Pipeface

Overview

Pipefaceee.

Nextflow pipeline to align, variant call (SNP's, indels's, SV's) and phase long read ONT and/or pacbio HiFi data.

Workflow

Overview

%%{init: {'theme':'dark'}}%%
flowchart LR

input_data("Input data: \n\n ONT fastq.gz \n and/or \n ONT fastq \n and/or \n ONT uBAM \n and/or \n pacbio HiFi uBAM")
merging{{"Merge runs (if needed)"}}
alignment{{"bam to fastq conversion (if needed), alignment, sorting"}}
depth{{"Calculate alignment depth"}}
snp_indel_calling{{"SNP/indel variant calling"}}
snp_indel_phasing{{"SNP/indel phasing"}}
haplotagging{{"Haplotagging bams"}}
sv_calling{{"Structural variant calling"}}

input_data-.->merging-.->alignment-.->snp_indel_calling-.->snp_indel_phasing-.->haplotagging-.->sv_calling
alignment-.->depth
alignment-.->haplotagging

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Detailed

%%{init: {'theme':'dark'}}%%
flowchart LR

ont_data_f1("Sample 1 \n\n Input data: \n\n ONT fastq.gz")
ont_data_f2("Sample 1 \n\n Input data: \n\n ONT fastq.gz")
pacbio_data_f3("Sample 2 \n\n Input data: \n\n Pacbio HiFi uBAM")
pacbio_data_f4("Sample 2 \n\n Input data: \n\n Pacbio HiFi uBAM")
ont_data_f5("Sample 3 \n\n Input data: \n\n ONT fastq")
ont_data_f6("Sample 4 \n\n Input data: \n\n ONT uBAM")

merging_m1{{"Description: merge runs \n\n Main tools: GNU coreutils \n\n Commands: cat"}}
merging_m2{{"Description: merge runs \n\n Main tools: Samtools \n\n Commands: samtools merge"}}

alignment_s1{{"Description: alignment, sorting \n\n Main tools: Minimap2 and Samtools \n\n Commands: minimap2 and samtools sort"}}
alignment_s2{{"Description: alignment, sorting \n\n Main tools: Minimap2 and Samtools \n\n Commands: minimap2 and samtools sort"}}
alignment_s3{{"Description: bam to fastq conversion, alignment, sorting \n\n Main tools: Minimap2 and Samtools \n\n Commands: minimap2 and samtools sort"}}
alignment_s4{{"Description: bam to fastq conversion, alignment, sorting \n\n Main tools: Minimap2 and Samtools \n\n Commands: minimap2 and samtools sort"}}

depth_s1{{"Description: calculate alignment depth \n\n Main tools: Samtools \n\n Commands: samtools depth"}}
depth_s2{{"Description: calculate alignment depth \n\n Main tools: Samtools \n\n Commands: samtools depth"}}
depth_s3{{"Description: calculate alignment depth \n\n Main tools: Samtools \n\n Commands: samtools depth"}}
depth_s4{{"Description: calculate alignment depth \n\n Main tools: Samtools \n\n Commands: samtools depth"}}

snp_indel_calling_s1{{"Description: SNP/indel variant calling \n\n Main tools: Clair3 or DeepVariant (NVIDIA Parabricks) \n\n Commands: run_clair3.sh or pbrun deepvariant"}}
snp_indel_calling_s2{{"Description: SNP/indel variant calling \n\n Main tools: Clair3 or DeepVariant (NVIDIA Parabricks) \n\n Commands: run_clair3.sh or pbrun deepvariant"}}
snp_indel_calling_s3{{"Description: SNP/indel variant calling \n\n Main tools: Clair3 or DeepVariant (NVIDIA Parabricks) \n\n Commands: run_clair3.sh or pbrun deepvariant"}}
snp_indel_calling_s4{{"Description: SNP/indel variant calling \n\n Main tools: Clair3 or DeepVariant (NVIDIA Parabricks) \n\n Commands: run_clair3.sh or pbrun deepvariant"}}

