Oxford Nanopore MinION Used To Produce Most Complete Human Genome

By Bio-IT World Staff 

January 29, 2018 A paper published today in Nature Biotechnology (doi:10.1038/nbt.4060), reports Oxford Nanopore sequencing resulting in the most complete human genome ever assembled with a single technology.

The work offers evidence that portable, biological nanopore sequencers could be used to sequence, assemble, and provisionally analyze structural variants and detect epigenetic marks, in point-of-care human genomics applications in the future, the study authors, led by Matt Loose and Nick Loman, write.

Using a MinION, the researchers generated 91.2 Gb of sequence data, representing ∼30× theoretical coverage. “The final assembled genome was 2,867 million bases in size, covering 85.8% of the reference,” the authors write in the paper abstract. “Assembly accuracy, after incorporating complementary short-read sequencing data, exceeded 99.8%.”

The authors are from UC Santa Cruz; the U.S. National Human Genome Research Institute (NHGRI); the University of Nottingham, University of Birmingham, and University of East Anglia in the U.K.; and the University of Salt Lake City, University of British Columbia, and the Ontario Institute for Cancer Research in Toronto. The paper lists seven lead authors who contributed equally to the paper.

"The ability to get long reads is one of the strengths of this technology, and as a result this is the most contiguous human genome assembly ever done," said co-first author Miten Jain, a postdoctoral researcher in biomolecular engineering at UC Santa Cruz in a press release.

MinION Improvements

Oxford Nanopore’s MinION sequencer was first used to sequence and assemble microbial genomes, but a new protein pore that Oxford Nanopore announced in September 2016 along with improvements in library prep, sequencing speed, and control software increased throughput. “[S]o we hypothesized that whole-genome sequencing (WGS) of a human genome might be feasible using only a MinION nanopore sequence,” the authors write.

Five laboratories used 53 MinION flowcells to sequence and assemble reference human genome for GM12878 from the Utah/CEPH pedigree, using MinION R9.4 1D chemistry. The sequencing was done without PCR to preserve epigenetic modifications such as DNA methylation, and produced ultra-long reads up to 882 kb in length.

The increased single-molecule read length enabled the authors to analyze regions of the human genome that were previously intractable with state-of-the-art sequencing methods, they write. “For example, we were able to phase megabase regions of the human genome in single contigs, to more accurately estimate telomere lengths, and to resolve complex repeat regions.”

"About 8 percent of the human genome has yet to be assembled, mostly in long, complex regions with lots of repetitive DNA sequences. With repetitive sequences, if you only have short reads it's hard to piece them together, and you can't tell how much there is," Jain said in the same press release. "If you can cover that region with a few long reads, however, it's a much easier puzzle to solve."

“If you imagine the process of assembling a genome together is like piecing together a jigsaw puzzle, the ability to produce extremely long sequencing reads is like finding very large pieces of the puzzle, which makes the process far less complex,” said Nick Loman of University of Birmingham, a corresponding author, in the release.

But this proof-of-concept does highlight some remaining challenges for nanopore sequencing. The team had to write custom software and algorithms, called poredb, to track the large number of reads, store each read as an individual file, and enable use of cloud-based pipelines for our analyses.

“Improvements in real-time base-calling are needed to simplify the workflow. More compact and convenient formats for storing raw and base-called data are urgently required, ideally employing a standardized, streaming compatible serialization format such as BAM/CRAM,” the authors wrote 

“We hope that a pocket-size sequencer is going to give us the ability to bring sequencing much closer to the patient,” Loman said in a press release. “At the moment sequencing is quite laborious and occurs in expensively equipped laboratories, but in the future we can imagine sequencing using pocket-size devices in GP surgeries, in clinics, and even in people’s own homes. The ability to sequence and assemble even very large complex genomes may have value one day in diagnostics and monitoring the evolution of diseases such as cancer and a wide range of infections.”