Nanopore long-read DNA sequencing data analysis pipeline

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nanopore_dna-seq
nanopore_dna-seq
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Description

This snakemake pipeline is for Nanopore long-read DNA sequencing data analysis. The pipeline may be run on an HPC or in a local environment.

Major steps in this workflow include:

  1. Modified basing calling using Megalodon

  2. Structural variant calling using Nanopore pipeline-structural-variation

  3. Haplotype-aware variant calling using PEPPER-Margin-DeepVariant

Software Requirements

User's guide

I. Input requirements

  • Edited config/config.yaml

  • Demultiplexed Nanopore fast5 data

  • Reference genome file

II. Editing the config.yaml

Basic parameters:

  • raw: Path to the directory where demultiplexed fast5 data is stored. The name of sub-directory for each sample should be in this format: {sampleID}_fast5

  • out: Output directory, default without input: "output" in working directory

  • reference: Path to primary reference genome fasta file

  • pepper: Path to the directory where PEPPER-Margin-DeepVariant singularity is downloaded

  • bind: Singularity bind mounting

  • snpeff: Path to SNPEFF installed location

Parameters for modified base calling:

  • outputs: Desired outputs

  • base_conv: modified base conversion

  • motif: Restrict modified base results to the specified motifs

  • thresh: Hard threshold for modified base aggregation (probability of modified/canonical base), default 0.75

  • config: Guppy model config file

Parameters for SV calling:

  • min_sv_length: Minimum SV length

  • max_sv_length: Maximum SV length

  • min_read_length: Minimum read length

  • min_read_mapping_quality: Minimum mapping quality

  • min_read_support: Minimum read support required to call a SV (auto for auto-detect)

  • snpsift: Run snpsift filtering or not

  • snpsift_filter: SnpSift filtering parameters if the input of the above parameter is "yes"

  • target_bed: (Optional) BED file containing targeted regions where SVs will only be called

Parameters for Haplotype-aware variant calling:

  • snpsift2: Run snpsift filtering or not

  • snpsift_filter2: SnpSift filtering parameters if the input of the above parameter is "yes"

Optional parameters to run the full analysis on HPV genomes:

(This function was originally designed for the HPV project, however, it can be applied to any secondary genome analysis of any species with references.)

  • hpv: Run the analysis on HPV genomes or not

  • hpv_ref: Path to HPV reference genome fasta file

  • snpeff_hpv: Name of the pre-build HPV snpEff annotation

  • snpeff_config: Path to the HPV snpEff config file

III. To run

  • Clone the repository to your working directory 

    git clone https://github.com/NCI-CGR/Nanopore_DNA-seq.git

  • Install required software

  • Edit and save config/config.yaml

  • To run on an HPC using slurm job scheduler like NIH Biowulf (Pascal GPU or higher architecture required):

    Edit config/cluster_config.yaml according to your HPC information and adjust the computing resources as needed. Run sbatch.sh to initiate pipeline running. 

    bash sbatch.sh

  • To run in a local environment: 

    snakemake -p --cores 16 --keep-going --rerun-incomplete --jobs 300 --latency-wait 120 all

  • Look in log directory for logs for each rule

  • To view the snakemkae rule graph: 

    snakemake --rulegraph | dot -T png > nanopore.png

    dag

IV Output directory structure 

{output directory}
├── mbc # Modified base calling on primary (eg human) genome
│ ├── basic # using the default model
│ │ └── {sampleID}
│ └── model_based # using a specific model
│ └── {sampleID}
├── mbc_hpv # Modified base calling on secondary (eg HPV) genome
│ ├── basic # using the default model
│ │ └── {sampleID}
│ └── model_based # using a specific model
│ └── {sampleID}
├── sv # SV calling on primary genome
│ └── {sampleID}
├── sv_hpv # SV calling on secondary genome
│ └── {sampleID}
├── vc # Haplotype-aware variant calling on primary genome
│ └── {smapleID}
└── vc_hpv # Haplotype-aware variant calling on secondary genome
 └── {smapleID}

