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Chenglin20170390 / phased-assembly-check

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phased-assembly-check

1. Haplotype-resolved ctg

  • Trio
yak count -b37 -t16 -o pat.yak <(cat pat_1.fq.gz pat_2.fq.gz) <(cat pat_1.fq.gz pat_2.fq.gz)
yak count -b37 -t16 -o mat.yak <(cat mat_1.fq.gz mat_2.fq.gz) <(cat mat_1.fq.gz mat_2.fq.gz)
hifiasm -o HG002.asm -t32 -1 pat.yak -2 mat.yak HG002-HiFi.fa.gz
  • HiC
hifiasm -o HG002.asm -t32 --h1 read1.fq.gz --h2 read2.fq.gz HG002-HiFi.fq.gz
  • Evaluation
## yak (https://github.com/lh3/yak)

yak trioeval pat.yak mat.yak HG002.asm.trio.hap1.p_ctg.fa > HG002.asm.hap1.trioeval
yak trioeval pat.yak mat.yak HG002.asm.trio.hap2.p_ctg.fa > HG002.asm.hap2.trioeval

## Merqury.FK (https://github.com/thegenemyers/MERQURY.FK)

2. Haplotype-aware scaffolding

meryl-lookup -memory 2 -exclude -mers pat.meryl -sequence $read1 -sequence2 $read2 -r2
mat.R2.fastq.gz | pigz -c > mat.R1.fastq.gz
meryl-lookup -memory 2 -exclude -mers mat.meryl -sequence $read1 -sequence2 $read2 -r2
pat.R2.fastq.gz | pigz -c > pat.R1.fastq.gz 

Using https://github.com/marbl/merqury/blob/master/trio/exclude_reads.sh

bash /data/software/Merqury/merqury/trio/exclude_reads.sh mat.hapmer.meryl f1.hic.R1.fq.gz f1.hic.R2.fq.gz pat

  • Scaffold two haplotype together and check (recommend)

    • run juicer + 3d-dna / AllHiC

    • Loading into JuiceBox for visualization

      • False duplication (Examples are based on the hifiasm 0.15.4 for high het plant genome with trio data)

        • Fig1

          Fig1

          seqName #matKmer #patKmer #pat-pat #pat-mat #mat-pat #mat-mat seqLen
          h1tg000041l 160 10644 77 83 82 10561 6152553
          h1tg000053l 50105 62 50059 45 45 17 5159680
          h1tg000050l 17945 27 17924 20 20 7 2082689

          Conclusion: Remove the h1tg000041l

        • Fig2

          Fig2

          seqName #patKmer #patKmer #pat-pat #pat-mat #mat-pat #mat-mat seqLen
          h1tg000015l 87 164 51 35 36 128 2770227
          h1tg000078l 42 66 24 18 18 47 1476449
          h1tg000028l 7532 87 7478 53 53 34 3799565

          Conclusion: Remove the h1tg000015l and h1tg000028l

        • Fig3

          Fig3

          seqName #patKmer #patKmer #pat-pat #pat-mat #mat-pat #mat-mat seqLen
          h1tg000054l 6908 10 6899 8 8 2 1004624
          h1tg000013l 25643 330 25547 95 96 234 8177266
          h1tg000059l 57 245 25 32 32 212 1963293
          h1tg000056l 12 29 4 7 7 22 685156
          h1tg000052l 25905 219 25816 88 88 131 5467035

          Conclusion: Remove the h1tg000059l and h1tg000056l, 52l vs (54l+13l) maybe real

        • Fig4

          Fig4

          seqName #patKmer #patKmer #pat-pat #pat-mat #mat-pat #mat-mat seqLen
          h1tg000031l 17138 12244 17075 63 63 12180 4988700
          h1tg000029l 44167 139 44067 99 99 40 7648617

          Conclusion: utg overlap. Find the utg in the gfa and remove rerun the utg trioeval and decide.

        • Fig5

          Fig5

          seqName #patKmer #patKmer #pat-pat #pat-mat #mat-pat #mat-mat seqLen
          h1tg000017l 39991 89 39929 61 61 28 5312873
          h1tg000064l 1572 5187 1540 31 32 5155 1725115

          Conclusion: utg overlap. Find the utg in the gfa and remove.

    • Misplaced haplotype If you find the hap1 ctg have more strong interaction with the hap2, and trioeval support it, you need manually move this hap1 ctg to hap2

    • Haplotype-specific inversion MQ>1 filter for haplotype interaction, kepp the heatmap inside the haplotype is normal. Fig6

3. Quality check

  • Trioeval for hamming error and switch error
Type Hap Switch Hamming
Trio (0.14.2) hap1 0.49% 0.59%
Trio (0.14.2) hap2 0.56% 1.13%
Trio-check hap1 0.44% 0.39%
Trio-check hap2 0.55% 1.12%
Trio(0.16.1) hap1 0.46% 0.46%
Trio (0.16.1) hap2 0.55% 0.57%
HiC (0.16.1) hap1 0.51% 0.46%
HiC (0.16.1) hap2 0.49% 0.51%
dual (0.16.1) hap1 0.51% 8.51%
dual (0.16.1) hap2 0.50% 13.69%
  • QV estimation for two haplotype
  • Check the haplotype synteny and confirm the utg (chhylp123/hifiasm#159)
  • Check the coverage of two haplotype

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