No more original Truseq kits

[murpheli]

Wondering if anyone is piloting NEB or Kapa library preparation kits for Illumina sequencing? Heard that the original Truseq library prep kit is being discontinued at the end of the year, and remaining kits are not useful for target capture. They are pushing toward using Nextera for these applications, but we tried it and had very poor luck, likely due to fragment size range after enzymatic fragmentation. Would love any pointers/input on using kits from these other companies.
Thanks.

[NextGenSeq]

We got good results with Nextera whole exome capture. The tagmentation reaction is very sensitive to variations in the amount of input DNA. You have to use exactly 50 ng for each genomic DNA sample.

[murpheli]

Sorry I should have been more clear, I'm asking in relation to using a non-Illumina custom enrichment procedure. Nimblegen and IDT capture protocols require an up front Library preparation and Nextera does not work well for these. Truseq works great but with them discontinuing it and not making a comparable product available we need to try one of these other kits (ie - NEB or Kapa).

[kcchan]

You should be able to use the TruSeq PCR free kit in conjunction with an additional PCR step to generate the libraries that you need for the subsequent exome capture. If you're not a fan of custom protocols, the Agilent sells their SureSelectXT reagent kits that can be used with either TruSeq exome or their own Sureselect capture protocols.

[Jon_Keats]

Kapa works the best of all things we have tested. TruSeqDNA, Agilent XT, Agilent XT2, NEB Ultra. Oddly Agilent XT2 actually worked best at 500ng. If you want our protocol see www.keatslab.org

[mcnelson.phd]

I'm not involved in these types of projects so I can't comment on kits from other suppliers, but in reference to your saying that there's no replacement for the standard TruSeq kits Illumina has put out what they call their TruSeq Nano DNA kits.

I haven't used it, but I've read over the manual and it's pretty much the same as the standard TruSeq DNA kit with lower input requirements and a bead based size selection instead of the gel method.

The Nano has a higher cost/sample based on the kit price, but when you factor in hands on time and all the AMPure cleanups involved in the old TruSeq DNA kits the Nano is pretty comparable.

[murpheli]

Thanks everyone for the replies. Unfortunately both the Truseq Nano and PCR-free are stated on the Illumina website as not being useful for downstream target capture. I'm sure if I had the time (and patience of my boss) there is a way to tweak them, but this is just not an option in my lab.

[buckybadger]

I don't know if this will be helpful, but here's a link to a poster from AGBT_2013 (it's posted on the Kapa Biosystems site) showing a comparison of their libraries to the TruSeq libraries for use in Nimblegen enrichment:

http://www.kapabiosystems.com/public/pdfs/miscellaneous/AGBT_2013_Burgess_RocheNimbleGen.pdf

Cheers,

--BB

[addyblanch]

+1 for KAPA kits

[buckybadger]

Nimblegen-Kapa Biosystems Partnership

http://www.nimblegen.com/news/press_rel/news_2013_12_18.html

Roche NimbleGen and Kapa Biosystems Team Up to Provide Optimized Next-Generation Sequencing (NGS) Target Enrichment Workflow Solution
Madison, WI, USA, December 18, 2013

Roche (SIX: RO, ROG; OTCQX: RHHBY) and Kapa Biosystems announced today a partnership to provide an optimized NGS target enrichment workflow solution. Kapa Biosystems will custom manufacture NGS library preparation products for Roche NimbleGen to distribute to its customers.

Roche NimbleGen's new offering combines KAPA Library Preparation Kits with Roche NimbleGen sequence capture products for complete workflow coverage, prior to sequencing. This validated protocol provides customers with the ability to work with low DNA quantity or quality, including FFPE DNA for whole exome or targeted sequencing. Compared to other workflows, this offering significantly reduces GC bias and obtains better molecular complexity, resulting in further improved enrichment performance.

[etc.]