snp_indel_phasing_s1{{"Description: SNP/indel phasing \n\n Main tools: WhatsHap \n\n Commands: whatshap phase"}}
snp_indel_phasing_s2{{"Description: SNP/indel phasing \n\n Main tools: WhatsHap \n\n Commands: whatshap phase"}}
snp_indel_phasing_s3{{"Description: SNP/indel phasing \n\n Main tools: WhatsHap \n\n Commands: whatshap phase"}}
snp_indel_phasing_s4{{"Description: SNP/indel phasing \n\n Main tools: WhatsHap \n\n Commands: whatshap phase"}}

haplotagging_s1{{"Description: haplotagging bams \n\n Main tools: WhatsHap \n\n Commands: whatshap haplotag"}}
haplotagging_s2{{"Description: haplotagging bams \n\n Main tools: WhatsHap \n\n Commands: whatshap haplotag"}}
haplotagging_s3{{"Description: haplotagging bams \n\n Main tools: WhatsHap \n\n Commands: whatshap haplotag"}}
haplotagging_s4{{"Description: haplotagging bams \n\n Main tools: WhatsHap \n\n Commands: whatshap haplotag"}}

sv_calling_s1{{"Description: structural variant calling \n\n Main tools: Sniffles2 and/or cuteSV \n\n Commands: sniffles and/or cuteSV"}}
sv_calling_s2{{"Description: structural variant calling \n\n Main tools: Sniffles2 and/or cuteSV \n\n Commands: sniffles and/or cuteSV"}}
sv_calling_s3{{"Description: structural variant calling \n\n Main tools: Sniffles2 and/or cuteSV \n\n Commands: sniffles and/or cuteSV"}}
sv_calling_s4{{"Description: structural variant calling \n\n Main tools: Sniffles2 and/or cuteSV \n\n Commands: sniffles and/or cuteSV"}}

ont_data_f1-.->merging_m1-.->alignment_s1-.->snp_indel_calling_s1-.->snp_indel_phasing_s1-.->haplotagging_s1-.->sv_calling_s1
ont_data_f2-.->merging_m1
ont_data_f5-.->alignment_s2-.->snp_indel_calling_s2-.->snp_indel_phasing_s2-.->haplotagging_s2-.->sv_calling_s2
ont_data_f6-.->alignment_s3-.->snp_indel_calling_s3-.->snp_indel_phasing_s3-.->haplotagging_s3-.->sv_calling_s3

pacbio_data_f3-.->merging_m2-.->alignment_s4-.->snp_indel_calling_s4-.->snp_indel_phasing_s4-.->haplotagging_s4-.->sv_calling_s4
pacbio_data_f4-.->merging_m2

alignment_s1-.->depth_s1
alignment_s2-.->depth_s2
alignment_s3-.->depth_s3
alignment_s4-.->depth_s4

alignment_s1-.->haplotagging_s1
alignment_s2-.->haplotagging_s2
alignment_s3-.->haplotagging_s3
alignment_s4-.->haplotagging_s4

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Main analyses

• ONT and pacbio HiFi data
• WGS and targeted
• hg38 or hs1 reference genome

Main tools

• Minimap2
• Clair3 OR DeepVariant (wrapped in NVIDIA Parabricks)
• WhatsHap
• Sniffles2 AND/OR cuteSV
• Samtools

Main input files

Required

• ONT/pacbio HiFi FASTQ (gzipped or uncompressed) or unaligned BAM
• Indexed reference genome
• Clair3 models (if running Clair3)

Optional

• Regions of interest BED file
• Tandem repeat BED file

Main output files

• Aligned, sorted and haplotagged bam
• Clair3 or DeepVariant phased SNP/indel VCF file
• Clair3 or DeepVariant SNP/indel gVCF file
• Phased Sniffles2 and/or un-phased cuteSV SV VCF file

Assumptions

• Running pipeline on Australia's National Computational Infrastructure (NCI)
• Access to if89 project on National Computational Infrastructure (NCI)
• Access to pipeline dependencies:
  • Nextflow and it's java dependency. Validated to run on:
    • Nextflow 24.04.1
    • Java version 17.0.2

Run it!

See a walkthrough for how to run pipeface on NCI.