genomic-medicine-sweden/nallo: Output

Introduction

This document describes the pipeline output files and the tools used to generate them.

Aligned reads

Minimap2 is used to map the reads to a reference genome. The aligned reads are sorted, merged and indexed using samtools. If the pipeline is run with phasing, the aligned reads will be happlotagged using the active phasing tool.

Path	Description	Alignment	Alignment & phasing
aligned_reads/minimap2/{sample}/*.bam	Alignment file in bam format
aligned_reads/minimap2/{sample}/*.bai	Index of the corresponding bam file

Path	Description	Alignment	Alignment & phasing
aligned_reads/{sample}/{sample}_haplotagged.bam	BAM file with haplotags
aligned_reads/{sample}/{sample}_haplotagged.bam.bai	Index of the BAM file

Assembly

Hifiasm is used to assemble genomes. The assembled haplotypes are then converted to fasta files using gfastats. A deconstructed version of dipcall is used to map the assembled haplotypes back to the reference genome.

Path	Description
assembly_haplotypes/gfastats/{sample}/*hap1.p_ctg.fasta.gz	Assembled haplotype 1
assembly_haplotypes/gfastats/{sample}/*hap2.p_ctg.fasta.gz	Assembled haplotype 2
assembly_haplotypes/gfastats/{sample}/*.assembly_summary	Summary statistics
assembly_variant_calling/dipcall/{sample}/*hap1.bam	Assembled haplotype 1 mapped to the reference genome
assembly_variant_calling/dipcall/{sample}/*hap1.bai	Index of the corresponding BAM file for haplotype 1
assembly_variant_calling/dipcall/{sample}/*hap2.bam	Assembled haplotype 2 mapped to the reference genome
assembly_variant_calling/dipcall/{sample}/*hap2.bai	Index of the corresponding BAM file for haplotype 2

Methylation pileups

Modkit is used to create methylation pileups, producing bedMethyl files for both haplotagged and ungrouped reads. Additionally, methylation information can be viewed in the BAM files, for example in IGV. When phasing is on, modkit outputs pileups per haplotype.

Path	Description	Alignment	Alignment & phasing
methylation/modkit/pileup/{sample}/*.modkit_pileup_phased_*.bed.gz	bedMethyl file with summary counts from haplotagged reads
methylation/modkit/pileup/{sample}/*.modkit_pileup_phased_ungrouped.bed.gz	bedMethyl file for ungrouped reads
methylation/modkit/pileup/{sample}/*.modkit_pileup.bed.gz	bedMethyl file with summary counts from all reads
methylation/modkit/pileup/{sample}/*.bed.gz.tbi	Index of the corresponding bedMethyl file

MultiQC

MultiQC generates an HTML report summarizing all samples' QC results and pipeline statistics.

Path	Description
multiqc/multiqc_report.html	HTML report summarizing QC results
multiqc/multiqc_data/	Directory containing parsed statistics
multiqc/multiqc_plots/	Directory containing static report images

Pipeline Information

Nextflow generates reports for troubleshooting, performance, and traceability.

Path	Description
pipeline_info/execution_report.html	Execution report
pipeline_info/execution_timeline.html	Timeline report
pipeline_info/execution_trace.txt	Execution trace
pipeline_info/pipeline_dag.dot	Pipeline DAG in DOT format
pipeline_info/pipeline_report.html	Pipeline report
pipeline_info/software_versions.yml	Software versions used in the run

Phasing

LongPhase, WhatsHap, or HiPhase are used for phasing.

Path	Description
aligned_reads/{sample}/{sample}_haplotagged.bam	BAM file with haplotags
aligned_reads/{sample}/{sample}_haplotagged.bam.bai	Index of the BAM file
phased_variants/{sample}/*.vcf.gz	VCF file with phased variants
phased_variants/{sample}/*.vcf.gz.tbi	Index of the VCF file
qc/phasing_stats/{sample}/*.blocks.tsv	Phase block file
qc/phasing_stats/{sample}/*.stats.tsv	Phasing statistics file

QC

FastQC, cramino, mosdepth, and somalier are used for read quality control.

FastQC

FastQC provides general quality metrics for sequenced reads, including information on quality score distribution, per-base sequence content (%A/T/G/C), adapter contamination, and overrepresented sequences. For more details, refer to the FastQC help pages.

Path	Description
qc/fastqc/{sample}/*_fastqc.html	FastQC report containing quality metrics
qc/fastqc/{sample}/*_fastqc.zip	Zip archive with the FastQC report, data files, and plot images

Mosdepth

Mosdepth is used to report quality control metrics such as coverage and GC content from alignment files.

