Because HPV has such a small and relatively simple genome (8,000 bp encoding 8 genes) it is possible to screen for genetic variation that may underly carcinogenesis. A team from the National Cancer Institute (NCI) has just published the sequencing of over 5,570 human cell and tissue samples – “the largest case-control study to date of variant sublineage risk of precancer and cancer” .
The work uses the methods published in Cullen et al., 2015. This is an Ion Torrent AmpliSeq library prep, which uses a panel of 47 overlapping amplicons ranging in size from 181 bp to 375 bp, amplifed as two overlapping primer pools. 1 ul of partially purified total DNA from exfoliated cervical cells is used as the template for each pooled PCR with two rounds of SPRI-bead cleanup to remove input DNA and unincorporated primers from the amplicons.
Another recent paper I’ve been talking to people about is the SLIMamp PCR method described in Schenk et al 2017. This is a neat trick that enables PCR of overlapping amplicons without the amplification being dominated by PCR artefacts. Read the paper…it’s a good one.
A potential method for field testing of HPV via WGS
Upon reading the Cell HPV paper I did wonder if the group might look into SLIMamp. An even better amplification of the SLIMamp technology might be to combine it with Twist DX’s TwistAMP chemistry to make a single-tube PCR assay ready for WGS. That would be about the most field portable amplification method I can imagine.
I’ll be writing this up over the next couple of days in more detail…
The Cell paper discusses the importance of APOBEC3 in fighting HPV infection. The authors report how women with benign infections have mutations in HPV E7 that impair the virus’s ability to survive in human cells, and are possibly the reason the infection is benign.
APOBEC3 works by inducing cytidine deamination thereby causing hypermutation. APOBECs were presented by Ted Davis from NEB, as a possible alternative to the harsh bisulfite chemistry used in Methylation sequencing.