Code Snippets

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shell:
     """
     megalodon {input[0]} \
               --guppy-server-path ${{MEGALODON_GUPPY_PATH}} \
               --outputs {params.outputs} \
               --output-directory {out}mbc/basic/{wildcards.sample} \
               --overwrite \
               --reference {input[1]} \
               --mod-map-emulate-bisulfite \
               --mod-map-base-conv {params.base_conv} \
               --mod-map-base-conv Z C \
               --mod-motif m CG 0 \
               --mod-binary-threshold {params.thresh} \
               --devices ${{CUDA_VISIBLE_DEVICES}} \
               --processes ${{SLURM_CPUS_PER_TASK}} 2>log/{wildcards.sample}_basic.err
     touch {output}
     """
SnakeMake From line 15 of modules/Snakefile_MBC 
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shell:
      """
      megalodon {input[0]} \
                --guppy-server-path ${{MEGALODON_GUPPY_PATH}} \
                --guppy-config {params.config} \
                --outputs {params.outputs} \
                --overwrite \
                --output-directory {out}mbc/model_based/{wildcards.sample} \
                --reference {input[1]} \
                --mod-map-emulate-bisulfite \
                --mod-map-base-conv {params.base_conv} \
                --mod-map-base-conv Z C \
                --mod-motif m CG 0 \
                --mod-binary-threshold {params.thresh} \
                --devices ${{CUDA_VISIBLE_DEVICES}} \
                --processes ${{SLURM_CPUS_PER_TASK}} 2>log/{wildcards.sample}_model.err
      touch {output}
      """
SnakeMake From line 45 of modules/Snakefile_MBC 
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shell:
      """
      samtools sort {out}mbc/basic/{wildcards.sample}/mappings.bam > {out}mbc/basic/{wildcards.sample}/mappings_sorted.bam 2>log/{wildcards.sample}_basic_bam_sort1.err
      samtools index {out}mbc/basic/{wildcards.sample}/mappings_sorted.bam 2>log/{wildcards.sample}_basic_bam_index1.err
      samtools sort {out}mbc/basic/{wildcards.sample}/mod_mappings.5mC.bam > {out}mbc/basic/{wildcards.sample}/mod_mappings.5mC_sorted.bam 2>log/{wildcards.sample}_basic_bam_sort3.err
      samtools index {out}mbc/basic/{wildcards.sample}/mod_mappings.5mC_sorted.bam 2>log/{wildcards.sample}_basic_bam_index3.err
      """
SnakeMake SAMtools From line 72 of modules/Snakefile_MBC 
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shell:
      """
      samtools sort {out}mbc/model_based/{wildcards.sample}/mappings.bam > {out}mbc/model_based/{wildcards.sample}/mappings_sorted.bam 2>log/{wildcards.sample}_model_based_bam_sort1.err
      samtools index {out}mbc/model_based/{wildcards.sample}/mappings_sorted.bam 2>log/{wildcards.sample}_model_based_bam_index1.err
      samtools sort {out}mbc/model_based/{wildcards.sample}/mod_mappings.5mC.bam > {out}mbc/model_based/{wildcards.sample}/mod_mappings.5mC_sorted.bam 2>log/{wildcards.sample}_model_based_bam_sort3.err
      samtools index {out}mbc/model_based/{wildcards.sample}/mod_mappings.5mC_sorted.bam 2>log/{wildcards.sample}_model_based_bam_index3.err
      """
SnakeMake SAMtools From line 88 of modules/Snakefile_MBC 
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shell:
     """
     megalodon {input[0]} \
               --guppy-server-path ${{MEGALODON_GUPPY_PATH}} \
               --outputs {params.outputs} \
               --output-directory {out}mbc_hpv/basic/{wildcards.sample} \
               --overwrite \
               --reference {input[1]} \
               --mod-map-emulate-bisulfite \
               --mod-map-base-conv {params.base_conv} \
               --mod-map-base-conv Z C \
               --mod-motif m CG 0 \
               --mod-binary-threshold {params.thresh} \
               --devices ${{CUDA_VISIBLE_DEVICES}} \
               --processes ${{SLURM_CPUS_PER_TASK}} 2>log/{wildcards.sample}_basic_hpv.err
     touch {output}
     """
SnakeMake From line 15 of modules/Snakefile_MBC_hpv 
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shell:
      """
      megalodon {input[0]} \
