Sample preparation and library information for HPV cervical swabs

[Shamol]

Hello all,

My project involves sequencing the complete genomes of local Human Papillomavirus isolates. The isolates were collected from three kinds of samples:

1. Tissue samples from cancer patients
2. Cervical swabs from cancer patients
3. Cervical swabs from asymptomatic HPV careers

Most of the samples were of type 3 above. I have been trying to sequence some of these genomes using the PCR amplification followed by Sanger sequencing, but very, very few of the samples could be amplified to begin with (presumably because the DNA concentration especially in the swab samples were relatively low). My supervisor suggested I look into NGS as an alternative due to its high resolution.

I'm from Bangladesh, and this technology is virtually unheard of here. I've been spending the past two weeks poring over papers on NGS and applications thereof in virology. I've picked up the general skeletal protocol, but there's only so much you can learn from papers and at some point you need directed expert advice. I'm currently looking for guidance on these three issues related sample preparation:

1. Since the samples I'm working with contain host DNA, should I have a physical enrichment process (e.g. centrifugation and/or DNAse/RNAse treatment to remove unprotected nucleic acid) incorporated in the sample preparation?

2. Given the low quantity of DNA in my samples, there's a chance that the amount of viral DNA would be very low. Is there a particularly high resolution library preparation service which can solve this problem? If so, what would be lowest quantity of DNA required?

3. In the cancerous tissue samples (and cancerous cervical swabs), the HPV genome stays integrated into the host (human) genome, precluding the possibility of physical enrichment. How would the sample preparation work in this case?

Thanks in advance for the replies, I'd appreciate them greatly.

[HESmith]

Your problem is not the sequencing method; it's the inability to amplify your samples by PCR. NGS will not solve that problem, b/c it also requires PCR amplification of low abundance DNA samples. You need to troubleshoot the PCR protocol.

[Shamol]

Thanks for your reply. Are you saying the problem I've been having with trying to amplify DNA with PCR (probably due to low quality of DNA in the original samples) will be there in an NGS platform as well? The whole reason I'm considering NGS is because my supervisor thought this could bypass the need for PCR, which has been really inconsistent with the samples we've been working with. Thanks again for the replies in advance.