Strategic collaboration between two organisations represents largest nanopore sequencing project to date, capitalising on recent developments bringing ultra-high yields with PromethION

GrandOmics, a leading sequencing company in China, has announced a populational-scale project, dbSV100k, to understand the impact of structural variation on human disease. The project aims to sequence 20,000 human genomes in 2019, with a goal of 50,000 by the end of 2020 and total of 100,000 by the end of 2021. This uses high-throughput nanopore sequencing on the PromethION device and the project will explore viable opportunities for the development of clinical applications, with a view to ultimately providing clinical genetic diagnosis services in the future.

This research project aims to gain a complete understanding of genetic variations associated with human health and disease. Oxford Nanopore has entered into a Memorandum of Understanding with GrandOmics in support of their plan to build genome medicine services at an affordable price with high-quality that is accessible for anyone, anywhere.

The highest yields, with long reads

The performance of PromethION, the newest nanopore sequencing device, has now accelerated sufficiently to allow >7Tb on human samples using a complete set of 48 flow cells – the equivalent of 86Gb of real time sequencing data per hour, or ~1 human genome at 30X coverage.

PromethION, as with other nanopore sequencers, sequences the complete nucleic acid fragment and therefore provides very long reads – the current record is 2.3Mb in a single read and this represents the full fragment rather than multiple repeat passes of a smaller fragment.  With real time data and modular flow cells, the performance of the technology has developed while flow cell costs have remained the same. Flow cells now deliver ultra-high yields, and the latest R10 nanopore has delivered Q50 (99.999% consensus accuracy) in a small genome in internal company experiments. R10 is now being trialled by GrandOmics.

GrandOmics: delivering high throughput nanopore genomics

To date, GrandOmics has sequenced more than a thousand human genome samples (G1k) and several hundreds of plants, animals, and microbial genomes using nanopore technology, servicing a network of over a thousand customers throughout the world. This has resulted in new discoveries in genome science and genomic medicine, in more than 50 publications for the last two years. This includes publications using nanopore technology, with more prospect papers in the near future.

This high-throughput infrastructure will be expanded to deliver dbSV-100K.

“GrandOmics’s mission is to provide innovative and precise genomics solutions for disease research and diagnosis. We have been running our PromethION devices to full capacity, and have developed our own bioinformatics pipelines to drive breakthrough projects to achieve new standards.” Said Mr. Depeng Wang, CEO of GrandOmics.

Dr. Gordon Sanghera, CEO of Oxford Nanopore said: “We are glad to form a strategic and ongoing collaboration with GrandOmics to support genomic medicine and personalized medicine for China. At Oxford Nanopore last week, the PromethION 48 has achieved 7.3Tb of data using a single set of 48 flow cells, and our development pathway offers higher future capacity. We are excited to see GrandOmics drive innovative solutions for offering accessible ‘de novo personal genomes’ to customers using high-throughput PromethION sequencing.”

“This is so far the largest high-throughput nanopore sequencing project in the world, and is the outcome of extensive effort between two parties in developing a strategic alliance for the last several months. This is achieved by a shared vision to create an opportunity to revolutionize genome medicine in the UK, China and the rest of the world.” Min S. Park, Director of GrandOmics Institute.

About GrandOmics

GrandOmics is a world-leading sequencing company in Beijing, China. It was one of the earliest to apply nanopore technology for commercial DNA sequencing in the world, and also the first in China and the second in the world to be certified to provide PromethION sequencing services using Nanopore technology.

GrandOmics provides comprehensive solutions for genome science and genomic medicine. The company aims to utilise advanced sequencing-based solutions for rare genetic diseases, complex diseases, microbial diseases, and cancer, by integrating nanopore long reads and optical mapping to detect and characterize complex mutations in clinical samples of patient genomes. GrandOmics has been establishing one of the world’s largest nanopore long-reads based structural variation databases (www.dbsv.com).

NextOmics, a subsidiary company of GrandOmics, has one of the most complete third-generation sequencing, optical and 3D mapping platforms. NextOmics delivers genome assemblies and analysis pipelines for animal, plant, microbial and human genomes, in research areas such as transcriptomics, metagenomics and epigenetics.

About Oxford Nanopore 

Oxford Nanopore Technologies aims to disrupt the paradigm of biological analysis by making high performance, novel DNA/RNA sequencing technology that is accessible and easy to use. Our goal is to enable the genetic analysis of any living thing, by any person, in any environment.

Our novel, electronics-based DNA/RNA sequencing technology is being used in more than 80 countries, for a range of biological research applications and is also being explored beyond research.

Oxford Nanopore’s proprietary technology is fully scalable for any requirement. Small formats such as Flongle address the need for on-demand, rapid, smaller tests or experiments, and can be used in labs or in the field. The pocket-sized MinION is a powerful and portable sequencing device that can deliver high volumes of long read sequence data. The benchtop GridION X5 can run up to five MinION Flow Cells at a time, on-demand, for larger genomics projects. The recently launched PromethION is the largest format for nanopore sequencing, designed to offer on-demand use of up to 24 or 48 flow cells – each of which can offer more than 100Gb of sequencing data in field.