                --guppy-server-path ${{MEGALODON_GUPPY_PATH}} \
                --guppy-config {params.config} \
                --outputs {params.outputs} \
                --overwrite \
                --output-directory {out}mbc_hpv/model_based/{wildcards.sample} \
                --reference {input[1]} \
                --mod-map-emulate-bisulfite \
                --mod-map-base-conv {params.base_conv} \
                --mod-map-base-conv Z C \
                --mod-motif m CG 0 \
                --mod-binary-threshold {params.thresh} \
                --devices ${{CUDA_VISIBLE_DEVICES}} \
                --processes ${{SLURM_CPUS_PER_TASK}} 2>log/{wildcards.sample}_model_hpv.err
      touch {output}
      """
SnakeMake From line 45 of modules/Snakefile_MBC_hpv 
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shell:
      """
      samtools sort {out}mbc_hpv/basic/{wildcards.sample}/mappings.bam > {out}mbc_hpv/basic/{wildcards.sample}/mappings_sorted.bam 2>log/{wildcards.sample}_basic_bam_sort1_hpv.err
      samtools index {out}mbc_hpv/basic/{wildcards.sample}/mappings_sorted.bam 2>log/{wildcards.sample}_basic_bam_index1_hpv.err
      samtools sort {out}mbc_hpv/basic/{wildcards.sample}/mod_mappings.5mC.bam > {out}mbc_hpv/basic/{wildcards.sample}/mod_mappings.5mC_sorted.bam 2>log/{wildcards.sample}_basic_bam_sort3_hpv.err
      samtools index {out}mbc_hpv/basic/{wildcards.sample}/mod_mappings.5mC_sorted.bam 2>log/{wildcards.sample}_basic_bam_index3_hpv.err
      """
SnakeMake SAMtools From line 72 of modules/Snakefile_MBC_hpv 
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shell:
      """
      samtools sort {out}mbc_hpv/model_based/{wildcards.sample}/mappings.bam > {out}mbc_hpv/model_based/{wildcards.sample}/mappings_sorted.bam 2>log/{wildcards.sample}_model_based_bam_sort1_hpv.err
      samtools index {out}mbc_hpv/model_based/{wildcards.sample}/mappings_sorted.bam 2>log/{wildcards.sample}_model_based_bam_index1_hpv.err
      samtools sort {out}mbc_hpv/model_based/{wildcards.sample}/mod_mappings.5mC.bam > {out}mbc_hpv/model_based/{wildcards.sample}/mod_mappings.5mC_sorted.bam 2>log/{wildcards.sample}_model_based_bam_sort3_hpv.err
      samtools index {out}mbc_hpv/model_based/{wildcards.sample}/mod_mappings.5mC_sorted.bam 2>log/{wildcards.sample}_model_based_bam_index3_hpv.err
      """
SnakeMake SAMtools From line 88 of modules/Snakefile_MBC_hpv 
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shell:
      """
      lra index -ONT {input} 2>log/index_lra.err
      """
SnakeMake From line 24 of modules/Snakefile_SV 
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shell:
      """
      catfishq -r {out}mbc/basic/{wildcards.sample}/ | seqtk seq -A - | lra align -ONT -t 16 {input.REF} - -p s | samtools addreplacerg -r \"@RG\tID:{wildcards.sample}\tSM:{wildcards.sample}\" - | samtools sort -@ 16 -T {wildcards.sample} -O BAM -o {output.BAM} - && samtools index -@ 16 {output.BAM} 2>log/{wildcards.sample}_lra_map.err
      """
SnakeMake SAMtools seqtk From line 40 of modules/Snakefile_SV 
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shell:
      """
      cuteSV -t 16 \
             --min_size {params.min_size} \
             --max_size  {params.max_size} \
             -S {wildcards.sample} \
             --retain_work_dir \
             --report_readid \
             --min_support {params.min_read_support} \
             --genotype {input.BAM} {input.REF} {output.VCF} \
             {out}sv/{wildcards.sample}/sv_calls/ 2>log/{wildcards.sample}_sv_call.err
      """
SnakeMake cuteSV From line 60 of modules/Snakefile_SV 
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shell:
      """
      mkdir -p {output.DIR}
      mosdepth -x -t 16 -n -b {params.BED} {output.DIR}/{wildcards.sample} {input.BAM} 2>log/{wildcards.sample}_depth.err
      """
SnakeMake mosdepth From line 82 of modules/Snakefile_SV 
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shell:
      """
      vcfsort {input.VCF} > {output.VCF} 2>log/{wildcards.sample}_sort_vcf.err
      """
SnakeMake From line 111 of modules/Snakefile_SV 
