Tips for finishing genomes
Ideally, a Unicycler hybrid assembly will result in a completed bacterial genome all by itself. But if it doesn't, then the genome might need 'manual completion'. This page contains some tips and tricks to help you along.
In a completed assembly, each chromosome/plasmid in the genome is represented by a single contig. How do you tell if a Unicycler assembly is complete? Unicycler's output/log might help. In the 'Bridged assembly graph' section towards the end of Unicycler's pipeline, it will summarise the graph components:
Component Segments Links Length N50 Longest segment Status
total 7 7 5,676,472 5,583,468 5,583,468
1 1 1 5,583,468 5,583,468 5,583,468 complete
2 1 1 71,104 71,104 71,104 complete
3 1 1 6,657 6,657 6,657 complete
4 1 1 5,783 5,783 5,783 complete
5 1 1 3,514 3,514 3,514 complete
6 1 1 3,223 3,223 3,223 complete
7 1 1 2,723 2,723 2,723 complete
Unicycler considers a component complete if it is circular: one segment and one link. This doesn't quite apply if your bacterial genome has linear chromosomes/plasmids, in which case a complete component would have no links.
You could also view the assembly graph (assembly.gfa) in Bandage and check that each contig is circular:
If that's what your graph looks like, then Unicycler completed the assembly on its own!
But what if it's not complete? The Unicycler log might have something like this:
Component Segments Links Length N50 Longest segment Status
total 23 29 5,819,363 5,242,094 5,242,094
1 1 1 5,242,094 5,242,094 5,242,094 complete
2 1 1 252,269 252,269 252,269 complete
3 1 1 130,933 130,933 130,933 complete
4 1 1 110,494 110,494 110,494 complete
5 1 1 69,826 69,826 69,826 complete
6 1 1 5,783 5,783 5,783 complete
7 17 23 7,964 1,023 3,382 incomplete
and the Bandage graph might look like this:
Yuck! This genome needs some manual completion...
There are many reasons why Unicycler might fail to complete a hybrid assembly, and so there is no single easy method for manual completion. You'll need to rely on detective work and bioinformatics-know-how. Some general methods which may help are:
- Using Bandage to visualise the assembly graphs from various stages of the Unicycler pipeline.
- Gathering long reads for incomplete regions of the assembly (see Read extraction) and BLASTing them to the graphs.
- Aligning short and/or long reads to the assembly and examining the alignments in IGV or Artemis.
- Using other assemblers (e.g. Canu) on the reads and comparing the results to Unicycler's assembly.
Helpful software:
To get you going, here are some real-world examples of assemblies which failed to complete and how I went about manual completion: