Technical Details

Technical details are regarding the internals to the off-the-shelf LENS workflow are described below.

Off-the-shelf defaults

Default references

Workflow

Reference type

Reference

DNA alignment

Genomic reference

Homo_sapiens.assembly38.no_ebv.fa

DNA alignment post-processing

Genomic reference

Homo_sapiens.assembly38.no_ebv.fa

DNA alignment post-processing

BED

hg38_exome.bed

DNA alignment post-processing

Known sites VCF

Homo_sapiens_assembly38.dbsnp138.vcf.gz

RNA alignment

Genomic reference

Homo_sapiens.assembly38.no_ebv.fa

RNA alignment

GTF

gencode.v37.annotation.with.hervs.gtf

Transcript quantification

Genomic reference

Homo_sapiens.assembly38.no_ebv.fa

Transcript quantification

GTF

gencode.v37.annotation.with.hervs.gtf

Somatic variant calling

Genomic reference

Homo_sapiens.assembly38.no_ebv.fa

Somatic variant calling

BED

hg38_exome.bed

Somatic variant calling

Panel of normals VCF

1000g_pon.hg38.vcf.gz

Somatic variant calling

Allele frequencies VCF

af-only-gnomad.hg38.vcf.gz

Somatic variant calling

Known sites VCF

small_exac_common_3.hg38.vcf.gz

Variant annotation

snpEff annotation file

GRCh38.GENCODEv37

Germline variant calling

Genomic reference

Homo_sapiens.assembly38.no_ebv.fa

Germline variant calling

BED

hg38_exome.bed

Variant phasing

Genomic reference

Homo_sapiens.assembly38.no_ebv.fa

Variant phasing

GTF

gencode.v37.annotation.with.hervs.gtf

Splice variant calling

Tool-specific reference

snaf-data

Virus detection

Virus-specific (no Homo sapiens homology) sequences

virus_masked_hg38.fa

Virus detection

Virus-specific sequences

virus.cds.2024f2.fa

Fusion detection

Genomic reference

Homo_sapiens.assembly38.no_ebv.fa

Fusion detection

Tool-specific reference

GRCh38_gencode_v37_CTAT_lib_Mar012021.plug-n-play

Tumor purity detection

Genomic reference

Homo_sapiens.assembly38.no_ebv.fa

Tumor purity detection

BED

hg38_exome.bed

Copy number variant detection

Genomic reference

Homo_sapiens.assembly38.no_ebv.fa

Copy number variant detection

BED

hg38_exome.bed

CTA pMHC generation

Genomic reference

Homo_sapiens.assembly38.no_ebv.fa

CTA pMHC generation

GTF

gencode.v37.annotation.with.hervs.gtf

CTA pMHC generation

CTA gene list

cta_and_self_antigen.homo_sapiens.gene_list

ERV pMHC generation

Genomic reference

Homo_sapiens.assembly38.no_ebv.fa

ERV pMHC generation

ERV annotations

Hsap38.txt

SNV pMHC generation

Genomic reference

Homo_sapiens.assembly38.no_ebv.fa

SNV pMHC generation

GTF

gencode.v37.annotation.with.hervs.gtf

SNV pMHC generation

Canonical protein reference

gencode.v37.pc_translations.fa

InDel pMHC generation

Genomic reference

Homo_sapiens.assembly38.no_ebv.fa

InDel pMHC generation

GTF

gencode.v37.annotation.with.hervs.gtf

InDel pMHC generation

Canonical protein reference

gencode.v37.pc_translations.fa

Fusion pMHC generation

Genomic reference

Homo_sapiens.assembly38.no_ebv.fa

Fusion pMHC generation

GTF

gencode.v37.annotation.with.hervs.gtf

pMHC characterization

Tool-specific reference

mhcflurry

CTA annotation

CTA metadata

canonical_txs.mtec.norm.subcell.annot.tsv

ERV annotation

ERV metadata

erv_scores.25SEP2023.tsv

Sample swap detection

Genomic reference

Homo_sapiens.assembly38.no_ebv.fa

Sample swap detection

Known sites VCF

somalier.sites.hg38.vcf.gz

Default tools

Workflow

Tool

Tool version

DNA alignment

fastp

v0.23.1

DNA alignment

bwa-mem2

v2.2.1

DNA alignment post-processing

samblaster

v0.1.26

DNA alignment

fastp

v0.23.1

RNA alignment

star

v2.7.0f

Transcript quantification

salmon

v1.1.0

Somatic variant calling

mutect2

v4.1.6.0

Somatic variant calling

varscan2

v2.1.1

Somatic variant calling

strelka2

v2.2.9

Somatic variant filtering

bcftools

v1.19

Variant annotation

snpeff

v4.3k

Somatic SNV/InDel filtering

snpsift

v4.3k

Variant unionizing

jacquard

v1.1.4

Germline variant calling

deepvariant

v1.1.0

Somatic and germline merging

jacquard

v1.1.4

Variant phasing

whatshap

v1.2.1

HLA Typing

seq2hla

v2.2

Splice variant calling

snaf

v0.7.0

Fusion detection

starfusion

v1.10.1

Tumor purity detection

sequenza

v3.0.0

Copy number variant detection

cnvkit

v0.9.9

Copy number variant detection

cnvkit

v0.9.9

CTA pMHC generation

lenstools

v1.3

ERV pMHC generation

lenstools

v1.3

SNV pMHC generation

lenstools

v1.3

InDel pMHC generation

lenstools

v1.3

Viral pMHC generation

lenstools

v1.3

Splice pMHC generation

lenstools

v1.3

Fusion pMHC generation

lenstools

v1.3

pMHC characterization

mhcflurry

v2.0.6

Sample swap detection

somalier

v0.2.17

Default parameters

Workflow

Tool

Parameters

DNA alignment

fastp

--in1 ${fq1} --out1 ${dataset}-${pat_name}-${run}_1.trimmed.fq.gz --in2 ${fq2} --out2 ${dataset}-${pat_name}-${run}_2.trimmed.fq.gz  --failed_out ${dataset}-${pat_name}-${run}.fastp_fails.fq.gz --thread ${task.cpus}

