Correcting Modification-Mediated Errors in Nanopore Sequencing by Nucleotide Demodification and in silico Correction

Abstract

The accuracy of Oxford Nanopore Technology (ONT) sequencing has significantly improved thanks to new flowcells, sequencing kits, and basecalling algorithms. However, novel modifications untrained in the basecalling models can seriously reduce the quality. This paper reports a set of ONT-sequenced genomes with unexpected low quality (∼Q30) due to extensive new modifications. Demodification by whole-genome amplification (WGA) significantly improved the quality of all genomes (∼Q50-60) while losing the epigenome. We developed a computational method, Modpolish, for correcting modification-mediated errors without WGA. Modpolish produced high-quality genomes and uncovered the underlying modification motifs without loss of epigenome. Our results suggested that novel modifications are prone to ONT errors, which are correctable by WGA or Modpolish without additional short-read sequencing.

Accurate bacterial outbreak tracing with Oxford Nanopore sequencing and reduction of methylation-induced errors

“…We found no coherent methylation motifs in the literature that would fit the observed pattern. Nevertheless, it has been reported that methylated bases can affect the raw signal in the surrounding region [22]. Thus, we can not determine whether multiple methylation motifs are the cause or if an unknown motif is present.…”

“…Given the notable strides made in direct methylation calling techniques, Oxford Nanopore [22]. Nevertheless, we strongly recommend constantly testing and evaluating reconstructed prokaryotic genomes to avoid erroneous conclusions based on these ambiguous positions introduced by unknown…”

“…The recently introduced research model reduces the ambiguous positions in direct comparison, but still a few remain (see Supplementary Table 4; Researchmodel “res_dna_r10.4.1_e8.2_400bps_sup” available on github.com/nanoporetech/rerio, not yet implemented in MinKnow version 23.07.12, as of 30.11.2023). Further advances that reduce methylation-induced errors for certain specific motifs are being developed, as shown for Listeria monocytogenes [22]. Nevertheless, we strongly recommend constantly testing and evaluating reconstructed prokaryotic genomes to avoid erroneous conclusions based on these ambiguous positions introduced by unknown and not yet considered methylation motifs.…”

The usefulness of Nanopore Sequencing in Whole-Genome Sequencing-Based Genotyping ofListeria monocytogenesandSalmonella entericaserovar Enteritidis

“…For the R10.4.1 flow cells (FLO-MIN114), the Rapid Barcoding Kit (SQK-RBK114.24 kit) was used to construct libraries for the rapid sequencing method, and the Native Barcoding Kit (SQK-NBD114.24) was used to construct libraries for the ligation-duplex method. Nanopore raw signal data were basecalled using Dorado 0.5.0 dna_r9.4.1_e8_sup@v3.3, dna_r10.4.1_e8.2_400bps_sup@v4.2.0, and dna_r10.4.1_e8.2_400bps_sup@v4.3.0 models, with or without modification-mediated error correction using the Modpolish toolkit (8).…”

“…Studies have suggested that base modifications can contribute significantly to base calling errors in ONT sequencing (7,8). Recent developments by ONT, including the introduction of Flowcells (R10.4.1) and Chemistry (SQK-NBD114.24), have demonstrated raw read accuracy exceeding 99.1% (9).…”