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Introduction

genomic-medicine-sweden/nallo is a bioinformatics analysis pipeline for long-reads from both PacBio and (targeted) ONT-data, focused on rare-disease. Heavily influenced by best-practice pipelines such as nf-core/sarek, nf-core/raredisease, nf-core/nanoseq, PacBio Human WGS Workflow, epi2me-labs/wf-human-variation and brentp/rare-disease-wf.

genomic-medicine-sweden/nallo workflow

Pipeline summary

QC

Read QC with FastQC, cramino and mosdepth

Alignment & assembly

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

Variant calling

Call SNVs & joint genotyping with deepvariant and GLNexus
Call SVs with Severus or Sniffles2
Call CNVs with HiFiCNV
Call tandem repeats with TRGT (HiFi only)
Call paralogous genes with Paraphase
Call variants from assembly with dipcall (HiFi only)

Phasing and methylation

Phase and haplotag reads with LongPhase, whatshap or HiPhase
Create methylation pileups with modkit

Annotation

Annotate SNVs and INDELs with databases of choice, i.e. gnomAD, CADD etc. with echtvar and VEP
Annotate repeat expansions with stranger
Annotate SVs with SVDB and VEP

Ranking

Rank SNVs with 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
NIST,HG002,/path/to/HG002.fastq.gz,FAM1,HG003,HG004,1,2
NIST,HG005,/path/to/HG005.bam,FAM1,HG003,HG004,2,1

Supply a reference genome with --fasta and choose a matching --preset for your data (revio, pacbio, ONT_R10). Now, you can run the pipeline using:

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

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

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

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

If you use genomic-medicine-sweden/nallo for your analysis, please cite it using the following doi: 10.5281/zenodo.13748210.

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.