ABSTRACTThe genome of Bordetella pertussis is complex, with high GC content and many repeats, each longer than 1,000 bp. Short-read DNA sequencing is unable to resolve the structure of the genome; however, long-read sequencing offers the opportunity to produce single-contig B. pertussis assemblies using sequencing reads which are longer than the repetitive sections. We used an R9.4 MinION flow cell and barcoding to sequence five B. pertussis strains in a single sequencing run. We then trialled combinations of the many nanopore-user-community-built long-read analysis tools to establish the current optimal assembly pipeline for B. pertussis genome sequences. Our best long-read-only assemblies were produced by Canu read correction followed by assembly with Flye and polishing with Nanopolish, whilst the best hybrids (using nanopore and Illumina reads together) were produced by Canu correction followed by Unicycler. This pipeline produced closed genome sequences for four strains, revealing inter-strain genomic rearrangement. However, read mapping to the Tohama I reference genome suggests that the remaining strain contains an ultra-long duplicated region (over 100 kbp), which was not resolved by our pipeline. We have therefore demonstrated the ability to resolve the structure of several B. pertussis strains per single barcoded nanopore flow cell, but the genomes with highest complexity (e.g. very large duplicated regions) remain only partially resolved using the standard library preparation and will require an alternative library preparation method. For full strain characterisation, we recommend hybrid assembly of long and short reads together; for comparison of genome arrangement, assembly using long reads alone is sufficient.DATA SUMMARYFinal sequence read files (fastq) for all 5 strains have been deposited in the SRA, BioProject PRJNA478201, accession numbers SAMN09500966, SAMN09500967, SAMN09500968, SAMN09500969, SAMN09500970A full list of accession numbers for Illumina sequence reads is available in Table S1Assembly tests, basecalled read sets and reference materials are available from figshare: https://figshare.com/projects/Resolving_the_complex_Bordetella_pertussis_genome_using_barcoded_nanopore_sequencing/31313Genome sequences for B. pertussis strains UK36, UK38, UK39, UK48 and UK76 have been deposited in GenBank; accession numbers: CP031289, CP031112, CP031113, QRAX00000000, CP031114Source code and full commands used are available from Github: https://github.com/nataliering/Resolving-the-complex-Bordetella-pertussis-genome-using-barcoded-nanopore-sequencingIMPACT STATEMENTOver the past two decades, whole genome sequencing has allowed us to understand microbial pathogenicity and evolution on an unprecedented level. However, repetitive regions, like those found throughout the B. pertussis genome, have confounded our ability to resolve complex genomes using short-read sequencing technologies alone. To produce closed B. pertussis genome sequences it is necessary to use a sequencing technology which can generate reads longer than these problematic genomic regions. Using barcoded nanopore sequencing, we show that multiple B. pertussis genomes can be resolved per flow cell. Use of our assembly pipeline to resolve further B. pertussis genomes will advance understanding of how genome-level differences affect the phenotypes of strains which appear monomorphic at nucleotide-level.This work expands the recently emergent theme that even the most complex genomes can be resolved with sufficiently long sequencing reads. Additionally, we utilise a more widely accessible alternative sequencing platform to the Pacific Biosciences platform already used by large research centres such as the CDC. Our optimisation process, moreover, shows that the analysis tools favoured by the sequencing community do not necessarily produce the most accurate assemblies for all organisms; pipeline optimisation may therefore be beneficial in studies of unusually complex genomes.
Nanopype: a modular and scalable nanopore data processing pipeline