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One command pipeline of C-Phasing

Pipeline

The -n 8:4 parameter of the following commands means assembling a tetraploid (4) with 8 chromosome basic numbers. If you set -n 0:0 means partition in both rounds automatically, also support it set to -n 8:0 or -n 0:4.

Note

CPhasing also support the monoploid scaffolding, when you set one group number, e.g. -n 8. The pipeline will automatically skip the step 1.alleles, and only run one round partition.

Note

If the user's genome is an allopolyploid with low subgenome similarity, the initial grouping may not be optimal. In such cases, users should adjust the -n parameter based on the ploidy and genome structure:

  • For allotetraploids (2n = 4x = 32), the genome can be treated as a diploid assembly. Use -n 16 if you want to assemble two subgenomes, set -n 16:2, if you want to phase the subgenome, respectively.

  • For allohexaploids:

    • AAABBB type (2n = 6x = 48): use -n 16:3.
    • AABBCC type (2n = 6x = 48): use -n 24:2.

Tip

For hifiasm-generated contigs, the p_utg.fasta is recommended for phased genome assembly. Optionally, users may concatenate the hap*.p_ctg.fasta outputs from hifiasm's Hi-C mode.

Start from a pore-c data:

cphasing pipeline -f draft.asm.fasta -pcd sample.fastq.gz -t 10 -n 8:4

Start from multiple Pore-C data:

specify multiple -pcd parameters. 

cphasing pipeline -f draft.asm.fasta -pcd sample1.fastq.gz -pcd sample2.fastq.gz -t 10 -n 8:4

Note

If you want to run on cluster system and submit them to multiple nodes, you can use cphasing mapper and cphasing-rs porec-merge to generate the merged porec.gz file and input it by -pct parameter. Please check the doc:Mapper

Start from a Pore-C table (.porec.gz):

which is generated by cphasing mapper. 

cphasing pipeline -f draft.asm.fasta -pct sample.porec.gz -t 10 -n 8:4

Start from CiFi data

Run pipeline or mapper with --mm2-params "-x cifi" parameter. And the output similar to the results of pore-c data. 

cphasing pipeline -f draft.asm.fasta -pcd hific.fastq.gz --mm2-params "-x cifi "  -t 10 -n 8:4

Note

The mapping results of CiFi is similar to Pore-C, such as output suffix with porec.gz, and process it use porec-merge, porec-intersect, et al.

Start from Hi-C/Omni-C data

cphasing pipeline -f draft.asm.fasta -hic1 Lib_R1.fastq.gz -hic2 Lib_R2.fastq.gz -t 10 -n 8:4

Note

  • 1 | If you want to run multiple samples, you can specified mutiple -hic1 and -hic2 parameters (e.g., -hic1 sample1_R1.fastq.gz -hic1 sample2_R1.fastq.gz -hic2 sample1_R2.fastq.gz -hic2 sample2_R2.fastq.gz)
  • 2 | If the total length of your input genome is larger than 8 Gb, the -hic-mapper-k 27 -hic-mapper-w 14 should be specified, to avoid the error of chromap.

  • 3 | You can switch the Hi-C aligner using the -hic-aligner option. Supported aligners include: - _chromap (default): Modified version of Chromap bundled with C-Phasing. - chromap: Official version of Chromap. - bwa-mem2: High-performance BWA-MEM. - minibwa: A faster BWA-like mapper.

    Example using minibwa: 

    cphasing pipeline -f draft.asm.fasta -hic1 Lib_R1.fastq.gz -hic2 Lib_R2.fastq.gz --hic-aligner minibwa -t 10 -n 8:4

Start from 4DN pairs (pairs.pqs or pairs.gz) file

cphasing pipeline -f draft.asm.fasta -prs sample.pairs.pqs -t 10 -n 8:4

cphasing pipeline -f draft.asm.fasta -prs sample.pairs.gz -t 10 -n 8:4

Skip some steps

## skip steps 1.alleles and 2.prepare steps 
cphasing pipeline -f draft.asm.fasta -pct sample.porec.gz -t 10 -ss 1,2

Perform only specified steps

## run 3.hyperpartition 
cphasing pipeline -f draft.asm.fasta -pct sample.porec.gz -t 10 -s 3

Improve performance

Use the -hcr parameter to filter greedy contacts (regions with excessive genome-wide interactions), which can improve phasing quality.

For restriction enzyme-based Hi-C libraries, set -p AAGCTT to normalize depth calculations based on restriction site density (AAGCTT is the restriction enzyme recognition sequence). This option is not needed for Omni-C data. 

cphasing pipeline -f draft.asm.fasta -pct sample.porec.gz -t 10 -hcr -p AAGCTT

Collapsed rescue

  • Identify collapsed unitigs from a Pore-C PAF file (including singletons): 
    cphasing pipeline -f draft.asm.fasta -paf sample.paf.gz -t 10 -hcr -p AAGCTT --collapsed-rescue
  • Or identify collapsed unitigs from a hifiasm GFA file, which contains the read depth of the unitigs: 
    cphasing pipeline -f draft.asm.fasta -paf  sample.paf.gz -t 10 -hcr -p AAGCTT --collapsed-rescue --gfa draft.asm.p_utg.noseq.gfa
  • Alternatively, provide a customized collapsed unitig table (format: contig\tcoverage\tcopynumber, details see the collapse): 
    cphasing pipeline -f draft.asm.fasta -paf  sample.paf.gz -t 10 -hcr -p AAGCTT --collapsed-contigs collapsed.contig.list

Curation by Juicebox [Or directly run 6.curation/curation.cmd.sh]

  • generate .assembly and .hic, depend on 3d-dna 
cphasing pairs2mnd sample.pairs.gz -o sample.mnd.txt
cphasing utils agp2assembly groups.agp > groups.assembly
bash ~/software/3d-dna/visualize/run-assembly-visualizer.sh sample.assembly sample.mnd.txt

Note

if chimeric corrected, please use groups.corrected.agp and generate a new corrected.pairs.pqs by cphasing-rs pairs-break

  • After curation 
    ## convert assembly to agp
cphasing utils assembly2agp groups.review.assembly -n 8:4 
## or haploid or a homologous group
cphasing utils assembly2agp groups.review.assembly -n 8
## extract contigs from agp 
cphasing agp2fasta groups.review.agp draft.asm.fasta --contigs > contigs.fasta
## extract chromosome-level fasta from agp
cphasing agp2fasta groups.review.agp draft.asm.fasta > groups.review.asm.fasta

Rename

Rename and orient chromosome according a monoploid reference (or genome of closely related species). More details please check Rename 

cphasing rename -r mono.fasta -f draft.asm.fasta -a groups.review.agp -t 20

Note

To reduce the time consumed, we only align the first haplotype (g1) to the monoploid, which the orientation among different haplotypes has already been set to the same in the scaffolding step. If not, you can set —-unphased to align all haplotypes to the monoploid to adjust the orientation.

Heatmap plotting

Please check the doc: Plot