Introduction

This document describes the output produced by the pipeline. Most of the plots are taken from the MultiQC report, which summarises results at the end of the pipeline.

The directories listed below will be created in the results directory after the pipeline has finished. All paths are relative to the top-level results directory.

Pipeline overview

The pipeline is built using Nextflow and processes data using the following steps:

• Preprocessing
  • FastP - trim low-quality reads, umi-barcodes, adapters
• Denovo Reference Construction
  • Prepare forward reads - combine forward and reverse sequences separated by ‘NNNNNNNNNN’
  • Combine uniqe reads - retain reads present n number of times between and across individuals
  • Seqtk - write dummy fasta file
  • Denovo FastP - trim adapters
  • CD-HIT-EST - cluster similar sequences
  • CD-HIT-EST to Rainbow div - convert cdhit file output into rainbow div input file format
  • Rainbow div - distiguish sequence errors from heterozygote or variants between repetitive sequences
  • Rainbow merge - merge potential heterozygous clusters
  • Write Fasta - convert rainbow merge file output into fasta format
• Alignment
  • SAMtools - Sort, index and obtain alignment statistics
  • BWA - short read aligner
  • BWAMEM2 - a faster short read aligner
  • UMI-tools dedup - UMI-based deduplication
• Freebayes Intervals
  • BEDtools bamtobed: converts bam file into bed datastructure
  • BEDOPS merge: merge indvidual bed files into a single bed file
  • BEDtools sort: sorts bed files
  • BEDtools coverage: counts read depth
  • BEDtools merge: merges indv bed files and takes sum the read depths
  • BEDtools makewindows: split regions with coverage above --max_read_coverage_to_split to half read length
  • BEDtools intersect: removes any overlapping regions between split reads and all merged reads
  • Create intervals: write regions for input to freebayes
• Variant Calling
  • FreeBayes - a bayesian genotyper tool
• Quality Control and Preprocessing
  • FastQC - Raw read QC
  • MultiQC - Aggregate report describing results and QC from the whole pipeline
  • Pipeline information - Report metrics generated during the workflow execution

Directory Structure

The default directory structure is as follows. This includes output from both denovo and reference methods.

{outdir}
├── denovo
│   ├── alignments
│   │   └── bwamem2
│   │       ├── bam
│   │       ├── index
│   │       ├── intervals
│   │       │   ├── bedops_merge
│   │       │   ├── bedtools_bamtobed
│   │       │   ├── bedtools_coverage
│   │       │   ├── bedtools_intersect
│   │       │   ├── bedtools_makewindows
│   │       │   ├── bedtools_merge
│   │       │   ├── bedtools_sort
│   │       │   └── create_intervals
│   │       ├── samtools_index
│   │       ├── samtools_merge
│   │       ├── samtools_stats
│   │       └── umitools_dedup
│   │           └── stats
│   ├── reference
│   │   ├── cdhit
│   │   ├── cdhit_to_rbdiv
│   │   ├── fastp
│   │   ├── rainbow_div
│   │   ├── rainbow_merge
│   │   ├── samtools
│   │   │   └── index
│   │   ├── seqtk
│   │   ├── unique_sequences
│   │   └── write_fasta
│   ├── trim_fastp
│   └── variant_calling
│       └── intervals
├── fastp
├── fastqc
├── multiqc
│   └── multiqc_data
├── pipeline_info
└── reference
    ├── alignments
    │   └── bwamem2
    │       ├── bam
    │       ├── index
    │       ├── intervals
    │       │   ├── bedops_merge
    │       │   ├── bedtools_bamtobed
    │       │   ├── bedtools_coverage
    │       │   ├── bedtools_intersect
    │       │   ├── bedtools_makewindows
    │       │   ├── bedtools_merge
    │       │   ├── bedtools_sort
    │       │   └── create_intervals
    │       ├── samtools_index
    │       ├── samtools_merge
    │       ├── samtools_stats
    │       └── umitools_dedup
    │           └── stats
    ├── samtools
    │   └── index
    └── variant_calling
        └── intervals

Pre-Processing

Radseq pre-processes reads prior to the alignment step.

FastP

FastP is a tool designed to be an all-in-one preprocessor for FastQ files. You can enable the saving of trimmed fq files in output directory through --save_trimmed=true.

Denovo Reference Construction

Radseq supports the construction of psuedoreference using a conglomerate of open source tools. By default the resulting fasta file is only outputed to enable the printing of intermediate files into {outdir}/denovo/reference/ use --denovo_intermediate_files=true.

