sequencing of circulating tumor DNA from plasma

[bluebaby]

Hi all,

I have a few questions that I am very much hoping you will be able to answer.
I am about to start a new project that involves next generation sequencing using Hiseq2500.

The project involves sequencing of a few exons of one specific gene. The source of DNA is circulating tumor DNA extracted from plasma of the cancer patients and the healthy controls (30 samples in total).
The average size of DNA fragments that I and others according to available literature are able to extract do not exceed 160 bp (140-180 bp). Therefore I can't really use Nextera XT kit for my library preparation (it requires the amplicon size of >300 bp for the optimal performance). Please correct me if I am wrong? The total amount of DNA per sample is around 50 ng.

My questions:

1. what approach would you suggest to use to generate my library?
2. I tried to use DesignStudio to design my primers but when I uploaded a file with my specific sequence (that didn't exceed 300 bp) it failed to design any primers.
3. Should I just design my own primers, get amplicons and then somehow attach adapters to them? If so could anyone outline some steps for me how I can do it? Sorry I am just getting in the field of NGS and have very limited knowledge!
Hopefully my questions make some sense!
Thank you very much!

[nucacidhunter]

To suggest any approach, the amount of DNA available and number of samples should be specified.

[bluebaby]

So far I was able to extract about 50 ng of DNA in total per sample.
We are planning to sequence 30 samples in total.

[nucacidhunter]

That amount of DNA (sorry I missed it in your first post) is enough to prepare libraries using TruSeq Nano or any other Illumina DNA library kit (some can be done with ultra low amount of DNA, please see this thread for more info: http://seqanswers.com/forums/showthr...ight=low+input. You just need to skip DNA shearing (as it is already small fragments) and size selection step (depending on kit it may not be in workflow) and prepare a library. That basically will include end-repair, A tailing and adapter ligation followed by PCR amplification. This libraries can be sequenced as shotgun libraries and then you can fish out your exons of interest bioinformatically. Other option which will reduce required sequencing, would be custom sequence capture which you have to use libraries made from highest input DNA. There is an Agilent kit for sequence capture that requires 200 ng input. But because your target region is small, you can amplify your library more to obtain the required input library into those capture reactions just to obtain target regions for sequencing.

[ejan]

Your best option is Rubicon Genomics ThruPLEX DNA-seq Kit. Requires only 50 pg to 50 ng of input. Compatible with SureSelect and SeqCap EZ. Provides equal or better target enrichment performance from 10 ng input as the standard SureSelect or SeqCap EZ protocol at 100 ng input.

http://rubicongenomics.com/products/picoplex-dna-seq-kit/

[ejan]

See paper below for plasma-seq using ThruPLEX at 1 ng and 10 ng inputs

Evaluation of Exome Sequencing to Estimate Tumor Burden in Plasma
Karolinska Institutet, Stockholm, Sweden
August, 2014

http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0104417

[SNPsaurus]

I sequenced some tumor plasma DNA and the fragment size was closer to 100 bp. We did a whole exome capture of regular TruSeq (it was a while ago). Maybe an oligo pulldown like IDT Lockdown probes? I don't have experience with those specific oligos but based on other pulldown approaches using limited baits the off-target rate will be very high but the coverage of the regions of interest will also be high since it is such a small region.