Path	Description	With --target_regions	Without --target_regions
qc/mosdepth/{sample}/*.mosdepth.global.dist.txt	Cumulative distribution of bases covered for at least a given coverage value, across chromosomes and the whole genome
qc/mosdepth/{sample}/*.mosdepth.summary.txt	Mosdepth summary file
qc/mosdepth/{sample}/*.mosdepth.region.dist.txt	Cumulative distribution of bases covered for at least a given coverage value, across regions
qc/mosdepth/{sample}/*.regions.bed.gz	Depth per region
qc/mosdepth/{sample}/*.regions.bed.gz.csi	Index of the regions.bed.gz file

Cramino

cramino is used to analyze both phased and unphased reads.

Path	Description
qc/cramino/phased/{sample}/*.arrow	Read length and quality in Apache Arrow format
qc/cramino/phased/{sample}/*.txt	Summary information in text format
qc/cramino/unphased/{sample}/*.arrow	Read length and quality in Apache Arrow format
qc/cramino/unphased/{sample}/*.txt	Summary information in text format

Somalier

somalier checks relatedness and sex.

Path	Description
pedigree/family/{family).ped	PED file updated with somalier-inferred sex per family
qc/somalier/relate/{project}/{project}.html	HTML report
qc/somalier/relate/{project}/{project}.pairs.tsv	Information about sample pairs
qc/somalier/relate/{project}/{project}.samples.tsv	Information about individual samples

DeepVariant

vcf_stats_report.py from DeepVariant is used to generate a html report per sample.

Path	Description
qc/deepvariant_vcfstatsreport/{sample}/${sample}.visual_report.html	Visual report of SNV calls from DeepVariant

Variants

In general, annotated variant calls are output per family while unannotated calls are output per sample.

Paralogous genes

Paraphase is used to call paralogous genes.

Path	Description
paraphase/{sample}/*.bam	BAM file with reads from analysed regions
paraphase/{sample}/*.bai	Index of the BAM file
paraphase/{sample}/*.json	Summary of haplotypes and variant calls
paraphase/{sample}/{sample}_paraphase_vcfs/{sample}_{gene}_vcf.gz	VCF file per gene
paraphase/{sample}/{sample}_paraphase_vcfs/{sample}_{gene}_vcf.gz.tbi	Index of the VCF file

Repeats

TRGT is used to call repeats.

Path	Description	Call repeats	Call & annotate repeats
repeats/family/{family}/{family}_repeat_expansions.vcf.gz	Merged VCF file per family
repeats/family/{family}/{family}_repeat_expansions.vcf.gz.tbi	Index of the VCF file
repeats/sample/{sample}/{sample}_sorted.vcf.gz	VCF file with called repeats for a sample
repeats/sample/{sample}/{sample}_sorted.vcf.gz.tbi	Index of the VCF file
repeats/sample/{sample}/{sample}_spanning_sorted.bam	BAM file with sorted spanning reads
repeats/sample/{sample}/{sample}_spanning_sorted.bai	Index of the BAM file

Stranger is used to annotate repeats.

Path	Description	Call repeats	Call & annotate repeats
repeat_expansions/family/{family}/{family}_repeat_expansions_annotated.vcf.gz	Merged, annotated VCF file per family
repeat_expansions/family/{family}/{family}_repeat_expansions_annotated.vcf.gz.tbi	Index of the VCF file

SNVs

DeepVariant is used to call variants, while bcftools and GLnexus are used for merging variants.

Path	Description	Call SNVs	Call & annotate SNVs	Call, annotate and rank SNVs
snvs/sample/{sample}/{sample}_snv.vcf.gz	VCF file containing called variants with alternative genotypes for a sample
snvs/sample/{sample}/{sample}_snv.vcf.gz.tbi	Index of the corresponding VCF file
snvs/stats/sample/*.stats.txt	Variant statistics
qc/deepvariant_vcfstatsreport/{sample}/${sample}.visual_report.html	Visual report of SNV calls from DeepVariant
snvs/family/{family}/{family}_snv.vcf.gz	VCF file containing called variants for all samples
snvs/family/{family}/{family}_snv.vcf.gz.tbi	Index of the corresponding VCF file

Annotation

Echtvar and VEP are used for annotating SNVs, while CADD is used to annotate INDELs with CADD scores.

Path	Description	Call SNVs	Call & annotate SNVs	Call, annotate and rank SNVs
snvs/sample/{sample}/{sample}_snvs_annotated.vcf.gz	VCF file containing annotated variants with alternative genotypes for a sample
snvs/sample/{sample}/{sample}_snvs_annotated.vcf.gz.tbi	Index of the annotated VCF file
snvs/family/{family}/{family}_snvs_annotated.vcf.gz	VCF file containing annotated variants per family
snvs/family/{family}/{family}_snvs_annotated.vcf.gz.tbi	Index of the annotated VCF file

Ranking

GENMOD is used to rank the annotated SNVs and INDELs.

