I'm currently trying to assembly a genome from a rodent parasite, Nippostrongylus brasiliensis. This genome does have an existing reference genome, but it is highly fragmented. Here are some continuity statistics for the scaffolds of the current Nippo reference genome (assembled from Illumina reads):
Total sequences: 29375 Total length: 294.400206 Mb Longest sequence: 394.171 kb Shortest sequence: 500 b Mean Length: 10.022 kb Median Length: 2.682 kb N50: 2024 sequences; L50: 33.527 kb N90: 11638 sequences; L90: 4.263 kb
This genome is most likely difficult to assemble because of the highly repetitive nature of the genomic sequences. These repetitive sequences come in (at least) three classes:
- Tandem repeats with a repeat unit length greater than the read length of Illumina sequencers (e.g. 171bp)
- Tandem repeats with a cumulative length greater than the fragment length of Illumina sequencers, or the template length for linked reads (e.g. 20kb)
- Complex (i.e. non-repetitive) sequence that appears at multiple places throughout the genome
Canu seems to deal quite well with the first two types of repeats, despite the abundance of repetitive structure in the genome. Here's the unitigging summary produced by Canu on one of the assemblies I've attempted. Notice that about 30% of the reads either span or contain a long repeat:
category reads % read length feature size or coverage analysis ---------------- ------- ------- ---------------------- ------------------------ -------------------- middle-missing 694 0.07 7470.92 +- 5552.00 953.06 +- 1339.13 (bad trimming) middle-hump 549 0.05 3770.05 +- 3346.10 74.23 +- 209.86 (bad trimming) no-5-prime 3422 0.33 6711.32 +- 5411.26 70.92 +- 272.99 (bad trimming) no-3-prime 3161 0.30 6701.35 +- 5739.86 87.41 +- 329.42 (bad trimming) low-coverage 27158 2.59 3222.51 +- 1936.79 4.99 +- 1.79 (easy to assemble, potential for lower quality consensus) unique 636875 60.76 6240.20 +- 3908.44 25.22 +- 8.49 (easy to assemble, perfect, yay) repeat-cont 48398 4.62 4099.55 +- 3002.72 335.54 +- 451.43 (potential for consensus errors, no impact on assembly) repeat-dove 135 0.01 16996.33 +- 6860.08 397.37 +- 319.52 (hard to assemble, likely won't assemble correctly or even at all) span-repeat 137927 13.16 9329.94 +- 6906.27 2630.06 +- 3539.53 (read spans a large repeat, usually easy to assemble) uniq-repeat-cont 155725 14.86 6529.83 +- 3463.16 (should be uniquely placed, low potential for consensus errors, no impact on assembly) uniq-repeat-dove 28248 2.70 12499.99 +- 8446.95 (will end contigs, potential to misassemble) uniq-anchor 5721 0.55 8379.86 +- 4575.71 3166.22 +- 3858.35 (repeat read, with unique section, probable bad read)
However, the third type of repeat is giving me a bit of grief. Using the above assembly, here are the continuity parameters from the assembled contigs:
Total sequences: 3505 Total length: 322.867456 Mb Longest sequence: 1.762243 Mb Shortest sequence: 2.606 kb Mean Length: 92.116 kb Median Length: 42.667 kb N50: 417 sequences; L50: 194.126 kb N90: 1996 sequences; L90: 35.634 kb
It's not a bad assembly, particularly given the complexity of the genome, but I feel like it could be improved by tackling the complex genomic repeats in some fashion. About 60Mb of the contigs in this assembly are linked with each other in a huge web (based on the GFA output from Canu):
The repetitive regions are typically over 500bp in length, average about 3kb, and I've seen at least one case which seems to be a 20kb sequence duplicated in multiple regions.
The Canu defaults seem to give the best assembly results for the few parameters that I've tried, with one exception: trimming. I've tried playing around a little bit with the trimming parameters, and curiously a trimming coverage of 5X (with overlap of 500bp) seems to give a more contiguous assembly than with a trimming coverage of 2X (with the same overlap).
If anyone is interested in having a look at these data themselves, called FASTQ files from Nippo sequencing runs can be found here. Raw nanopore signal files are available within ENA project PRJEB20824. There's also a Zenodo archive here that contains the GFA and assembly contigs.
I can use Illumina data to correct the Canu assembly, but that doesn't help with resolving the "type 3" repeats. The regions are sufficiently similar that illumina reads get mapped to multiple points in the genome. The Illumina contigs are high quality (i.e. they have good BUSCO scores, indicating few variant errors), but quite short. Any sniff of a repeat and the contig ends. I've got more than a few examples of regions that would make an Illumina read (even 10x linked reads) cower in fear.
Does anyone have any other suggestions on how I could resolve these complex repeats?
Computational solutions would be preferred, but resequencing is not out of the question.