Prepare forward reads

Prior to clustering forward and reverse reads are joined into one sequence and seperated with a NNNNNNNNNN. Reads are reduced based on number of presences within an individual and among all individuals and combined into one file.

Seqtk seq

Seqtk is a tool for processing FASTA or FASTQ file formats.

Denovo FastP

Denovo Fastp uses FastP as a tool to trim any unwanted sequences prior to clustering like adapter content.

Cdhit est

CD-HIT is used for clustering and comparing nucleotide sequences.

CD-HIT to Rainbow div

This module converts CD-HIT output into input for Rainbow div.

Rainbow div

Rainbow div is a tool used to divide heterozygote calls into putative haplotypes based on minimum thresholds passed as arguments.

Rainbow merge

Rainbow merge is a tool used to merge reads from Rainbow div and assemble into contigs based on minimum and maximum thresholds that are passed as arguments.

Write fasta

This module converts Rainbow merge into fasta format. These files are outputed by default to:

Alignment

Indices

enable the saving of reference indices with --save_reference_indices true generate from samtools and bwa for variant calling and short-read alignment respectiviely.

samtools faidx

bwa index

Not Working

Aligners

To enable the output of bam files use --save_bam_files=true. Output of bam files from different alignment methods follow the same output structure.

BWA

BWA is a tool for mapping sequencies with low divergence against a reference genome. Aligned reads are then potentially filtered and coordinate-sorted using samtools.

BWA-mem2

BWA-mem2 is a tool next version of bwa-mem for mapping sequencies with low divergence against a reference genome with increased processing speed (~1.3-3.1x). Aligned reads are then potentially filtered and coordinate-sorted using samtools.

UMI-tools dedup

UMI-tools dedup is a tool for deduplicating reads and ensuring only a single representative read is retained in the bam file. Files are outputed using --save_bam_files=true as a result of many individual bed files being passed through.

FreeBayes Interval Construction

radseq supports multithreading of FreeBayes through the bam_intervals_bedtools.nf subworkflow that uses a collection of BEDtools software and BEDOPS merge.

To enable the saving of files generated for interval construction in the output-folder set --save_bed_intervals=true.

For large datasets, it may be useful to randomly subsample the number of individuals going into bam_intervals_bedtools.nf subworkflow using --subset_intervals_channel=<integer>. Particularly at the BEDtools merge module the memory footprint can be large.

BEDtools bamtobed

Convert individual binary alignment files to bed format

BEDOPS merge

Merge individual bed files

BEDtools sort

Sort the single merged bed file based on reference index

BEDtools coverage

Calculate number of reads across regions in a single individual bed file.

BEDtools merge cov

Determine the sum of coverage across all regions and individuals.

BEDtools makewindows

Split regions with more than --splitByReadCoverage into half the total read length.

BEDtools intersect

Joins and removes any sites overlapping in the single merged bed file with split regions.

Create intervals

Splits bed files into regions for input to FreeBayes.

Variant Calling

FreeBayes

FreeBayes is a bayesian genetic variant detector capable of genotyping SNPs, indels, MNPs, and complex events smaller than the length of short-read sequencing alignment.

By default radseq will output a single vcf joined from independent runs. To enable the outputting of VCF files based on regions passed to FreeBayes use --save_freebayes_intervals=true.

Quality Control and Visualization

FastQC

FastQC gives general quality metrics about your sequenced reads. It provides information about the quality score distribution across your reads, per base sequence content (%A/T/G/C), adapter contamination and overrepresented sequences. For further reading and documentation see the FastQC help pages.

NB: The FastQC plots displayed in the MultiQC report shows untrimmed reads. They may contain adapter sequence and potentially regions with low quality.

FastP

FastP is a tool designed to be an all-in-one preprocessor for FastQ files. Statistics are passed to MultiQC.

samtools

Alignment statistics may be generated from BAM files with samtools stats, samtools flagstat, samtools idxstats.

bcftools stats

bcftools stats is a tool for collecting statistics from a VCF or BCF file that can be interpretted by MultiQC.

MultiQC

MultiQC is a visualization tool that generates a single HTML report summarising all samples in your project. Most of the pipeline QC results are visualised in the report and further statistics are available in the report data directory.

Results generated by MultiQC collate pipeline QC from supported tools e.g. FastQC. The pipeline has special steps which also allow the software versions to be reported in the MultiQC output for future traceability. For more information about how to use MultiQC reports, see http://multiqc.info.

Pipeline information

Nextflow provides excellent functionality for generating various reports relevant to the running and execution of the pipeline. This will allow you to troubleshoot errors with the running of the pipeline, and also provide you with other information such as launch commands, run times and resource usage.