Oxford-Nanopore eBook - 5

carried out on a single MinION flow cell. The Beggs lab basecalls the run in real time with Guppy, and finally HLA assembly
and calling is carried out with HLA*LA, which uses reference
graph assembly. The total workflow takes just 5.5 hours.
Testing this process on 33 reference samples, Beggs and
colleagues found it outperformed current technologies,
with 100% concordance for class I calls, and only one sample
with a second field mismatch. In that case, it turned out that
the nanopore sequencing result was correct. Beggs says
"[Nanopore's] accuracy at the moment outperforms current
state of the art."

C linical whole-genome
sequencing is likely to
transform patient care.

For class II calls, concordance was also 100% for the first field.
Haplotyping was performed using the WhatsHap genomic
variant phasing tool, while runs of homozygosity could also
be called as part of the HLA algorithm used.

Copy That
Another approach Beggs' group is taking is copy number
variation (CNV) calling on the Flongle, which is an adapter
for Oxford Nanopore's MinION or GridION instruments that
enables direct, real-time DNA or RNA sequencing on smaller,
single-use flow cells.
Many human diseases and cancers are caused by germline
CNVs, such as EGFR amplification in lung cancer. Taking
1 µg input DNA from blood or tumor samples, Beggs' lab
performed an 8-hour Flongle run, yielding ~0.05x depth of
coverage of the whole human genome.
Early results from this work on a few colorectal cancer samples,
using Sniffles for structural variation (SV) calling and QDNASeq/
Bioconductor for CNV calling, showed concordance between
the nanopore and short-read whole genome sequencing (WGS)
data. The Birmingham team detected known translocations and
deletions as well as loss of heterozygosity.
"Clinical whole-genome sequencing is likely to transform
patient care," says Beggs. Many patients with advanced
metastatic disease have already had treatment changes due
to WGS findings. However, the workflow of the UK 100,000
Genomes Project (UK 100K GP) is relatively slow-with an
average turnaround time of 4-6 weeks-which Beggs points
out is "too slow for patient care." Short-read WGS, which was
used for the UK 100K GP, also struggles to provide high-quality SV calls due to read length.

Beggs has also considered an approach to clinical WGS and
variant calling using Oxford Nanopore's PromethION sequencing platform. With 3 µg DNA from Genomics England samples,
they performed library prep with the Ligation Sequencing Kit,
followed by 72-hour sequencing runs and a custom bioinformatics pipeline that included alignment (Minimap2), variant
calling with various tools (Clair, Longshot and Sniffles), and
methylation calling (Nanopolish).
So far, a dozen samples have been processed via this pipeline
(48 will be processed in total), with a median flow cell output
of 100 Gbases and a longest read of 1.14 Mbp.
Beggs and his team have observed a reduced output with
very long read lengths, but shearing before library prep
increased the yield. In terms of variant calling, single nucleotide variant (SNV) accuracy was comparable to short-read
sequencing, and many SVs were identified in cancer samples
that were not seen in the short-read WGS data. Typically,
Beggs observed mostly intronic variants. CNVs were "relatively
straightforward" to call on the PromethION data, including
complex CNVs and loss of heterozygosity, with binning
reduced down to 15 kb, and using only the tools QDNAseq
and Bioconductor for calling.
This approach "can detect fusions much more easily at the
DNA level" compared to short-read sequencing, Beggs
says. Fusions were detected with the Sniffles tool, although
Beggs thinks it may be preferable to detect fusions from RNA
sequencing data. Nanopolish and MethplotLib were used to
call methylation. Hypomethylation of MLH1 near its promoter,
as is commonly observed in colorectal cancer, was detected.
(Nanopolish is a suite of tools designed for working with
signal-level nanopore data. It is used for consensus calling,
methylation detection, reference-based SNP calling, and
signal alignment.)
According to Beggs, PromethION provides much higher
resolution "compared to anything else we do" in terms of
methylation calling. It was a "piece of cake" and "in fact we are
going to move all our methylation assays onto PromethION
and Nanopolish. PromethION has the potential to be game
changing," he says, although his team are still in the "beta"
stage of its application. To make the transition to the clinic, he
says, "we need better variant calling tools, a clinical pipeline,
and ISO accreditation." n
*Nanopore Community Meeting, hosted by Oxford Nanopore
Technologies; New York: December 5-6, 2019.

Watch Andrew's full talk

https://nanoporetech.com/andrewbeggs
clinicalomics.com

https://www.nanoporetech.com/andrewbeggs http://www.clinicalomics.com

Oxford-Nanopore eBook

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