Ratatosk – Hybrid error correction of long reads enables accurate variant calling and assembly

Abstract

Motivation: Long Read Sequencing (LRS) technologies are becoming essential to complement Short Read Sequencing (SRS) technologies for routine whole genome sequencing. LRS platforms produce DNA fragment reads, thousands to millions bases long, allowing the resolution of numerous uncertainties left by SRS reads for genome reconstruction and analysis. In particular, LRS characterizes long and complex structural variants undetected by SRS due to short read length. Furthermore, assemblies produced with LRS reads are considerably more contiguous than with SRS while spanning previously inaccessible telomeric and centromeric regions. However, a major challenge to LRS reads adoption is their much higher error rate than SRS of up to 15%, introducing obstacles in downstream analysis pipelines. Results: We present Ratatosk, a new error correction method for erroneous long reads based on a compacted and colored de Bruijn graph built from accurate short reads. Short and long reads color paths in the graph while vertices are annotated with candidate Single Nucleotide Polymorphisms. Long reads are subsequently anchored to the graph using exact and inexact k-mer matches to find paths corresponding to corrected sequences. We demonstrate that Ratatosk can reduce the raw error rate of Oxford Nanopore reads 6-fold on average with a median error rate as low as 0.28%. Ratatosk corrected data maintain nearly 99% accurate SNP calls and increase indel call accuracy by up to about 40% compared to the raw data. An assembly of the Ashkenazi individual HG002 created from Ratatosk corrected Oxford Nanopore reads yields a contig N50 of 43.22 Mbp and less misassemblies than an assembly created from PacBio HiFi reads. Availability: https://github.com/DecodeGenetics/Ratatosk Contact: guillaume{dot}holley{at}decode{dot}is

Telomere length de novo assembly of all 7 chromosomes and mitogenome sequencing of the model entomopathogenic fungus, Metarhizium brunneum, by means of a novel assembly pipeline

“…All assemblers were run with default parameters (flagging raw or corrected reads depending on read input, Raven was run with the --weaken flag when corrected reads were used). Additional Flye assemblies were performed using both Canu and NECAT self-corrected read sets and an additional short-read corrected read set corrected with Ratatosk version 0.1 [49], in order to assess read correction strategy performance. The Ratatosk corrected reads were Canu trimmed using the same settings as for the FMLRC corrected read set.…”

GraphUnzip: unzipping assembly graphs with long reads and Hi-C

“…Assembling the full dataset with hifiasm would yield an oversized assembly (258 Mb without overlaps). Low-accuracy long reads (ONT or PacBio CLR) were corrected with the Illumina reads using Ratatosk [17] to generate high-accuracy long reads, with default parameters. These corrected long reads were assembled with Flye using the same parameters as for HiFi assemblies.…”

Telomere Length De Novo Assembly of all 7 Chromosomes and Mitogenome Sequencing of the Model Entomopathogenic Fungus, Metarhizium Brunneum, by Means of a Novel Assembly Pipeline

“…All assemblers were run with default parameters ( agging raw or corrected reads depending on read input, Raven was run with theweaken ag when corrected reads were used). Additional Flye assemblies were performed using both Canu and NECAT self-corrected read sets and an additional short-read corrected read set corrected with Ratatosk version 0.1 (40), in order to assess read correction strategy performance. The Ratatosk corrected reads were Canu trimmed using the same settings as for the FMLRC corrected read set.…”

“…Different isolates (variants) of the same species have been found to vary greatly in their phenotypes (16), but due to the relatively small number of isolates sequenced, the extent of genomic variation between strains is poorly understood. Owing to their genomes having multiple chromosomes that contribute to their relatively large genome sizes (30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41)(42)(43)(44)(45) in comparison to bacterial microbes (around 5 Mb), de novo genome assemblies of Metarhizium spp. using rst generation sequencing is very costly, and second-generation sequencing results in assemblies that are highly contiguous, falling apart around repeat rich and homologous regions of the genome.…”