Jared Simpson: Signal analysis using nanopolish

Jared Simpson, Principal Investigator at the Ontario Institute for Cancer Research is well known amongst the nanopore community having developed nanopolish – one of the foremost tools for the analysis of nanopore data. As Jared explained, nanopolish is essentially a suite of tools for working with signal-level nanopore data and includes consensus calling, methylation detection, reference-based SNP calling and signal alignment. Jared kicked off his presentation by discussing the use of nanopolish for the detection of base modifications. He explained that this is a particularly exciting area as nanopore sequencing allows direct detection of methylated bases without the need for chemical treatment such as bisulfite conversion. Much of the early work was done in collaboration with Winston Timp, who developed the method in which training data is generated to inform the nanopolish calling. This proof of principle work was published in nature methods in 2017. More recently, as part of the nanopore human genome sequencing project the team used nanopolish to call methylated based with high concordance to bisulfite sequencing. Jared mentioned that a lot of research is now taking place in this area and pointed out some ongoing work studying imprinting. Nanopolish also allows the detection of other nucleotide analogues, including synthetic bases such as BrdU. This work is being done in collaboration with Conrad Nieduszynski at University of Oxford. They have shown that it is possible to detect BrdU in replicating yeast cells to identify origins of replication across the yeast chromosome.

Jared then moved on to consensus calling. The tool uses a hidden Markov model (HMM) to examine the raw data for possible improvements to the consensus sequence. In collaboration with Nick Loman and Josh Quick, Jared has used nanopolish to calculate consensus sequences from a mock microbial community, containing 8 bacterial and 2 yeast genomes. When used on reads obtained using PCR-based library preparation, nanopolish (after miniasm and 2 rounds of Racon polishing) significantly improved assembly accuracy. For native DNA, Jared noted that E. coli and Salmonella concensus calling accuracy didn’t improve as drastically as for other species. This is due to the high level of methylation in these two organisms. In light of this, Jared built methylation aware-polishing into nanopolish, which significantly improves the consensus calling of methylated DNA. Closing his presentation, Jared shared data generated just this week, on the use of nanopolish with data obtained by combining the R9 and R10 pores, which revealed over 99.9% consensus accuracy.