Nanopore long-read DNA sequencing data analysis pipeline

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:

Modified basing calling using Megalodon
Structural variant calling using Nanopore pipeline-structural-variation
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

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}

NCI-CGR / Nanopore_DNA-seq