Path	Description	Call SNVs	Call & annotate SNVs	Call, annotate and rank SNVs
snvs/sample/{sample}/{sample}_snvs_annotated_ranked.vcf.gz	VCF file with annotated and ranked variants for a sample
snvs/sample/{sample}/{sample}_snvs_annotated_ranked.vcf.gz.tbi	Index of the ranked VCF file
snvs/family/{family}/{family}_snvs_annotated_ranked.vcf.gz	VCF file with annotated and ranked variants per family
snvs/family/{family}/{family}_snvs_annotated_ranked.vcf.gz.tbi	Index of the ranked VCF file

Filtering

Filter_vep and bcftools can be used to filter variants. These will be output if either of --filter_variants_hgnc_id and --filter_snvs_expression has been used, and only family VCFs are filtered.

Path	Description
snvs/{family}/{family}_*_filtered.vcf.gz	VCF file with filtered variants for a family
snvs/{family}/{family}_*_filtered.vcf.gz.tbi	Index of the filtered VCF file

Tip

Filtered variants are output alongside unfiltered variants as additional files.

SVs (and CNVs)

Severus or Sniffles are used to call structural variants, while HiFiCNV is used to call CNVs. HiFiCNV also produces copy number, depth, and MAF visualization tracks.

Variant merging strategies

SV and CNV calls are output unmerged per sample, while the family files are first merged between samples for SVs and CNVs separately, then the merged SV and CNV files are merged again, with priority given to coordinates from the SV calls.

Path	Description	Call SVs	Call CNVs	Call SVs & CNVs
svs/sample/{sample}/{sample}_svs.vcf.gz	VCF file with SVs per sample
svs/sample/{sample}/{sample}_svs.vcf.gz.tbi	VCF file with SVs per sample
svs/sample/{sample}/{sample}_cnvs.vcf.gz	VCF file with CNVs per sample
svs/sample/{sample}/{sample}_cnvs.vcf.gz.tbi	VCF file with CNVs per sample
svs/family/{family_id}/{family_id}_svs_merged.vcf.gz	VCF file with merged SVs per family
svs/family/{family_id}/{family_id}_svs_merged.vcf.gz.tbi	Index of the merged VCF file
svs/family/{family_id}/{family_id}_cnvs_svs_merged.vcf.gz	VCF file with merged CNVs and SVs per family
svs/family/{family_id}/{family_id}_cnvs_svs_merged.vcf.gz.tbi	Index of the merged VCF file

Annotation

SVDB and VEP are used to annotate structural variants.

Path	Description	Call & annotate SVs	Call & annotate SVs & CNVs
svs/family/{family_id}/{family_id}_cnvs_svs_merged_annotated.vcf.gz	VCF file with merged and annotated CNVs and SVs per family
svs/family/{family_id}/{family_id}_cnvs_svs_merged_annotated.vcf.gz.tbi	Index of the merged VCF file
svs/family/{family_id}/{family_id}_svs_merged_annotated.vcf.gz	VCF file with merged and annotated SVs per family
svs/family/{family_id}/{family_id}_svs_merged_annotated.vcf.gz.tbi	Index of the merged VCF file

Ranking

GENMOD is used to rank the annotated SVs.

Path	Description	Rank SVs	Rank SVs & CNVs
svs/family/{family_id}/{family_id}_cnvs_svs_merged_annotated_ranked.vcf.gz	VCF file with merged, annotated and ranked CNVs and SVs per family
svs/family/{family_id}/{family_id}_cnvs_svs_merged_annotated_ranked.vcf.gz.tbi	Index of the merged VCF file
svs/family/{family_id}/{family_id}_svs_merged_annotated_ranked.vcf.gz	VCF file with merged, annotated and ranked SVs per family
svs/family/{family_id}/{family_id}_svs_merged_annotated_ranked.vcf.gz.tbi	Index of the merged VCF file

Filtering

Filter_vep and bcftools can be used to filter variants. These will be output if either of --filter_variants_hgnc_id and --filter_svs_expression has been used, and only family VCFs are filtered.

Path	Description
svs/{family}/{family}_*_filtered.vcf.gz	VCF file with filtered variants for a family
svs/{family}/{family}_*_filtered.vcf.gz.tbi	Index of the filtered VCF file

Tip

Filtered variants are output alongside unfiltered variants as additional files.

Visualization Tracks

HiFiCNV is used to call CNVs, but it also produces copy number, depth, and MAF tracks that can be visualized in for example IGV.

Path	Description
visualization_tracks/{sample}/*.copynum.bedgraph	Copy number in bedgraph format
visualization_tracks/{sample}/*.depth.bw	Depth track in BigWig format
visualization_tracks/{sample}/*.maf.bw	Minor allele frequencies in BigWig format