Introduction

genomic-medicine-sweden/nallo 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.

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 (HiFi only) (TRGT)
• Assembly based variant calls (HiFi only) (dipcall)
• CNV-calling (HiFiCNV)
• Call paralogous genes (Paraphase)

Phasing and methylation

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

Annotation

• Annotate SNVs and INDELs with database(s) of choice, i.e. gnomAD, CADD etc. (echtvar and VEP)
• Annotate repeat expansions with stranger

Filtering and ranking

• Rank variants (GENMOD)

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.

Prepare a samplesheet with input data:

samplesheet.csv

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

Now, you can run the pipeline using:

nextflow run genomic-medicine-sweden/nallo -profile YOURPROFILE \
    --input samplesheet.csv \
    --preset <revio/pacbio/ONT_R10> \
    --fasta <reference.fasta> \
    --outdir <OUTDIR>

For more details and further functionality, please refer to the usage documentation.

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.

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

nf-core download genomic-medicine-sweden/nallo

Credits

genomic-medicine-sweden/nallo 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.

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.