alignment-nf

The alignment-nf pipeline performs alignment for wild isolate sequence data at the strain level, and outputs BAMs and related information. Those BAMs can be used for downstream analysis including variant calling, concordance analysis, wi-gatk-nf (variant calling) and other analyses.

This page details how to run the pipeline and how to add new wild isolate sequencing data. You can also find more information on the Andersen Lab dry guide.

Pipeline overview

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	parameters              description                                 Set/Default
	==========              ===========                                 ========================
	--debug                 Use --debug to indicate debug mode          null
	--sample_sheet          See test_data/sample_sheet for example      null
	--species               Species to map: 'ce', 'cb' or 'ct'          null
	--fq_prefix             Path to fastq if not in sample_sheet        /vast/eande106/data/{species}/WI/fastq/dna/
	--kmers                 Whether to count kmers                      false
	--reference             genome.fasta.gz to use in place of default  defaults for c.e, c.b, and c.t
	--output                Output folder name.                         alignment-{date}

	HELP: http://andersenlab.org/dry-guide/pipelines/pipeline-alignment/

Software requirements

• Nextflow v23+ (see the dry guide on Nextflow here or the Nextflow documentation here). On Rockfish, you can access this version by loading the nf23_env conda environment prior to running the pipeline command:

module load python/anaconda
source activate /data/eande106/software/conda_envs/nf23_env

• Singularity. On Rockfish, you can get this with module load singularity before running

Note: previous versions of pipeline used conda environments on QUEST installed at /projects/b1059/software/conda_envs/ but this will no longer be maintained

Usage

Testing on Rockfish

This command uses a test dataset

nextflow run -latest andersenlab/alignment-nf --debug

Running on Rockfish

You should run this in a screen or tmux session.

Note: if you are having issues running Nextflow or need reminders, check out the Nextflow page.

nextflow run -latest andersenlab/alignment-nf --sample_sheet <path_to_sample_sheet> --species c_elegans

Parameters

-profile

There are three configuration profiles for this pipeline.

• rockfish - Used for running on Rockfish (default).
• quest - Used for running on Quest.
• local - Used for local development.

Note

If you forget to add a -profile, the rockfish profile will be chosen as default

--sample_sheet

The sample sheet for alignment is the output from the trim-fq-nf pipeline. The sample sheet must be tsv formatted, is the full path to the sample sheet (even if it is in your current directory), and has the following columns:

• strain - the name of the strain. Multiple sequencing runs of the same strain are merged together.
• id - A unique ID for each sequencing run. This must be unique for every single pair of FASTQs.
• lb - A library ID. This should uniquely identify a DNA sequencing library.
• fq1 - The path to FASTQ1
• fq2 - The path to FASTQ2

Note

Remember that in --debug mode the pipeline will use the sample sheet located in test_data/sample_sheet.tsv.

The library column is a useful tool for identifying errors by variant callers. For example, if the same library is sequenced twice, and a variant is only observed in one sequencing run then that variant may be excluded as a technical / PCR artifact depending on the variant caller being used.

Important

The alignment pipeline will merge multiple sequencing runs of the same strain into a single bam. However, summary output is provided at both the strain and id level. In this way, if there is a poor sequencing run it can be identified and removed from a collection of sequencing runs belonging to a strain. For this reason, it is important that each id be unique and not just the strain name

Note

The sample sheet is a critical tool. It allows us to associated metadata with each sequencing run (e.g. isotype, reference strain, id, library). It also allows us to quickly verify that all results have been output. It is much easier than working with a list of files!

--debug (optional)

You should use --debug for testing/debugging purposes. This will run the debug test set (located in the test_data folder) using your specified configuration profile (e.g. rockfish / quest / local).

For example:

nextflow run -latest andersenlab/alignment-nf --debug -resume

Using --debug will automatically set the sample sheet to test_data/sample_sheet.tsv

--species (optional)

Defaults to "c_elegans", change to "c_briggsae" or "c_tropicalis" to select correct reference file. If species == "c_elegans", a check will be run for the npr-1 allele. Note: this process used to happen later in concordance-nf, however it was moved up to alignment-nf to avoid having to rerun the long wi-gatk process if an incorrect strain is included.

--fq_prefix (optional)

Within a sample sheet you may specify the locations of FASTQs using an absolute directory or a relative directory. If you want to use a relative directory, you should use the --fq_prefix to set the path that should be prefixed to each FASTQ.

Note

Previously, this option was --fqs_file_prefix

--kmers (optional)

default = false

Toggles kmer-analysis

--reference (optional)

A fasta reference indexed with BWA. WS245 is packaged with the pipeline for convenience when testing or running locally.

