PolkaPox :microbe: :dna: :accordion:

Introduction

PolkaPox is a workflow for filtering, trimming, QC, reference-based analysis, and de novo assembly of Illumina short sequencing reads from orthopoxviruses.

The pipeline is built using Nextflow, a workflow tool to run tasks across multiple compute infrastructures in a very portable manner. It uses Docker/Singularity containers making installation trivial and results highly reproducible. The Nextflow DSL2 implementation of this pipeline uses one container per process which makes it much easier to maintain and update software dependencies. Where possible, these processes have been submitted to and installed from nf-core/modules in order to make them available to all nf-core pipelines, and to everyone within the Nextflow community!

Pipeline summary

Read pre-processing 1. Filter raw reads to capture only Orthopox Virus sequences. (Kraken + SeqTK) 1. Trim and filter reads for adapter contamination and quality, summarize QC (Fastp)

Reference-based analyses 1. Align reads to reference genome. (BWA) 1. Variant calling and target gene mutation summary. (iVar) 1. Consensus sequence generation. (iVar)

Reference-free analyses 1. De novo assembly. (Unicycler) 1. Calculate assembly quality metrics. (QUAST) 1. Assembly graph resolution. (AssemblyGraph_gfaPy.py) 1. Align reads to assembled genome. (BWA) 1. Correct assembly errors and ambiguities. (iVar) 1. Quantify assembly corrections. (MUMmer)

Performance summary 1. Visualize reads metrics and summary of software versions. (MultiQC) 1. Compile various QC metrics. (summarize_qc.py)

Quick Start

1. Install Nextflow (>=21.10.3)

2. Install any of Docker, Singularity (you can follow this tutorial), Podman, Shifter or Charliecloud for full pipeline reproducibility (you can use Conda both to install Nextflow itself and also to manage software within pipelines. Please only use it within pipelines as a last resort; see docs).

   • Note: if running this pipeline on CDC infrascructure (aspen, biolinux), use singularity containers.

3. Clone this repo to your working environment:

console git clone git@github.com:CDCgov/polkapox.git

1. Prepare your samples. You have two options: > (A) Make a sample sheet which will act as pipeline input. The samplesheet should consist of three columns with sample ID, R1 and R2 specified. If you are using single-end data, only one fastq can be specified and the pipeline will auto detect this. Example:

console sample,fastq_1,fastq_2 CONTROL_REP1,AEG588A1_S1_L002_R1_001.fastq.gz,AEG588A1_S1_L002_R2_001.fastq.gz

(B) Pass a directory of FastQ files as your input and the pipeline will auto-create the samplesheet. See Usage for more details.

1. Start running your own analysis!

console nextflow run polkapox/main.nf --input {SAMPLESHEET.csv OR input_directory} --outdir {OUTDIR} --fasta {REF.fa} -profile sge,singularity --kraken_db {PATH/TO/DB} --gff {ANNOTATION.gff} --workflow {WORKFLOW} --filter {true/false}

note: If you do not provide --fasta, --gff, or --kraken_db, they will default to a Clade II reference and gff in the assets folder of this repo, and a kraken db will be downloaded from s3://io-pe1-prod-ncezid-oamd-nextstrain/polkapox/orthopox_kdb/. If you do not specify --filter then it will default to true. See nextflow.config for details. Add --file_levels {top (default)/nested} if passing a directory as input. See usage for details.

Pipeline configuration

Note that some form of configuration will be needed so that Nextflow knows how to fetch the required software. You can chain multiple config profiles in a comma-separated string.

• The pipeline comes with config profiles called docker, singularity, podman, shifter, charliecloud and conda which instruct the pipeline to use the named tool for software management. For example, -profile test,docker.
• If you are using singularity, please use the nf-core download command to download images first, before running the pipeline. Setting the NXF_SINGULARITY_CACHEDIR or singularity.cacheDir Nextflow options enables you to store and re-use the images from a central location for future pipeline runs.
• If you are using conda, it is highly recommended to use the NXF_CONDA_CACHEDIR or conda.cacheDir settings to store the environments in a central location for future pipeline runs.

Running on CDC cluster

For use on an HPC cluster (rosalind, aspen) the workflow can be run as a job by the following steps:

1) After logging in, activate the nextflow environment:

```console
module load nextflow
```

2) Point to the cdc.config file, which contains custom profiles for the CDC HPC clusters. Submit individual processes as jobs to the scheduler using a profile defined in cdc.config. For example, to run a job on rosalind:

```consol
nextflow run main.nf --input {SAMPLESHEET.csv} --outdir {OUTDIR} --fasta {REF.fa} -profile rosalind,singularity --kraken_db {PATH/TO/DB} --gff {ANNOTATION.gff} -config /scicomp/reference/nextflow/configs/cdc.config
```

Documentation

Basic usage

The PolkaPox pipeline requires two inputs: [1] a samplesheet (or input directory) --input and [2] a --workflow definition. Additional details are provided in usage.

Output

Pipeline outputs are organized into sub-directories for each step in the selected workflow. All paths are relative to the top-level results directory --outdir and additional details are provided in output.

${outdir}/ bwa bandage fastp final_assembly graph_recon graph_recon_mapping ivar ivar_variants kraken2 multiqc mummer pipeline_info quast sample_summary.tsv samtools seqtk unicycler variant_summaries

De novo assembly

The PolkaPox pipeline includes de novo assembly optimized for the linear genome architecture of orthopoxviruses. Additional details are provided in de novo.

Credits

Contributors:\ Kyle O'Connell | Michael Weigand | Jessica Rowell | Shatavia Morrison\ Kristen Knipe | Ethan Hetrick | Crystal Gigante | Lynsey Kovar\ Hunter Seabolt | Dhwani Batra | Daisy McGrath\ Yesh Kulasekarapandian | Jason Caravas

Contributions and Support

If you would like to contribute to this pipeline, please see the contributing guidelines.

Citations

An extensive list of references for the tools used by the pipeline can be found in the CITATIONS.md file.

You can cite the nf-core publication as follows:

The nf-core framework for community-curated bioinformatics pipelines.

Philip Ewels, Alexander Peltzer, Sven Fillinger, Harshil Patel, Johannes Alneberg, Andreas Wilm, Maxime Ulysse Garcia, Paolo Di Tommaso & Sven Nahnsen.

Nat Biotechnol. 2020 Feb 13. doi: 10.1038/s41587-020-0439-x.

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Repository Metadata

Organization: NCEZID-OAMD contact email: ncezid_shareit@cdc.gov exemption status: NA exemption justification: NA description fields: Nextflow workflow for the assembly of Orthopox virus sequences