Viralrecon

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Introduction

nf-core/viralrecon is a bioinformatics analysis pipeline used to perform assembly and intra-host/low-frequency variant calling for viral samples. The pipeline supports both Illumina and Nanopore sequencing data. For Illumina short-reads the pipeline is able to analyse metagenomics data typically obtained from shotgun sequencing (e.g. directly from clinical samples) and enrichment-based library preparation methods (e.g. amplicon-based: ARTIC SARS-CoV-2 enrichment protocol; or probe-capture-based). For Nanopore data the pipeline only supports amplicon-based analysis obtained from primer sets created and maintained by the ARTIC Network.

On release, automated continuous integration tests run the pipeline on a full-sized dataset on the AWS cloud infrastructure. This ensures that the pipeline runs on AWS, has sensible resource allocation defaults set to run on real-world datasets, and permits the persistent storage of results to benchmark between pipeline releases and other analysis sources. The results obtained from running the full-sized tests individually for each --platform option can be viewed on the nf-core website and the output directories will be named accordingly i.e. platform_illumina/ and platform_nanopore/.

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

The pipeline has numerous options to allow you to run only specific aspects of the workflow if you so wish. For example, for Illumina data you can skip the host read filtering step with Kraken 2 with --skip_kraken2 or you can skip all of the assembly steps with the --skip_assembly parameter. See the usage and parameter docs for all of the available options when running the pipeline.

The SRA download functionality has been removed from the pipeline (>=2.1) and ported to an independent workflow called nf-core/fetchngs. You can provide --nf_core_pipeline viralrecon when running nf-core/fetchngs to download and auto-create a samplesheet containing publicly available samples that can be accepted directly by the Illumina processing mode of nf-core/viralrecon.

A number of improvements were made to the pipeline recently, mainly with regard to the variant calling. Please see Major updates in v2.3 for a more detailed description.

Illumina

1. Merge re-sequenced FastQ files (cat)
2. Read QC (FastQC)
3. Adapter trimming (fastp)
4. Statistics/removal of host reads (Kraken 2; optional)
5. Variant calling
   1. Read alignment (Bowtie 2)
   2. Sort and index alignments (SAMtools)
   3. Primer sequence removal (iVar; amplicon data only)
   4. Duplicate read marking (picard; optional)
   5. Alignment-level QC (picard, SAMtools)
   6. Genome-wide and amplicon coverage QC plots (mosdepth)
   7. Choice of multiple variant callers (iVar variants; default for amplicon data || BCFTools; default for metagenomics data)
      • Variant annotation (SnpEff, SnpSift)
   8. Choice of multiple consensus callers (BCFTools, BEDTools; default for both amplicon and metagenomics data || iVar consensus)
      • Consensus assessment report (QUAST)
      • Lineage analysis (Pangolin)
      • Clade assignment, mutation calling and sequence quality checks (Nextclade)
   9. Relative lineage abundance analysis from mixed SARS-CoV-2 samples (Freyja)
   10. Create variants long format table collating per-sample information for individual variants (BCFTools), functional effect prediction (SnpSift) and lineage analysis (Pangolin)
6. De novo assembly
   1. Primer trimming (Cutadapt; amplicon data only)
   2. Choice of multiple assembly tools (SPAdes || Unicycler || minia)
      • Blast to reference genome (blastn)
      • Contiguate assembly (ABACAS)
      • Assembly report (PlasmidID)
      • Assembly assessment report (QUAST)
7. Present QC and visualisation for raw read, alignment, assembly and variant calling results (MultiQC)

Nanopore

1. Sequencing QC (pycoQC)
2. Aggregate pre-demultiplexed reads from MinKNOW/Guppy (artic guppyplex)
3. Read QC (NanoPlot)
4. Statistics/removal of host reads (Kraken 2; optional)
5. Align reads, call variants and generate consensus sequence (artic minion)
6. Remove unmapped reads and obtain alignment metrics (SAMtools)
7. Genome-wide and amplicon coverage QC plots ([mosdepth](https:/