Hybracter: Enabling Scalable, Automated, Complete and Accurate Bacterial Genome Assemblies

Abstract

Improvements in the accuracy and availability of long-read sequencing mean that complete bacterial genomes are now routinely reconstructed using hybrid (i.e. short- and long-reads) assembly approaches. Complete genomes allow a deeper understanding of bacterial evolution and genomic variation beyond small nucleotide variants (SNVs). They are also crucial for identifying plasmids, which often carry medically significant antimicrobial resistance (AMR) genes. However, small plasmids are often missed or misassembled by long-read assembly algorithms. Here, we present Hybracter, method for fast, automatic and scalable recovery of near-perfect complete bacterial genomes using a long-read first assembly approach. We compared Hybracter to existing automated hybrid assembly tools using a diverse panel of samples with manually curated ground truth reference genomes. We demonstrate that Hybracter is more accurate and faster than the existing gold standard automated hybrid assembler Unicycler. We also show that Hybracter with long-reads only is comparable to hybrid methods in recovering small plasmids.

How low can you go? Short-read polishing of Oxford Nanopore bacterial genome assemblies

“…A combination of Pypolca-careful and Polypolish depending on estimated short-read depth has been implemented in our automated assembly tool Hybracter from v0.7.0 [ 15 ]. Depth is estimated inside Hybracter with Seqkit [ 23 24 ] based on the chromosome size parameter estimate provided by the user.…”

“…S1). We have incorporated the above recommendations into Hybracter’s [ 15 ] polishing logic from v0.7.0 and show that is it possible to consistently recover automated perfect genome assemblies when short-read depth is 25× or higher (Fig. S8).…”

“…ONT-only assemblies were created using Trycycler v0.5.4 and reoriented to begin with a consistent starting position using Dnaapler [ 14 ] v0.7.0 as described by us in previous studies using these data [ 1 15 ]. The number of errors in the ONT-only assemblies (compared to our previously published method for generating robust and curated bacterial reference genome assemblies [ 16 ]) ranged from 0 to 18 total errors, for a total of 37 errors across all nine genomes.…”

Bacterial genome sequences of uncharacterized Chitinophaga species isolated from the International Space Station

“…Hybrid genome assembly was carried out using Unicycler ( 11 ) v0.5.0 with default parameters guided by a long-read-only assembly using Flye ( 12 ) v2.9.2 and the --nano-hq parameter. Resulting assemblies were short-read polished using Polypolish ( 13 ) v0.5.0 and PyPolca ( 14 ) v0.2.0. Annotations were performed using Bakta ( 15 ) v1.8.2 in compliant mode using its full database (5.0).…”