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!
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!
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