DNA alignment

bwa-mem2

-R "@RG\\tID:${dataset}-${pat_name}-${run}\\tSM:${dataset}-${pat_name}-${run}\\tLB:NULL\\tPL:Illumina" ${fa} ${fq1} ${fq2} -t ${task.cpus - 1}

DNA alignment post-processing

samblaster

RNA alignment

fastp

--in1 ${fq1} --out1 ${dataset}-${pat_name}-${run}_1.trimmed.fq.gz --in2 ${fq2} --out2 ${dataset}-${pat_name}-${run}_2.trimmed.fq.gz --failed_out ${dataset}-${pat_name}-${run}.fastp_fails.fq.gz --thread ${task.cpus}

RNA alignment

star

-quantMode TranscriptomeSAM --outSAMtype BAM SortedByCoordinate --twopassMode Basic --outSAMunmapped Within

Transcript quantification

salmon

quant --threads ${task.cpus} -t ${fa} -l a -a ${aln} -o .

Somatic variant calling

mutect2

Placeholder

Somatic variant calling

strelka2

Placeholder

Somatic variant calling

varscan2

Placeholder

Somatic variant filtering (mutect2)

bcftools

FILTER="PASS"

Somatic variant filtering (strelka2)

bcftools

FILTER="PASS"

Somatic variant filtering (varscan2)

bcftools

FILTER="PASS"

Variant annotation

snpeff

Somatic SNV filtering

snpsift

ANN[*].EFFECT has 'missense_variant'

Somatic InDel filtering

snpsift

(ANN[*].EFFECT has 'conservative_inframe_insertion') || (ANN[*].EFFECT has 'conservative_inframe_deletion') || (ANN[*].EFFECT has 'disruptive_inframe_insertion') || (ANN[*].EFFECT has 'disruptive_inframe_deletion') || (ANN[*].EFFECT has 'frameshift_variant')

Variant unionizing

jacquard

--include_format_tags=\"GT,AF,AU,CU,GU,TU,TAR,TIR,FREQ,VAF

Germline variant calling

deepvariant

--model_type WES

Variant merging

jacquard

Variant phasing

whatshap

--ignore-read-groups

Splice variant filtering

split_snaf_by_sample

snaf_awk_filter: '\$5 == "True" && \$3 == 0.0', snaf_tumor_exp_threshold: '1000'

Fusion detection

star

--outReadsUnmapped None --twopassMode Basic --outSAMstrandField intronMotif --outSAMunmapped Within --chimSegmentMin 12 --chimJunctionOverhangMin 8 --chimOutJunctionFormat 1 --alignSJDBoverhangMin 10 --alignMatesGapMax 100000 --alignIntronMax 100000 --alignSJstitchMismatchNmax 5 -1 5 5 --chimMultimapScoreRange 3 --chimScoreJunctionNonGTAG -4 --chimMultimapNmax 20 --chimNonchimScoreDropMin 10 --peOverlapNbasesMin 12 --peOverlapMMp 0.1 --alignInsertionFlush Right --alignSplicedMateMapLminOverLmate 0 --alignSplicedMateMapLmin 30

Fusion detection

starfusion

--examine_coding_effect

Tumor purity detection

sequenza

sequenza_gc_wiggle: '-w 50', sequenza_bam2seqz: '', sequenza_seqz_binning: '-w 50'

Copy number variant detection

cnvkit

cnvkit_segment: '--drop-low-coverage', cnvkit_call: '--ploidy 2'

Expressed CTA detection

lenstools_filter_expressed_self_genes

-p 95

Expressed ERV detection

lenstools_filter_ervs_by_rna_coverage

--mean-depth 60

Expressed SNV detection

lenstools_filter_expressed_variants_parameters

-p 75

Expressed InDel detection

lenstools_filter_expressed_variants_parameters

-p 75

Expressed virus detection

lenstools_filter_viruses_by_rna_coverage

--coverage 25

pMHC characterization

mhcflurry

8,9,10,11

pMHC filtering

lenstools

<500 nM

LENS workflow flowchart

Somatic Nucleotide Variants (SNVs)

Somatic single-nucleotide variants (SNVs), or variants present within tumor tissue but absent from germline tissue, can be a source of tumor-specific immunogenic peptides.

Somatic Insertion and Deletion Variants (InDels)

Somatic insertion/deletion variants (InDels), or variants present within tumor tissue but absent from germline tissue, can be a source of tumor-specific immunogenic peptides.

Splice Variants

Fusion Events

Endogenous Retroviruses (ERVs)

Viruses

Cancer-testis Antigens (CTAs)

Aberrantly expressed genes (e.g. CTAs) can be a source of tumor-associated immunogenic peptides.

Determining expressed CTA and self-antigens

CTA/Self-antigens that are included in the user-provided list. The default list is available in /path/to/raft/references/homo_sapiens/cta_self/cta_and_self_antigen.homo_sapiens.gene_list. This list includes loci described in the CTDatabase (http://www.cta.lncc.br/). Targetable CTA/Self-antigen peptides are generated using the coding sequence of CTA transcripts that exceed the user-provided expression percentile (default: 95%).

Generating patient-specific CTA coding sequences

LENS performs germline variant calling (default: DeepVariant) as part of its workflow.

Reference generation notes