On Rockfish, the default references are here:

c_elegans: /vast/eande106/data/c_elegans/genomes/PRJNA13758/WS283/c_elegans.PRJNA13758.WS283.genome.fa.gz
c_briggsae: /vast/eande106/data/c_briggsae/genomes/QX1410_nanopore/Feb2020/c_briggsae.QX1410_nanopore.Feb2020.genome.fa.gz
c_tropicalis: /vast/eande106/data/c_tropicalis/genomes/NIC58_nanopore/June2021/c_tropicalis.NIC58_nanopore.June2021.genome.fa.gz

Note

A different --project and --wsbuild can be used with the --species parameter to generate the path to other reference genomes such as:

nextflow run -latest andersenlab/alignment-nf --species c_elegans --project PRJNA13758 --wsbuild WS280

--ncbi (optional)

Default - /vast/eande106/data/other/ncbi_blast_db/

Path to the NCBI blast database used for blobtool analysis. Should not need to change.

--blob (optional)

Defaults to true. Change to false if you don't need to run blobtool analysis on low coverage strains. This step can take a while, so if you don't need it you might want to exclude it.

--output (optional)

Default - alignment-YYYYMMDD

A directory in which to output results. If you have set --debug, the default output directory will be alignment-YYYYMMDD-debug.

Output

├── _aggregate
│   ├── kmers.tsv
│   └── multiqc
│       ├── strain_data/
│       │   ├── mqc_mosdepth-coverage-dist-id_1.txt
│       │   ├── mqc_mosdepth-coverage-per-contig_1.txt
│       │   ├── mqc_mosdepth-coverage-plot-id_1.txt
│       │   ├── mqc_picard_deduplication_1.txt
│       │   ├── mqc_samtools-idxstats-mapped-reads-plot_Counts.txt
│       │   ├── mqc_samtools-idxstats-mapped-reads-plot_Normalised_Counts.txt
│       │   ├── mqc_samtools_alignment_plot_1.txt
│       │   ├── multiqc.log
│       │   ├── multiqc_data.json
│       │   ├── multiqc_general_stats.txt
│       │   ├── multiqc_picard_dups.txt
│       │   ├── multiqc_qualimap_bamqc_genome_results.txt
│       │   ├── multiqc_samtools_flagstat.txt
│       │   ├── multiqc_samtools_idxstats.txt
│       │   ├── multiqc_samtools_stats.txt
│       │   └── multiqc_sources.txt
│       ├── strain_multiqc_report.html
│       ├── id_data/
│       │   └──... (same as strain_data/)
│       └── id_multiqc_report.html
├── bam
│   ├── [strain].bam
│   └── [strain].bam.bai
├── blobtools
│   ├── {strain}.*.blobplot.bam0.png
│   ├── {strain}.*.blobplot.read_cov.bam0.png
│   └── {strain}.*.blobplot.stats.txt
├── software_versions.txt
├── sample_sheet.tsv
├── strain_summary.tsv
├── stats_strain_all.tsv
├── stats_strains_with_low_values.tsv
├── sample_sheet_for_seq_sheet.tsv
├── sample_sheet_for_seq_sheet_ALL.tsv
├── low_map_cov_for_seq_sheet.Rmd
├── low_map_cov_for_seq_sheet.html
└── summary.txt

Most files should be obvious. A few are detailed below.

• software_versions.txt - Outputs the software versions used for every process (step) of the pipeline.
• summary.txt - Outputs a summary of the parameters used.
• sample_sheet.tsv - The sample sheet (input file) that was used to produce the alignment directory.
• strain_summary.tsv - A summary of all strains and bams in the alignment directory.
• aggregate - Stores data that has been aggregated across all strains or sequencing IDs.
• coverage - Contains coverage data at the strain or id level, presented in a variety of ways.
• low_map_cov_for_seq_sheet.(Rmd/html) - Report showing low coverage or problematic strains to remove.
• stats_strain_all.tsv - contains stats for all strains, with all replicates combined
• stats_strains_with_low_values.tsv - contains stats for strains with either (1) low number of reads, (2) low mapping rate, and/or (3) low coverage
• sample_sheet_for_seq_sheet.tsv - sample sheet to be added to google sheet, filtered to remove low coverage strains
• sample_sheet_for_seq_sheet_ALL.tsv - sample sheet to be added to google sheet, contains all strains (use this one)
• blobplot/ - contains plots for low coverage strains to see if they show contamination issues and if they should be resequenced.
• npr1_allele_strain.tsv - if species == c_elegans, this file will be output to show problematic strains that contain the N2 npr-1 allele and should be manually checked.

Relevant Docker Images

• andersenlab/alignment (link): Docker image is created within this pipeline using GitHub actions. Whenever a change is made to env/align.Dockerfile or .github/workflows/build_docker.yml GitHub actions will create a new docker image and push if successful
• andersenlab/blobtools (link): Docker image is created manually, code can be found in the dockerfile repo.
• andersenlab/multiqc (link): Docker image is created within the trim-fq-nf pipeline using GitHub actions. Whenever a change is made to env/multiqc.Dockerfile or .github/workflows/build_multiqc_docker.yml GitHub actions will create a new docker image and push if successful