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As genome sequencing technology advances, researchers are increasingly exploring emerging tools for a host of potential clinical applications. Andrew Beggs of the University of Birmingham is one of those pioneers. A surgeon as well as a researcher in cancer genetics, Beggs recently described several applications of advanced sequencing technology that are widely used in research but could, in the future, be applied in the clinic as well.*
Organ and stem cell transplantation, for example, are life-saving treatments for a variety of illnesses, including cancers and immune diseases. Approximately 140,000 solid organ transplants are carried out around the globe each year, as well as more than 50,000 stem cell transplants.
One of the critical steps in matching donor organs to patients is accurate human leukocyte antigen (HLA) typing. The HLA complex on chromosome 6 contains the most polymorphic genes in the human genome. HLA class I and II genes define specific adaptive immune responses and genetic variation in these genes is associated with susceptibility to autoimmune and infectious diseases. These genes also have a major role in transplantation and immunology.
Currently, HLA genes are characterized using Sanger or next-generation sequencing of a limited amplicon repertoire or labeled oligonucleotides for allele-specific sequences. But HLA typing is difficult for several reasons, including that the locus is highly polymorphic and is co-dominantly inherited. Plus, these sequencing techniques are expensive.
Beggs’ lab has been testing nanopore-based HLA typing using the portable MinION platform from Oxford Nanopore Technologies. This approach provides high resolution—4-field (8-digit)—while exploiting the inherent advantages of long reads. Compared to previous PCR assays, which used 96-well plates, Beggs says this is a “single-tube assay with a 150-minute turn-around.”
The nanopore assay costs about $100, with just 60 ng DNA required for the PCR reaction. Post-PCR, ligation library preparation is performed (nanopore library prep kit SQK-LSK109), 12-sample multiplexed sequencing is then carried out on a single MinION flow cell. The Beggs lab base-calls 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.”
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.
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.”
*Nanopore Community Meeting, hosted by Oxford Nanopore Technologies; New York: December 5-6, 2019.
Oxford Nanopore Technologies products are currently for research use only.