Oxford nanopore long-read sequencing enables the generation of complete bacterial and plasmid genomes without short-read sequencing

Abstract

IntroductionGenome-based analysis is crucial in monitoring antibiotic-resistant bacteria (ARB)and antibiotic-resistance genes (ARGs). Short-read sequencing is typically used to obtain incomplete draft genomes, while long-read sequencing can obtain genomes of multidrug resistance (MDR) plasmids and track the transmission of plasmid-borne antimicrobial resistance genes in bacteria. However, long-read sequencing suffers from low-accuracy base calling, and short-read sequencing is often required to improve genome accuracy. This increases costs and turnaround time.MethodsIn this study, a novel ONT sequencing method is described, which uses the latest ONT chemistry with improved accuracy to assemble genomes of MDR strains and plasmids from long-read sequencing data only. Three strains of Salmonella carrying MDR plasmids were sequenced using the ONT SQK-LSK114 kit with flow cell R10.4.1, and de novo genome assembly was performed with average read accuracy (Q > 10) of 98.9%.Results and DiscussionFor a 5-Mb-long bacterial genome, finished genome sequences with accuracy of >99.99% could be obtained at 75× sequencing coverage depth using Flye and Medaka software. Thus, this new ONT method greatly improves base-calling accuracy, allowing for the de novo assembly of high-quality finished bacterial or plasmid genomes without the need for short-read sequencing. This saves both money and time and supports the application of ONT data in critical genome-based epidemiological analyses. The novel ONT approach described in this study can take the place of traditional combination genome assembly based on short- and long-read sequencing, enabling pangenomic analyses based on high-quality complete bacterial and plasmid genomes to monitor the spread of antibiotic-resistant bacteria and antibiotic resistance genes.

Do we still need Illumina sequencing data?: Evaluating Oxford Nanopore Technologies R10.4.1 flow cells and v14 library prep kits for Gram negative bacteria whole genome assemblies

“…We investigated the difference in read quality metrics between RBKv10, RBKv14, and LSKv14 library preparation kits, and found that read Q-scores were highest with LSKv14 followed by RBKv14, but read length distribution was best with RBKv14. Both LSKv14 and RBKv14 had better read quality metrics than RBKv10, which agree with other studies 6,11,17,18 . While LSKv14 had higher Q-scores, the read length distribution for RBKv14 had a better representation of longer reads in the RBKv14 datasets, whereas the LSKv14 read size was skewed towards shorter reads, which has been observed previously with older kit/flowcell versions (SQK-LSK108 & SQK-RAD002 on R9.4.1 flow cells) 12,13 .…”

“…Genome contiguity becomes important when considering the mechanisms and drivers of antimicrobial resistance in bacterial pathogens. Plasmids are common vectors for horizontal gene transfer, and obtaining complete plasmid sequences is the most effective means to detect the coexistence of antimicrobial resistance genes on the same plasmid 6 , as well as the potential for that plasmid to transfer to other strains, species, or genera 7 . Long-read sequencing is usually essential to discern complete plasmid structures 6 , which often contain repetitive sequences that are difficult to resolve with short-read sequencing data alone 8 .…”

“…Plasmids are common vectors for horizontal gene transfer, and obtaining complete plasmid sequences is the most effective means to detect the coexistence of antimicrobial resistance genes on the same plasmid 6 , as well as the potential for that plasmid to transfer to other strains, species, or genera 7 . Long-read sequencing is usually essential to discern complete plasmid structures 6 , which often contain repetitive sequences that are difficult to resolve with short-read sequencing data alone 8 . Bacterial genome assemblies have been preferentially constructed with short-read-only data but are shifting to using short reads to polish long-read assemblies 9,10 .…”

“…Given the continual advances in ONT long-read sequencing, the need for additional short-read data for genome polishing of long-read assemblies is called into question. Several studies have examined bacterial genome assembly quality using the Ligation Sequencing Kit v14 (LSKv14) 6,17–19 , but no studies to date have examined assembly quality using the Rapid Barcoding Kit v14 (RBKv14). The objective of this project was to determine how much ONT read coverage produced by the Rapid Barcoding Kit v14 is necessary to generate high-quality hybrid and long-read-only genome assemblies.…”

A recently collectedXanthomonas translucensisolate encodes TAL effectors distinct from older, less virulent isolates

“…Nearly 80 Xt genomes are publicly available, and many of these have been assembled using longread sequencing, including PacBio and Oxford Nanopore Technology (ONT), which may or may not be corrected with short-read sequencing such as Illumina [10][11][12]. While long-read sequencing is useful in detecting large polymorphisms or structural variation, the base sequence accuracy is lower than with short-read sequencing [13].…”