Trycycler: consensus long-read assemblies for bacterial genomes

Abstract

While long-read sequencing allows for the complete assembly of bacterial genomes, long-read assemblies contain a variety of errors. Here, we present Trycycler, a tool which produces a consensus assembly from multiple input assemblies of the same genome. Benchmarking showed that Trycycler assemblies contained fewer errors than assemblies constructed with a single tool. Post-assembly polishing further reduced errors and Trycycler+polishing assemblies were the most accurate genomes in our study. As Trycycler requires manual intervention, its output is not deterministic. However, we demonstrated that multiple users converge on similar assemblies that are consistently more accurate than those produced by automated assembly tools.

Polypolish: short-read polishing of long-read bacterial genome assemblies

“…As error-free reference genomes are not available for real read sets, we instead used six isolates for which we had four independent sets of matched Illumina and ONT reads for each isolate ( Figure S8A-B ): A. baumannii J9, C. koseri MINF_9D, E. kobei MSB1_1B, Haemophilus M1C132_1, K. oxytoca MSB1_2C and K. variicola INF345 19 . For each isolate, we produced four long-read-only assemblies (one for each ONT read set) using Trycycler 3 and Medaka 8 ( Figure S8C ). We then polished each assembly with an independent set of Illumina data using each of the short-read polishing tools ( Figure S8D ).…”

“…For each hybrid read set, we performed a long-read-only assembly using Trycycler v0.5.0 3 and Medaka v1.4.3 8 , following the instructions in Trycycler’s documentation ( Figure S8C, Table S5 ). One of the ONT read sets for K. oxytoca MSB1_2C had very low depth (10×) and was therefore not able to yield a high-quality long-read-only assembly, leaving only three assemblies for this genome.…”

“…For bacterial genomes, reads from these platforms are often longer than the largest repeat in the genome, making complete assemblies (one contig per replicon) possible 1 . However, systematic errors in long reads can lead to hundreds of residual errors in long-read-only assemblies of bacterial genomes, most of which are indels in homopolymer sequences 2,3 . When these errors occur in protein-coding sequences, they cause frameshifts in the open reading frame, leading to problems with genome annotation and limiting the utility of long-read-only assemblies 4 .…”

“…Hybrid assembly can be carried out in a short-read-first manner, where long reads are used to scaffold short-read-based contigs into complete genomes, or a long-read-first manner, where short reads are used to correct errors in long-read-based contigs 6 . Given sufficient read depth, long-read-first hybrid assemblies can be more accurate than short-read-first hybrid assemblies, but errors often remain, particularly in repetitive regions of the genome 3 .…”

Complete Genome Sequences of Kinneretia sp. Strain XES5, Shinella sp. Strain XGS7, and Vogesella sp. Strain XCS3, Isolated from Xenopus laevis Skin

“…Reads passing QC were assembled with Trycycler (v0.5.0) ( 21 ) using Raven (v1.6.0) ( 22 ), Flye (v2.9-b1768) ( 23 ), and miniasm (v0.3-r179) ( 24 ). All assembled sequences were successfully circularized by Trycycler with the “reconcile” command.…”