Introduction

fellen31/skierfe is a bioinformatics analysis pipeline for long-read rare disease SV/SNV identification using both PacBio and (targeted) ONT-data. Heavily influenced by best-practice pipelines such as nf-core/nanoseq, nf-core/sarek, nf-core/raredisease, PacBio Human WGS Workflow, epi2me-labs/wf-human-variation and brentp/rare-disease-wf.

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

QC

• FastQC (FastQC)
• Aligned read QC (cramino)
• Depth information (mosdepth)

Alignment & assembly

• Align reads to reference (minimap2)
• Assemble (trio-binned) haploid genomes (HiFi only) (hifiasm)

Variant calling

• Short variant calling & joint genotyping of SNVs (deepvariant + GLNexus)
• SV calling and joint genotyping (sniffles2)
• Tandem repeats (TRGT)
• Assembly based variant calls (HiFi only) (dipcall)
• CNV-calling (HiFi only) (HiFiCNV)

Phasing and methylation

• Phase and haplotag reads (whatshap + hiphase)
• Methylation pileups (Revio/ONT) (modkit)

Annotation - SNV

1. Annotate variants with database(s) of choice, i.e. gnomAD, CADD etc. (echtvar)
2. Annotate variants (VEP)

Filtering

• TBD

Usage

Note If you are new to Nextflow and nf-core, please refer to this page on how to set-up Nextflow. Make sure to test your setup with -profile test before running the workflow on actual data.

1. Prepare a samplesheet with input data (gzipped fastq-files):

samplesheet.csv

sample,file,family_id,paternal_id,maternal_id,sex,phenotype
HG002,/path/to/HG002.fastq.gz,FAM1,HG003,HG004,1,1
HG005,/path/to/HG005.fastq.gz,FAM1,HG003,HG004,2,1

2. Optional inputs:

• Limit SNV calling to regions in BED file (--bed)
• If running dipcall, download a BED file with PAR regions (hg38)
• If running TRGT, download a BED file with tandem repeats (TRGT) matching your reference genome.
• If running SNV annotation, download VEP cache and prepare a samplesheet with annotation databases (echtvar encode):
• If running CNV-calling, expected CN regions for your reference genome can be downloaded from HiFiCNV GitHub

snp_dbs.csv

sample,file
gnomad,/path/to/gnomad.v3.1.2.echtvar.popmax.v2.zip
cadd,/path/to/cadd.v1.6.hg38.zip

Note If running dipcall, make sure chrY PAR is hard masked in reference.

3. Download the pipeline and test it on a minimal dataset run with a single command run:

   nextflow run fellen31/skierfe -r dev -profile YOURPROFILE \
     --input samplesheet.csv \
     --outdir <OUTDIR> \
     --dipcall_par hs38.PAR.bed \
     --fasta GRCh38_no_alt_analysis_set.fasta \
     --trgt_repeats repeat_catalog_and_pathogenic.bed \
     --snp_db snp_dbs.csv \
     --vep_cache /path/to/vep/cache/dir/ \
     --preset revio/pacbio/ONT_R10

To run in an offline environment, download the pipeline using nf-core download:

nf-core download fellen31/skierfe -r dev

• The pipeline comes with config profiles called docker, singularity, podman, shifter and charliecloud and which instruct the pipeline to use the named tool for software management. For example, -profile test,docker.
• Please check nf-core/configs to see if a custom config file to run nf-core pipelines already exists for your Institute. If so, you can simply use -profile <institute> in your command. This will enable either docker or singularity and set the appropriate execution settings for your local compute environment.
• 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.

Warning: Please provide pipeline parameters via the CLI or Nextflow -params-file option. Custom config files including those provided by the -c Nextflow option can be used to provide any configuration except for parameters; see docs.

For more information about pipeline parameters, see usage.

Credits

fellen31/skierfe was originally written by Felix Lenner.

We thank the following people for their extensive assistance in the development of this pipeline:

Contributions and Support

If you would like to contribute to this pipeline, please see the contributing guidelines any contribution is very welcome.

Citations

This pipeline uses code and infrastructure developed and maintained by the nf-core community, reused here under the MIT license.

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.

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