Table of Contents

• Introduction
• Installation
• Usage
• Workflow of dip3d
• A running example

Introduction

dip3d is a fast and effective high-order diploid human 3D genome construction method for Pore-C reads. The high data utilization and high-order chromosome fragment interactions captured by Pore-C reads enable us to explore diploid second-order and high-order 3D constructs of human genomes systematically with high resolution.

dip3d has many advantages:

• High speed. dip3d is capable of processing 574 Gbp Pore-C reads successfully in only one day.
• High SNP calling accuracy. SNPS called with 90X Pore-C reads achieve over 99% of recall, precision and F1-score.
• High SNP phasing accuracy. Using 90X Pore-C reads, the whole genome sequences are in phase. SNPS phased with Pore-C fragments achieve over 99% phasing resolutions. Switch errors are 0.05%~1.18%. Hamming distance are 0.03%~1.37%.
• High data utilization. Using contact rescue, over 63.3% Pore-C fragments are successfully phased and over 78% pairwise contacts are successfully phased.

Installation

prerequisites

dip3d depends on the following tools (parentheses are the versions that we used):

• falign (2.0.0)
• bgzip (1.15)
• tabix (1.15)
• bcftools (1.15)
• samtools (1.15)
• Clair3 (v0.1-r10, via docker, using this version is mandatory)
• HapCUT2
• whatshap(1.6)

We download dip3d in the /home/zyserver/chenying/tools directory:

$ pwd
/home/zyserver/chenying/tools

$ git clone https://github.com/xiaochuanle/dip3d.git
$ cd dip3d
$ cd models
$ tar xzvf clair3_v0.1-r10_guppy4_model.tar.gz 
$ cd ..

Usage

Remember that we download dip3d in directory /home/zyserver/chenying/tools.

We run dip3d in another directory /data1/chenying/hg001-3d-genome:

$pwd
/data1/chenying/hg001-3d-genome

First, we copy dip3d.sh in working directory:

$cp /data3/HG001-pore-c/dip3d/dip3d.sh .

We next edit dip3d.sh and fill in the following items:

### 1) Output directory of Dip3D
I_output=hg001-dip3d-output

### 2) Absolute path of reference genome
I_reference=/data1/chenying/dip3d-1/GCA_000001405.15_GRCh38_no_alt_plus_hs38d1_analysis_set.major.fna

### 3) Chromosome list
I_CHR_LIST=(chr1 chr2 chr3 chr4 chr5 chr6 chr7 chr8 chr9 chr10 chr11 chr12 chr13 chr14 chr15 chr16 chr17 chr18 chr19 chr20 chr21 chr22 chrX chrY)

### 4) Number of CPU threads used by Dip3D
I_threads=96

### 5) Directory storing Clair Pore-C model
###    The model is released together with Dip3D
I_clair3_model_directory=/data3/HG001-pore-c/dip3d/models/clair3_v0.1-r10_guppy4_model

### 6) Options for calling SNP by Clair3
###   1. Coverage of Pore-C data
I_snp_frag_coverage=90
###   2. Minimum Pore-C fragment length
I_snp_frag_length=100
###   3. Minimum alignment identities
I_snp_frag_identity=90.0
###   4. Minimum mapping qualities
I_snp_frag_mapQ=5

### 7) Options for select BAM for HapCUT2
I_hapcut_frag_coverage=30
I_hapcut_frag_length=100
I_hapcut_frag_identity=90.0
I_hapcut_frag_mapQ=5

### 7) Options for haplo-tagging Pore-C fragments with SNP
###   1. Number of flanking match residues in overlapped SNP sites
I_snp_tag_match_bases=3
###   2. Only haplo-tagging Pore-C fragments with at least this mapping qualities
I_snp_tag_mapQ=5
###   3. Only haplo-tagging Pore-C fragments with at least this aligment identites
I_snp_tag_identity=85.0

### 8) Options for haplotype imputation
### 1. Maximum genomic distance between adjacent fragment pairs
I_imputation_adj_dist=29500000
### 2. Maximum genomic distacne between non-adjacent fragment pairs
I_imputation_non_adj_dist=16500000

### 9) Absolute path list of Pore-C list
###     Please don't concat concat FASTQ files genergeted with different enzymes into one
I_pore_c_reads=( \
/data1/chenying/dip3d/pore-c/NA12878_Rep1/pass.gt7.fastq.gz
/data1/chenying/dip3d/pore-c/NA12878_Rep2/pass.gt7.fastq.gz
/data1/chenying/dip3d/pore-c/NA12878_Rep3/pass.gt7.fastq.gz
/data1/chenying/dip3d/pore-c/NA12878_Rep4/pass.gt7.fastq.gz
/data1/chenying/dip3d/pore-c/NA12878_Rep5/pass.gt7.fastq.gz
/data1/chenying/dip3d/pore-c/NA12878_Rep8/pass.gt7.fastq.gz
/data1/chenying/dip3d/pore-c/NA12878_Rep9/pass.gt7.fastq.gz
/data1/chenying/dip3d/pore-c/NA12878_Rep10/pass.gt7.fastq.gz
)

Finally, we save and run dip3d.sh:

./dip3d.sh

Workflow of dip3d

dip3d runs in three steps.

