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

Thu 6th April 2023

In a preprint released today, 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 the preprint 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.  

Using a single flow cell with the latest version of Oxford Nanopore’s ‘Q20+ chemistry’, the team stated, “we can detect SNPs with F1-score better than ...short read sequencing” and “further, we can discover structural variants with F1-scores comparable to state-of-the-art methods involving [alternative long read sequencing] and trio information (but at lower cost and greater throughput)”. The results with Oxford Nanopore’s R10 flow cells included SNP recall/precision of 0.9979/0.998, SV accuracy 0.9764/0.9835 and significant improvements to indel accuracy to 0.9948/0.9748 in coding regions. The study protocol is currently being used to sequence thousands of brain-based human genomes as a part of the NIH CARD initiative. 

The study showed that the latest R10.4.1 flow cells significantly improve reference-based indel calling, which is vital for small variant calling which, in regions not containing homopolymers or tandem repeats, achieves an F1-score of 0.997. 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. 

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 49001 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 platform. This shows that Oxford Nanopore now delivers comprehensive 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. We congratulate all the fantastic scientists that have contributed a huge amount to this significant project.   

Furthermore 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.”   

The preprint can be accessed in full here.


For more information on the NIH CARD programme, please join this Technology Networks webinar on 'Scalable Nanopore Sequencing for Alzheimer's Research', 26th April, 3.00pm BST. 

1. 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.