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shell:
      """
      cat {input.VCF} | bgziptabix {output.VCF} 2>log/{wildcards.sample}_index_vcf.err
      """
SnakeMake From line 123 of modules/Snakefile_SV 
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shell:
      """
      NanoPlot -t 16 \
               --bam {input.BAM} \
               --raw -o {output.DIR} \
               -p {wildcards.sample}_ \
               --N50 \
               --title {wildcards.sample} \
               --downsample 100000 2>log/{wildcards.sample}_qc.err
      """
SnakeMake NanoPlot From line 135 of modules/Snakefile_SV 
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shell:
      """
      cd {out}sv/{wildcards.sample}/sv_calls/ 
      java -Xmx16g -jar $SNPEFF_JAR -v hg38 {input.VCF} > {output.VCF} 2>{run}log/{wildcards.sample}_snpeff.err
      """
SnakeMake From line 152 of modules/Snakefile_SV 
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run:
      if config["snpsift"] == "yes":
         shell("""cat {input.VCF} | java -jar $SNPSIFT_JAR filter "{params.filter}" > {output.VCF} 2>log/{wildcards.sample}_snpsift.err""")
      else:
           shell("""touch {output.VCF}""")
SnakeMake From line 166 of modules/Snakefile_SV 
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shell:
      """
      lra index -ONT {input} 2>log/index_lra_hpv.err
      """
SnakeMake From line 28 of modules/Snakefile_SV_hpv 
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shell:
      """
      catfishq -r {out}mbc_hpv/basic/{wildcards.sample}/ | seqtk seq -A - | lra align -ONT -t 16 {input.REF} - -p s | samtools addreplacerg -r \"@RG\tID:{wildcards.sample}\tSM:{wildcards.sample}\" - | samtools sort -@ 16 -T {wildcards.sample} -O BAM -o {output.BAM} - && samtools index -@ 16 {output.BAM} 2>log/{wildcards.sample}_lra_map_hpv.err
      """
SnakeMake SAMtools seqtk From line 44 of modules/Snakefile_SV_hpv 
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shell:
      """
      cuteSV -t 16 \
             --min_size {params.min_size} \
             --max_size  {params.max_size} \
             -S {wildcards.sample} \
             --retain_work_dir \
             --report_readid \
             --min_support {params.min_read_support} \
             --genotype {input.BAM} {input.REF} {output.VCF} \
             {out}sv_hpv/{wildcards.sample}/sv_calls/ 2>log/{wildcards.sample}_sv_call_hpv.err
      """
SnakeMake cuteSV From line 64 of modules/Snakefile_SV_hpv 
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shell:
      """
      mkdir -p {output.DIR}
      mosdepth -x -t 16 -n -b {params.BED} {output.DIR}/{wildcards.sample} {input.BAM} 2>log/{wildcards.sample}_depth_hpv.err
      """
SnakeMake mosdepth From line 86 of modules/Snakefile_SV_hpv 
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shell:
      """
      vcfsort {input.VCF} > {output.VCF} 2>log/{wildcards.sample}_sort_vcf_hpv.err
      """
SnakeMake From line 115 of modules/Snakefile_SV_hpv 
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shell:
      """
      cat {input.VCF} | bgziptabix {output.VCF} 2>log/{wildcards.sample}_index_vcf_hpv.err
      """
SnakeMake From line 127 of modules/Snakefile_SV_hpv 
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shell:
      """
      NanoPlot -t 16 \
               --bam {input.BAM} \
               --raw -o {output.DIR} \
               -p {wildcards.sample}_ \
               --N50 \
               --title {wildcards.sample} \
               --downsample 100000 2>log/{wildcards.sample}_qc_hpv.err
      """
SnakeMake NanoPlot From line 138 of modules/Snakefile_SV_hpv 
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shell:
      """
      cd {out}sv_hpv/{wildcards.sample}/sv_calls/
      java -Xmx16g -jar {snpeff} -c {snpeff_config} -v {snpeff_hpv} {input.VCF} > {output.VCF} 2>{run}log/{wildcards.sample}_snpeff_hpv.err
      """
SnakeMake From line 155 of modules/Snakefile_SV_hpv 
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shell:
      """
      minimap2 -ax map-ont {input[1]} {out}mbc/basic/{wildcards.sample}/basecalls.fastq > {output} 2> log/{wildcards.sample}_minimap.err
