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kerryp 10-18-2017 09:41 AM

NexteraXT kit on cDNA amplicon?
Can I use the Nextera XT library prep kit on a 2kb cDNA amplicon? Just wanting to check that the tagmentation process will work ok on cDNA. I've only ever used it on gDNA for WGS.
Thanks in advance!

nucacidhunter 10-18-2017 01:16 PM

Yes, currently Smart-Seq, Drop-Seq, Fludigm single cell RNA-Seq, Illumina Bio-Rad single cell and some other techniques use Nextera to prep libraries from cDNA.

kerryp 10-19-2017 01:48 AM

Thanks nucacidhunter. Do you think I could prep a 2kb amplicon (amplified from cDNA) using Nextera XT? Basically treating it like gDNA?
I usually run WGS with Nextera XT and MiSeq v2 reagent kit, but I've been asked to sequence a bunch of 2kb viral cDNA amplicons. Just wondering if I could use Nextera XT, or if a different prep kit would be needed.

nucacidhunter 10-19-2017 03:55 AM

Generally tagmentation will work with any dsDNA. Two points to note:
1- Some sequences from both 3' and 5' end will not be sequenced because transposon requires some region to engage with DNA. It is around 30 bases.

2- Transposon have some bias which in whole genome does not make much difference but if the amplicon lacks the preference sites (it is not very restrict) some region might not get good coverage.

kerryp 10-19-2017 06:09 AM

Thanks - that's really helpful. I expect Illumina don't communicate precisely what their transposon sites are? :)
I suppose it'd just be a case of prepping a couple of amplicons to see how it goes.
Sorry to test your patience, but does tagmentation also work on eukaryotic DNA? You say it'll generally work with any dsDNA, but just checking.
Thanks again!

nucacidhunter 10-19-2017 11:33 AM

I am not sure if Nextera preference site has been studied in details. It just was a precautionary note and in most experiments it does not affect final results.

Nextera works on eukaryotic DNA as well.

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