NIH Center for Alzheimer’s and Related Dementias publication shows comprehensive, high accuracy sequencing approach, using new Oxford Nanopore sequencing chemistry

In a new publication in Nature Methods, a team contributing to the National Institutes of Health (NIH) Center for Alzheimer’s and Related Dementias (CARD) share an end-to-end pipeline that produces state-of-the-art single nucleotide polymorphism (SNP), structural variant and methylation calls, while being cost-effective and scalable for large projects.

Oxford Nanopore sequencing technology has been used by a team led by researchers at the National Institutes of Health Center for Alzheimer's and Related Dementias (CARD), the University of California, Santa Cruz and the National Cancer Institute, to develop a protocol for highly accurate whole human genome sequencing, at scale, that provides a comprehensive view of haplotype-resolved variation and methylation. In a new Nature Methods paper, the team describe how this makes large-scale, long, native DNA sequencing projects feasible due to the lower cost and higher throughput of Oxford Nanopore’s PromethION when compared with alternative sequencing methods.

According to the paper, “cost and scalability have remained prohibitive barriers to the use of long-read sequencing in population-scale studies...Here, we show that it is possible to achieve state-of-the-art small and structural calling performance using only [Oxford Nanopore] reads produced by a single flow cell at high throughput.”

The results with Oxford Nanopore’s Q20+ chemistry included a SNP F1-score of 0.998, and an SV F1-score of 0.978.

The study showed that the latest R10.4.1 flow cells significantly improve reference-based indel calling, which is vital for small variant calling, achieving an F1-score of 0.996 in regions not containing homopolymers or tandem repeats. The paper also describes how with nanopore-based phasing, it is possible to combine and phase small and structural variants at megabase scales, all of which combines to give the clearest picture yet of the whole genome. The study protocol is currently being used to sequence thousands of brain-based human genomes as a part of the NIH CARD initiative.

Base modifications provide critical insights into many biological questions and the paper states how methylation calls “were highly concordant with the standard bisulfite sequencing”, but in addition they could produce reliable haplotype-resolved methylation calls for even greater insight. The methylation data is produced during a standard sequencing run and not as the result of a separate process.

The team used Oxford Nanopore’s high-throughput sequencing device, the PromethION 48, which is capable of sequencing over 4900* genomes per year delivering scalability for large projects. Combined with the latest chemistry, Kit 14 and R10.4.1 flow cells, Oxford Nanopore now delivers the most complete and accurate genomic data, at scale. This combines very high single-molecule accuracy with the ability to reach all parts of the genome and characterise all types of genetic variation, through the ability to sequence any length fragments of native DNA/RNA.

The CARD initiative supports basic, translational, and clinical research on Alzheimer’s disease and related dementias. The CARD project aims to address the significant unmet needs in Alzheimer’s research by identifying the underlying mechanisms of disease and ageing, and to support the development of new interventions to delay or prevent disease progression.

Gordon Sanghera, CEO, Oxford Nanopore Technologies, commented:

“We are delighted to see this latest work from the NIH CARD team, whose approach demonstrates breakthrough accuracy with Oxford Nanopore’s newest Q20+ chemistry and the R10 nanopore, whilst using the same device. This shows that Oxford Nanopore now delivers comprehensive and accurate genomic data, at scale. We congratulate all the fantastic scientists that have contributed a huge amount to this significant project.

It’s fantastic to see this protocol being used in the CARD programme’s work in Alzheimer’s disease. As the paper states, a substantial part of the variation in the human genome is not accessible with short-reads, so we know that what’s missing matters. We look forward to seeing the outcomes and impact of the CARD programme in due course.”

* 4,900 genomes per year assumes sequencing one genome per flow cell across all 48 flow cells, twice a week for 52 weeks a year.  Users may choose to sequence two genomes per flow cell for twice the capacity.