Step One: Mapping

In this first step, dip3d maps Pore-C reads (provided via I_pore_c_reads) to the reference genome (provided via I_reference) using falign. The mapping results are in frag-bam format and saved in

/data1/chenying/hg001-3d-genome/dip3d-output/1-falign/frag-ref.bam

Note that this BAM file is not sorted.

Step Two: SNP calling and phasing

If you have previously created a phased SNP VCF file, you can skip this step by providing it to dip3d:

I_vcf=/absolute-path-to-a-previous-dip3d-output/2-snp/phased_snp.vcf

In this case dip3d will skip the whole step two.

dip3d extracts sequentially 270G (provided by I_snp_reads) Pore-C fragments from /data1/chenying/hg001-3d-genome/dip3d-output/1-falign/frag-ref.bam and saves the extracted fragments in

/data1/chenying/hg001-3d-genome/dip3d-output/2-snp/snp-frag-ref.bam

Note that this snp-frag-ref.bam is sorted.

Clair3 is used for calling variants with snp-frag-ref.bam and the reference genome (provided via I_reference). The variants called by Clair3 are saved in

/data1/chenying/hg001-3d-genome/dip3d-output/2-snp/clair3_output/merge_output.vcf.gz

SNPs are extracted from merge_output.vcf.gz and saved in

/data1/chenying/hg001-3d-genome/dip3d-output/2-snp/snp.vcf
/data1/chenying/hg001-3d-genome/dip3d-output/2-snp/snp.vcf.gz
/data1/chenying/hg001-3d-genome/dip3d-output/2-snp/snp.vcf.gz.tbi

snp.vcf is phased with snp-frag-ref.bam by HapCUT2 and whatshap. The largest phase blocks of each chromosome in the reference genome (provided via I_reference) are extracted from the phased SNPs and saved in

/data1/chenying/hg001-3d-genome/dip3d-output/2-snp/phased_snp.vcf
/data1/chenying/hg001-3d-genome/dip3d-output/2-snp/phased_snp.vcf.gz
/data1/chenying/hg001-3d-genome/dip3d-output/2-snp/phased_snp.vcf.gz.tbi

Step Three: fragment haplotyping

The frag-bam file output by Step One

/data1/chenying/hg001-3d-genome/dip3d-output/1-falign/frag-ref.bam

is sorted and saved in

/data1/chenying/hg001-3d-genome/dip3d-output/3-frag-hap/sorted-frag-ref.bam

This sorted frag-bam is then phased by Dip3d with phased_snp.vcf. The phased results are saved in

/data1/chenying/hg001-3d-genome/dip3d-output/3-frag-hap/chr1/snp-frag-hap.list
/data1/chenying/hg001-3d-genome/dip3d-output/3-frag-hap/chr2/snp-frag-hap.list
...

The untaged fragments are then imputed and saved in

/data1/chenying/hg001-3d-genome/dip3d-output/3-frag-hap/chr1/imputed-frag-hap.list
/data1/chenying/hg001-3d-genome/dip3d-output/3-frag-hap/chr2/imputed-frag-hap.list
...

The BAM file of each chromosome is also tagged and stored in

/data1/chenying/hg001-3d-genome/dip3d-output/3-frag-hap/chr1/tagged-chr1.bam
/data1/chenying/hg001-3d-genome/dip3d-output/3-frag-hap/chr2/tagged-chr2.bam
...

This is the final results output by dip3d. It is tagged in the same way as whatshap. That is, HP:i:1, HP:i:2 and untagged.

A running example

We run an example on the computer equipped with 96 1.5Ghz CPU threads and 256GB RAM.

datasets

We produce 8 flowcells of NA12878 Pore-C reads in house:

    /data1/chenying/dip3d/pore-c/NA12878_Rep1/pass.gt7.fastq.gz \
    /data1/chenying/dip3d/pore-c/NA12878_Rep2/pass.gt7.fastq.gz \
    /data1/chenying/dip3d/pore-c/NA12878_Rep3/pass.gt7.fastq.gz \
    /data1/chenying/dip3d/pore-c/NA12878_Rep4/pass.gt7.fastq.gz \
    /data1/chenying/dip3d/pore-c/NA12878_Rep5/pass.gt7.fastq.gz \
    /data1/chenying/dip3d/pore-c/NA12878_Rep8/pass.gt7.fastq.gz \
    /data1/chenying/dip3d/pore-c/NA12878_Rep9/pass.gt7.fastq.gz \
    /data1/chenying/dip3d/pore-c/NA12878_Rep10/pass \

There are 123081636 Pore-C reads and 574 Gbp (191.3 X).

wallclock time

dip3d took 25 hours to process the above Pore-C reads.

SNP calling performance

recall: 99.01%, precision: 99.25, F1-score: 99.13

SNP phasing performance

• resolutions (the proportion of heterozygous SNPs contained in the largest phased block): 97.6-99.6%
• switch error: 0.05-1.18%
• hamming distance: 0.03-1.37%

fragment phasing performance

• Total fragments: 564.8m
• phased fragments by H-snp: 134.2m (23.76%)
• phased fragments after imputation: 357.3m (63.3%)

contacts phasing performance

• Total contacts: 1325m

contacts phased by H-snp

• Total: 87.1m (6.6%)

phased contacts after imputation

• Total: 1033.5m (78%)
• H1: 513.2m
• H2: 512.9m