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Old 11-25-2019, 06:47 AM   #1
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Default Assembling haplotypes of a highly heterozygous gene cluster with canu

I am trying to assemble haplotypes for a peculiar region of the human genome that (1) has high heterozygosity, (2) has variation in presence or absence of entire genes, and (3) encompasses a gene cluster of highly similar paralogues. This is obviously making assembly difficult, since a paralogue on the same chromosome may have only 10% divergence from its duplicate, while the homologue on the other chromosome has 5% differences due to segregating polymorphism at that locus. Currently, I have nanopore and short reads from this region, both at approximately 30X coverage. I would like to use canu to assemble the nanopore reads, then short reads to polish, but I am getting nowhere near the full assembly. My command is

canu -p prefix -d canu_run genomeSize=250k correctedErrorRate=0.144 minOverlapLength=500 -nanopore-raw sample.fastq
Here sample.fastq are reads filtered for my region of interest, so its a fairly small total assembly. So far, I have tried varying the corrected error rate between 0.1 and 0.2, and the minOverlapLength between 500 and 1000 with no luck. Using BLAST, I can see large chunks of my genes of interest in the prefix.unassembled.fasta file. It seems varying error rates should help find a sweet spot of expected divergence between reads from the same allele at a locus, reads from different alleles at a locus, and reads from different paralogous loci - I'm wondering, is there any other parameters I can vary to try and get a more complete assembly? Is there any preprocessing I can do with the more accurate short reads to lead to a more complete assembly? Ideally, I eventually want phased haplotype information.
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Old 11-25-2019, 10:15 AM   #2
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This isn't so helpful to solve the asked problem, but PacBio HiFi reads might be a better choice since you can generate 15kb sequences at 1% or 0.1% error rates. They might more obviously segregate into 4 bins (2 haplotypes at a locus, 2 at paralogous locus).
Providing nextRAD genotyping and PacBio sequencing services.
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assembly, canu, heterozygous, nanopore, phase

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