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seq-ngs 10-23-2014 08:32 AM

Library Prep for Cancer NGS
 
Hi all,

I am designing several cancer panels to run on the MiSeq. Each panel consists of 600-800 amplicons - approximately 150 bp each. What I am pondering right now is the library prep method to use. There are 5 main options:

1. Illumina - TSCA
2. Life Tech - AmpliSeq
3. Agilent - Haloplex
4. NuGen
5. Qiagen

We deal with formalin-fixed, paraffin-embedded samples that are degraded and often of low DNA quantity/quality. I like Ampliseq better than TSCA because it is multiplex PCR rather than probe hybridization (like Illumina) but don't want to run Ampliseq on the MiSeq because the Life Tech people seem not to like that. Any thoughts on a good library prep method from the choices above for FFPE samples? Thanks so much for any input.

shrutimish@gmail.com 02-23-2015 08:02 AM

I would also like to know more about why Life Tech does not like Ampliseq on the Miseq?

pmiguel 02-24-2015 06:14 AM

Quote:

Originally Posted by shrutimish@gmail.com (Post 160792)
I would also like to know more about why Life Tech does not like Ampliseq on the Miseq?

What makes you think that this is the case?

Life Tech has their own sequencer, by the way.

--
Phillip

shrutimish@gmail.com 02-24-2015 06:56 AM

I asked this question "Life Tech does not like Ampliseq on the Miseq" because the previous person commented that. So, do you think it is not an issue? Can we actually use Ampliseq for target enrichment and run it on MiSeq?
Also, when you say life tech has their own sequencer- Are you talking about the Ion torrent sequencer?

Thanks in advance for answering these questions.

thomasblomquist 02-25-2015 08:47 AM

I do it all the time. I have my target specific primers with "tails on them." These tails are "universal" sequences alien to the genome your targeting. I then add on the "platform" specific P5/P7 tails with R1/R2 priming regions for Illumina, or the Forward and Reverse sequencing tails for Ion Torrent to complete the job.

Wham bam, the results that get spit out (for genotyping purposes) are pretty much similar.

Now, and this is a big IF, you are trying to QUANTIFY rare alleles, the detection limits for that give base variation between the different loci will be HIGHLY PLATFORM DEPENDENT.

For the most part Illumina Error profile is quite good. And cost wise, is easier/cheaper to get up and running. Ion Torrent with its heterodimer bead issues can create problems with similar templates that vary by a single allele; especially in low complexity libraries.

Ion torrent, if done right, can have a VERY QUICK turn around, and can be used for quick identification once the assay is well characterized.

Best of luck.

-Tom Blomquist

shrutimish@gmail.com 02-26-2015 01:01 PM

Thanks Tom for your reply. It is very useful to know this.


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