Oxford-Nanopore eBook - 9

The results speak for themselves. Iyer's team successfully
captured and sequenced BRCA1 end-to-end in a single,
ultra-long nanopore read of 198 kb. As far as she is aware,
this stands as the record for the longest read generated with
CRISPR-Cas9 enrichment. All of her group's long read
experiments were carried out using Oxford Nanopore's
FLO-MIN 106 cells on the GridION instrument.

Analysis of the different target lengths-spanning tens of
kilobases-determined that good end-to-end enrichment
is seen with the method up to ~100 kb. As there isn't always
enough sample to enable multiple preps and pooling prior
to sequencing, Iyer tested ACME using single-prep libraries,
which showed improved depth of coverage over the
non-ACME libraries.

Going Deep

Fixing Blind spots

Iyer was also interested in improving the depth of
coverage in regions of interest. Pooled libraries helped
but did not fully address the problem, so Iyer decided
to focus on the "background" problem. Was the background DNA competing and inhibiting the sequencing
of the ultra-long fragments? To tackle this, she used
the Circulomics Short Read Eliminator (SRE) Kit on the
CRISPR-Cas9-enriched libraries, prior to preparation for
sequencing. In an enriched sample of MCF 10A prepared
using this method, one 142-kb read was observed,
further bridging the target.

This advance is important, because while cancer genomics
is rapidly advancing, SVs are still often "blind spots," Iyer says.
To date, many thousands of tumors have been sequenced via
next-generation sequencing, enabling the discovery of different signatures and mutation rates across dozens of tumor
types, while revealing insights into clonal structure and tumor
evolution. Several hundred genes have been identified that
can cause cancer if mutated. Those genetic alterations can
vary from single point mutations to larger SVs, which
can affect one or multiple genes.

"

 his stands as the record for
T
the longest read generated with
CRISPR-Cas9 enrichment.

"

The team's next step was to enrich multiple targets, some of
which were below the length cut-off of the SRE Kit, meaning
that they would be removed by the process. To enable the
preservation of this enriched DNA whilst effectively removing
the background DNA, Iyer developed ACME (Affinity-based
Cas9-Mediated Enrichment). This method makes use of the
histidine tag present on the Cas9 enzyme, used to purify the
protein in its production, enabling the capture of Cas-bound
regions on Dynabeads. Iyer showed how libraries prepared
using ACME performed better in terms of depth of coverage,
but reads still did not fully span very large targets.
She then designed a cancer gene panel, targeting multiple
genes of different sizes to test the upper limit of the enrichment method; the genes selected were those where SVs had
been found in whole genome data. With more targets, more
DNA was pulled out using the ACME process. Iyer displayed
alignments showing end-to-end coverage of the 90-kb region
targeting the BRCA2 gene in both cell lines, with depth of
coverage much improved by ACME. For SK-BR-3, 99-fold enrichment, to a depth of coverage of 100x, was achieved with ACME.

Iyer's team is now focusing its efforts on targeted, longread sequencing to achieve the depth needed to identify
rare variants. The use of targeting strategies allows higher-throughput sequencing of key targets, improving depth
of coverage and allowing for the detection of rare alleles. As
targeting avoids having to sequence an entire genome to
generate sufficient depth of coverage,. However, until about
two years ago, Iyer says, there wasn't an effective method of
long-read target enrichment.
In the future, Iyer and her team plan to apply this long-read
method to the second version of their cancer panel, encompassing BRCA1, ERBB2, APAF1 and other Catalogue of Somatic
Mutations in Cancer (COSMIC) genes with evidence of SVs.
She also intends to compare the performance of SV detection
between targeted and whole genome strategies, among
other projects. n
*Nanopore Community Meeting, hosted by Oxford Nanopore
Technologies; New York: December 5-6, 2019.

Watch Shruti's full talk

https://nanoporetech.com/shrutiiyer

clinicalomics.com

9
https://www.nanoporetech.com/shrutiiyer http://www.clinicalomics.com
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