Oxford-Nanopore eBook - 8

New Tools for
Studying Genetic
Variation in
Cancer

Shruti Iyer explains the
advantages of targeted
nanopore sequencing
using long reads and
CRISPR-Cas9.

n cancer genomics, researchers are
"really missing a lot of things by not
looking for them in the right way," says
Shruti Iyer, a PhD candidate in the Genetics
Program at Stony Brook University. In
a recent talk in New York*, Iyer, who is
pursuing her doctoral research in Dr. W.
Richard McCombie's lab at Cold Spring
Harbor Laboratory, described how her
lab is using targeted long-read nanopore
sequencing to study previously undetected
structural variants (SVs) with sensitivity
and specificity of over 95%..
SSVs are DNA variants spanning more than 50 base pairs (bp),
including insertions, deletions, duplications and translocations. Because of their size, SVs can be very disruptive. Among

clinicalomics.com

their achievements, the McCombie group has identified
several thousand variants in the HER-2-amplified breast cell
line SK-BR-3 using long-read DNA and RNA sequencing in
addition to short reads. This study, Iyer says, "completely
highlighted the deficiency of short reads", prompting her
to look at developing targeted long read enrichment
approaches using CRISPR-Cas9.
The CRISPR-Cas9 method of PCR-free, long-read target enrichment begins with dephosphorylation of the ends of the sample
DNA. The CRISPR-Cas9 machinery (including the custom guide
RNA) is then added. The Cas9 nuclease is guided to the sites
flanking the target loci, where it induces double-stranded cuts
in the DNA, excising the region. Sequencing adapters can then
be ligated to these exposed phosphorylated ends, enabling
sequencing of the target regions.
The process has the advantage of dispensing with PCR and can
be used to enrich for very large regions. In an early test, Iyer
enriched the BRCA1 gene in the SK-BR-3 cell line. At the time,
she says, groups using CRISPR-Cas9 enrichment hadn't gone
beyond targeting and sequencing regions of 5-10 kilobases
(kb). Iyer wanted to push the process, aiming for 200 kb.

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