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Long-read assembly has come a long way in the last few years, and there are many good assemblers available, including Canu, Flye, Raven and Redbean. Since bacterial genomes are relatively simple (not too large, not too many repeats), a completed assembly (one contig per replicon) is often possible from a long-read set.
But even the best assemblers are not perfect! They often fail to circularise sequences, either duplicating or omitting sequence at the start/end of a contig. They sometimes produce spurious contigs, e.g. assembling a repetitive part of the chromosome into a separate contig. They sometimes omit entire replicons, e.g. failing to include a plasmid. They sometimes create medium-scale indel errors, e.g. deleting 50 bp from the genome. And they occasionally create large-scale misassemblies, e.g. a major structural rearrangement. Check out our paper comparing long-read assemblers for a more in-depth look at how assemblers perform.
So imagine that you've done long-read sequencing of a bacterial isolate and assembled the reads. The result looks like a nice completed assembly (e.g. a big circular contig for the chromosome and a couple smaller circular contigs for plasmids), but how can you be sure that it's free from the kinds of problems listed above?
Trycycler is a tool that takes as input multiple separate long-read assemblies of the same genome (e.g. from different assemblers and/or different subsets of the input reads) and produces a consensus long-read assembly.
In brief, Trycycler does the following:
- Clusters the contig sequences, so the user can distinguish nice and complete contigs (i.e. those that correspond to an entire replicon) from spurious and/or incomplete contigs.
- Reconciles the alternative contig sequences with each other and repairs circularisation issues.
- Performs a multiple sequence alignment (MSA) of the alternative sequences.
- Constructs a consensus sequence from the MSA by choosing between alternative options where the sequences differ.
The end result is a long-read assembly you can trust!
Trycycler does not ensure a perfect assembly of the underlying genome. This is because systematic basecalling errors can create small-scale sequence errors. Incorrect homopolymer lengths are a common example of this problem, e.g. AAAAAAAA becoming AAAAAAA (read more here).
But if all goes well when running Trycycler, small-scale errors will be the only type of error in its consensus long-read assembly. You can then polish your Trycycler assembly to repair these small-scale errors, e.g. long-read polishing with Medaka then short-read polishing with Pilon. A Trycycler-Medaka-Pilon approach to assembly can therefore yield the best possible bacterial genome: Trycycler fixes the medium-to-large-scale errors and Medaka/Pilon fix the small-scale errors.