower volume reactions rather than a single reaction with multiple samples. Of course, your results may vary. We've only done this a small amount with whole-exome and a bit more often with a custom design that was specifically designed to increase the number of probes per capture sequence to enable multiplexed capture.

[upenn_ngs]

interesting, i have heard rumors illumina is developing exome capture with pooled hybridization to lower cost. also makes the workflow easier with a high volume of samples.