Comparison of long-read sequencing technologies in interrogating bacteria and fly genomes

Abstract

The newest generation of DNA sequencing technology is highlighted by the ability to generate sequence reads hundreds of kilobases in length. Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT) have pioneered competitive long read platforms, with more recent work focused on improving sequencing throughput and per-base accuracy. We used whole-genome sequencing data produced by three PacBio protocols (Sequel II CLR, Sequel II HiFi, RS II) and two ONT protocols (Rapid Sequencing and Ligation Sequencing) to compare assemblies of the bacteria Escherichia coli and the fruit fly Drosophila ananassae. In both organisms tested, Sequel II assemblies had the highest consensus accuracy, even after accounting for differences in sequencing throughput. ONT and PacBio CLR had the longest reads sequenced compared to PacBio RS II and HiFi, and genome contiguity was highest when assembling these datasets. ONT Rapid Sequencing libraries had the fewest chimeric reads in addition to superior quantification of E. coli plasmids versus ligation-based libraries. The quality of assemblies can be enhanced by adopting hybrid approaches using Illumina libraries for bacterial genome assembly or polishing eukaryotic genome assemblies, and an ONT-Illumina hybrid approach would be more cost-effective for many users. Genome-wide DNA methylation could be detected using both technologies, however ONT libraries enabled the identification of a broader range of known E. coli methyltransferase recognition motifs in addition to undocumented D. ananassae motifs. The ideal choice of long read technology may depend on several factors including the question or hypothesis under examination. No single technology outperformed others in all metrics examined.

Accumulation of endosymbiont genomes in an insect autosome followed by endosymbiont replacement

“…Multiple Wolbachia genome equivalents have been transferred into D. ananassae We identified at least 4.9 Mbp of sequence as nuclear Wolbachia transfers (nuwts) in the D. ananassae genome (RefSeq: GCF_017639315.1) by assembling long-read sequences from a wAna-cured fly strain (Figure 1A; Table S1). Despite obtaining complete autosome and chromosome X arms, 10 the nuwts were assembled into nine contigs. Alignments between the 1.4 Mbp reference wAna Hawaii genome sequence (GenBank: CP042904.1) 11 and nuwt-containing D. ananassae contigs revealed the integration of an entire bacterial genome at least once, with most portions present at least twice and smaller portions present in three to ten copies (Figure 1A).…”

BLEND: A Fast, Memory-Efficient, and Accurate Mechanism to Find Fuzzy Seed Matches in Genome Analysis

“…To use realistic depth of coverage, we use SeqKit (Shen et al, 2016) to down-sample the original E. coli and D. ananassae reads to 100× and 50× sequencing depth of coverage, respectively. For D. ananassae and E. coli genomes, the reference genomes are the high-quality assemblies generated using the same read sets we use for these genomes (Tvedte et al, 2021). We evaluate BLEND based on two use cases: 1) finding overlapping reads and 2) read mapping to a reference genome.…”

Characterization and simulation of metagenomic nanopore sequencing data with Meta-NanoSim

“…However, the accuracy of these tools would be understandably impacted when applied on ONT reads, especially for lowly represented genomes, because of the variable lengths and relatively high error rates compared to Illumina reads (5 -15% depending on the flowcell chemistry and basecalling algorithm). In addition, ONT sequencing projects on genome, transcriptome, and metagenome, from prokaryotes to eukaryotes, were all reported to have certain problematic reads with gapped or chimeric alignments, likely generated due to library preparation or sequencing artifacts (17,(20)(21)(22)(23)(24)(25). Reference-based abundance estimation using merely primary alignments may further be affected by the presence of these chimeric reads, as well as reads that span the start position of a circular genome.…”