Introduction

CBRA (CIBERER Bioinformatics for Rare diseases Analysis) is a workflow optimized for the analysis of rare diseases, designed to detect SNVs, INDELs, CNVs and SVs in targeted sequencing data (CES/WES) as well as whole genome sequencing (WGS).

This pipeline is developed using Nextflow, a workflow management system that enables an easy execution across various computing environments. It uses Docker or Singularity containers, simplifying setup and ensuring reproducibility of results. The pipeline assigns a container to each process, which simplifies the management and updating of software dependencies. When possible, processes are sourced from nf-core/modules, promoting reusability across all nf-core pipelines and contributing to the broader Nextflow community.

Pipeline summary

The pipeline can perform the following steps:

• Mapping (mapping = true) of the reads to reference (BWA-MEM)

• Process BAM file (GATK MarkDuplicates, GATK BaseRecalibrator and GATK ApplyBQSR)

• Single Nucleotide Variant (SNVs) calling (variant_calling = true) with the following tools:

  • GATK4 Haplotypecaller (run_gatk = true). This subworkflow includes:

    • GATK4 Haplotypecaller.
    • Hard Filters and VarianFiltration to mark PASS variants. More information here.
    • Bcftools Filter to keep PASS variants on chr1-22, X, Y.
    • Split Multialletic.

  • Dragen (run_dragen = true). This subworkflow includes:

    • GATK4 Calibratedragstrmodel
    • GATK4 Haplotypecaller with --dragen-mode.
    • VarianFiltration with --filter-expression "QUAL < 10.4139" --filter-name "DRAGENHardQUAL"to mark PASS variants. More information here.
    • Bcftools Filter to keep PASS variants on chr1-22, X, Y.
    • Split Multialletic.

  • DeepVariant (run_deepvariant = true). This subworkflow includes:

    • DeepVariant makeexamples: Converts the input alignment file to a tfrecord format suitable for the deep learning model.
    • DeepVariant callvariants: Call variants based on input tfrecords. The output is also in tfrecord format, and needs postprocessing to convert it to vcf.
    • DeepVariant postprocessvariants: Convert variant calls from callvariants to VCF, and also create GVCF files based on genomic information from makeexamples. More information here.
    • Bcftools Filter to keep PASS variants on chr1-22, X, Y.
    • Split Multialletic.

  • In addition to these three variant callers, a trio analysis can be performed with option trio_analysis = true. Based on GATK guides. This subworkflow includes:

    • GATK4 Haplotypecaller.
    • GATK4 Genomicsdbimport to merge GVCFs from multiple samples
    • GATK4 Genotypegvcfs to perform joint genotyping
    • Hard Filters and VarianFiltration to mark PASS variants. More information here.
    • Bcftools Filter to keep PASS variants on chr1-22, X, Y.
    • GATK4 Calculategenotypeposteriors to calculate genotype posterior probabilities given the family
    • GATK4 Variantfiltration based on genotypeposterior GQ<20
    • GATK4 Variantannotator to annotate possible de novo mutations in trios
    • Filter proband ref: filter variants that are REF in the proband
    • Split Multialletic.

• Additional analysis: Expansion Hunter (--run_expansionhunter true) for targeted genotyping of short tandem repeats (STRs) and flanking variants.

• Merge and integration of the vcfs obtained with the different tools.

• Annotation of SNVs (annotation = true) of the variants:

  • Regions of homozygosity (ROHs) with AUTOMAP

  • Effect of the variants with Ensembl VEP using the flag --everything, which includes the following options: --sift b, --polyphen b, --ccds, --hgvs, --symbol, --numbers, --domains, --regulatory, --canonical, --protein, --biotype, --af, --af_1kg, --af_esp, --af_gnomade, --af_gnomadg, --max_af, --pubmed, --uniprot, --mane, --tsl, --appris, --variant_class, --gene_phenotype, --mirna

  • Postvep format VEP tab demilited output and filter variants by minor allele frequency (--maf).

  • You can enhance the annotation by incorporating gene rankings from GLOWgenes, a network-based algorithm developed to prioritize novel candidate genes associated with rare diseases. To add GLOWgenes, set the parameter --glowgenes true. GLOWgenes will run using the list of genes specified in the --gen_list parameter (an example here). If you already have a GLOWgenes ranking file, you can add it directly using --glowgenes_ranking. Precomputed rankings based on PanelApp gene panels are available here. To include a specific GLOWgenes ranking, use the option --glowgenes_ranking (path to the panel.txt), for example: --glowgenes_ranking https://raw.githubusercontent.com/TBLabFJD/GLOWgenes/refs/heads/master/precomputed_panelAPP/GLOWgenes_prioritization_Neurological_ciliopathies_GA.txt.

  • Additionally, you can include the Gene-Disease Specificity Score (SGDS) using: --sgds. This score ranges from 0 to 1, where 1 indicates a gene ranks highly for only a few specific diseases (high specificity), and 0 indicates the gene consistently ranks highly across many diseases (low specificity).

• Copy number variants (CNVs) calling (cnvs = true), with the following steps:

  • Bed file filtering: Module to filter the bed file used for targered sequencing, to keep only the regions with a length > --min_target (default 20) and to exclude the regions in --chromosomes (default 'chrX,X,chrY,Y,chrM,MT').
  • Software for detecting CNVs: These tools require a set of samples sequenced in the same batch in order to detect changes in coverage that indicate the presence of a CNV.
    • ExomeDepth (exomedepth = true).
    • panelcn.MOPS (panelcmops = true).
    • CoNVaDING (convading = true).
  • Combining the results from the various CNVs caller
  • CNVs Annotation: AnnotSV is used to annotate the merged results. AnnotSV needs the annotations files. They can be downloaded using annotsv_install_annotations = true. The path to the notes folder can be specified using --annotsv_annotations folder_path. If --annotsv_annotations is not specified, the annotations files will be downloadad directly.

Usage

First, prepare a samplesheet with your input data:

sample,fastq_1,fastq_2
SAMPLE_PAIRED_END,/path/to/fastq/files/AEG588A1_S1_L002_R1_001.fastq.gz,/path/to/fastq/files/AEG588A1_S1_L002_R2_001.fastq.gz

Each row represents a pair of paired end fastq files.

You can run the pipeline using:

nextflow run CBRA/main.nf \
   -profile  \
   --input samplesheet.csv \
   --outdir 

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

Pipeline output

For details about the output files and reports, please refer to the output documentation.

Credits

CBRA was developed within the framework of a call for intramural cooperative and complementary actions (ACCI) funded by CIBERER (Biomedical Research Network Centre for Rare Diseases).

Main Developer

• Yolanda Benítez Quesada

Coordinator

• Carlos Ruiz Arenas

Other contributors

• Graciela Uría Regojo
• Pedro Garrido Rodríguez
• Rafa Farias Varona
• Pablo Minguez
• Daniel Lopez
• Cristina Arias Sardá