The first >2Mb continuous DNA read sequence has been reported by Oxford Nanopore sequencing technology, which is regarded as another giant leap in the development of Nanopore sequencing. The result was published on BioRxiv by a team at the University of Nottingham, led by Alex Payne, etc.^[1]

The output data of NanoporeMinION sequencing is in fast5 format, which can be further converted into fastq format through the base-calling procedure. Previously, the MinKNOW was the conventional choice for MinION base-calling. But Alexander Payne found that the MinKNOW may interrupt long reads by error, while they eliminated this “bug”, the length of reads can achieve over 2Mb.

Why long reads?

Traditional short-read DNA sequencing technologies (also known as the Next-Generation Sequencing, NGS) may provide data about the small fragments of genomic DNA sequences, and it is a great challenge for researchers to assemble these large number of small pieces into a complete genome or dataset.

Nanopore sequencing technology has significant advantages in sequencing read length, including greatly improve the continuity of genome assembly and overcome the problem that caused by complex repeat sequences or structural variation which beyond the capabilities of short-read sequencing. The article recently published on Nature Biotechnology about human Y chromosome centromere sequences achieved by Nanopore sequencing have emphasized the abilities of Nanopore sequencing in solving complex repetitive regions. In addition, the identification of complex tandem rearrangement in the nematode genome and structural variations in the drosophila genome are all outstanding applications of Nanopore long reading sequencing. (For more details, see the “extended reading” at the end of this article)

As the first third-generation sequencing company with the Nanopore sequencing platform in China, NextOmics Biosciences have obtained a great amount of excellent data. Here we present you some results.

Case one: Assembly of an insect’s genome by Nanopore sequencing

The size of the insect genome was estimated to be ~ 330Mb based on k-mer analysis

Fig. 1 K-mer analysis

Qualified sample DNA was extracted, and 30Gb of third-generation data were sequenced on the Oxford Nanopore GridlON X5 platform, with a maximum reading length of 270Kb, and reads N50 length was 26.8kb. Long read length is a prerequisite for more accurate genome assembly.

Fig. 2 Distribution of read length

Genome assembly applying a variety of software and select the optimal scheme. The ultra-long read Nanopore sequencing matching super-computing platform enables genome assembly to be more continuous and faster. In this case, Contig N50 can be >7mb, which has reached the high-quality assembly level as insect model animal fruit flies.

Table1 The assembly results

The assembled genome was compared with the insect genome database by BUSCO to assess the integrity of the assembly of conservative genes, and the entire genome was reflected by indirect measurement f. The results show that after Nanopolish+Pilon (* 2) correction, BUSCO evaluation can reach ~ 98% and genome assembly integrity is good.

Table2 BUSCO assessment

Case 2: Ultra-long sequencing data from an animal

Nanopore ultra-long sequencing can achieve ultra-long reading length. Based on its unique transposase library, DNA sequencing library containing ultra-long fragments, and the ultra-long DNA sequence can be obtained by Nanopore sequencing. Ultra-long sequences will greatly facilitate the de novo assembly of genome and the identification of complex structural variations (SVs) of chromosomes.

Fig. 3 Process of Ultra-long library construction

NextOmics Bioscience conducted ultra-long library construction and sequencing on the blood of a mammal based on Nanopore sequencing platform. The reads N50 of multiple libraries were longer than 70kb, and the longest read length was more than 1Mb.

Fig. 4 Library reads N50 (partial)   

   Fig. 5 Distribution of read-length in a library

[1]Payne, A., Holmes, N., Rakyan, V. & Loose, M. Whale watching with BulkVis: A graphical viewer for Oxford Nanopore bulk fast5 files., doi:10.1101/312256 (2018).

Pacific Biosciences of California, Inc. formally announced a new version of Sequel® Software (V5.1) and a new polymerase on Mar. 7, 2018. Combined, these enhancements increase throughput and the overall performance of Single Molecule, Real-Time (SMRT®) Sequencing for key applications such as de novo assembly, structural variant detection, targeted sequencing, and RNA sequencing (Iso-Seq® method), making genomic research more economical.

With this release the Sequel System can achieve up to 10 Gb per SMRT Cell for genomic libraries, effectively doubling the throughput when using ultra-long inserts (>40 kb) for de novo genome assembly. For targeted and RNA sequencing, customers can achieve up to 20 Gb per SMRT Cell.

For human whole genome sequencing (WGS) studies, the new improvements support sensitive detection of structural variants with as little as 5- to 10-fold coverage per individual. As a result, customers can now complete low-cost WGS studies in thousands of individuals using fewer SMRT Cells.

For long amplicons (>3 kb), the new polymerase increases the number of high-quality sequences per SMRT Cell, reducing costs for HLA sequencing and other targeted applications. Further, software enhancements for multiplexed samples simplify the analytical workflow.

Appendix: Based on PacBio SMRT technology, NectOmics has assembled hundreds of genomes and authored mature process for full-length transcriptome, some published papers as follow:

Classic NextOmics genome-assembling projects

Classic NextOmics Full-length Transcriptome projects based on the Third Generation Sequencing Technology