      """
SnakeMake Minimap2 From line 12 of modules/Snakefile_VC 
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shell:
      """
      samtools view -b {input} | samtools sort > {output[0]} 2>log/{wildcards.sample}_minimap_sort.err
      samtools index {output} 2>log/{wildcards.sample}_minimap_index.err
      """
SnakeMake SAMtools From line 24 of modules/Snakefile_VC 
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shell:
     """
     {bind}
     singularity exec \
     {pepper}/pepper_deepvariant_r0.4.sif \
     run_pepper_margin_deepvariant call_variant \
               -b {input[0]} \
               -f {input[1]} \
               -p {wildcards.sample}_PEPPER_Margin_DeepVariant \
               -o {out}vc/{wildcards.sample} \
               -t 30 \
               --ont \
               --phased_output 2>log/{wildcards.sample}_pmd.err
     touch {output[1]}
     """
SnakeMake Singularity Hub From line 42 of modules/Snakefile_VC 
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shell:
      """
      cd {out}vc/{wildcards.sample}/
      java -Xmx16g -jar $SNPEFF_JAR -v hg38 {input} > {output} 2>{run}log/{wildcards.sample}_snpeff2.err
      """
SnakeMake From line 64 of modules/Snakefile_VC 
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run:
      if config["snpsift2"] == "yes":
         shell("""cat {input} | java -jar $SNPSIFT_JAR filter "{params.filter}" > {output} 2>log/{wildcards.sample}_snpsift2.err""")
      else:
           shell("""touch {output}""")
SnakeMake From line 78 of modules/Snakefile_VC 
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shell:
      """
      cd {out}vc/{wildcards.sample}/filter/
      java -Xmx16g -jar $SNPEFF_JAR -v hg38 {input} > {output} 2>{run}log/{wildcards.sample}_snpeff3.err
      """
SnakeMake From line 90 of modules/Snakefile_VC 
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shell:
      """
      minimap2 -ax map-ont {input[1]} {out}mbc_hpv/basic/{wildcards.sample}/basecalls.fastq > {output} 2> log/{wildcards.sample}_minimap_hpv.err
      """
SnakeMake Minimap2 From line 16 of modules/Snakefile_VC_hpv 
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shell:
      """
      samtools view -b {input} | samtools sort > {output[0]} 2>log/{wildcards.sample}_minimap_sort_hpv.err
      samtools index {output} 2>log/{wildcards.sample}_minimap_index_hpv.err
      """
SnakeMake SAMtools From line 28 of modules/Snakefile_VC_hpv 
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shell:
     """
     {bind}
     singularity exec \
     {pepper}/pepper_deepvariant_r0.4.sif \
     run_pepper_margin_deepvariant call_variant \
               -b {input[0]} \
               -f {input[1]} \
               -p {wildcards.sample}_PEPPER_Margin_DeepVariant \
               -o {out}vc_hpv/{wildcards.sample} \
               -t 16 \
               --ont \
               --phased_output 2>log/{wildcards.sample}_pmd_hpv.err
     touch {output}
     """
SnakeMake Singularity Hub From line 41 of modules/Snakefile_VC_hpv 
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shell:
      """
      cd {out}vc_hpv/{wildcards.sample}/

      java -Xmx16g -jar {snpeff} -c {snpeff_config} -v {snpeff_hpv} {params.vcf} > {output} 2>{run}log/{wildcards.sample}_snpeff2_hpv.err
      """
SnakeMake From line 65 of modules/Snakefile_VC_hpv
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Created: 1yr ago
Updated: 1yr ago
Maitainers: public
URL: https://github.com/NCI-CGR/Nanopore_DNA-seq
Name: nanopore_dna-seq
Version: 1
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Copyright: Public Domain
License: MIT License
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This Snakemake pipeline implements the GATK best-practices workflow for calling small germline variants. The usage of thi...
VCF raw sequence reads Variant calling genetic variants gatk rust-bio-tools snakemake-wrapper-utils tabix BCFtools BWA FastQC MultiQC Pandas Picard SAMtools Snakemake Trimmomatic Variant Effect Predictor (VEP) common matplotlib numpy seaborn DNA
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