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somAgg sample stats

There are 16,341 samples present in the somAgg. These include all samples that are present in cancer_analysis on the 100kGP Release v12 and are all the somatic samples that were successfully sequenced and interpreted.

Single sample sequencing and variant calling

somAgg combines the annotated somatic VCF files generated by Strelka and annotated with CellBase. Each tumour sample has a matched germline, both deep whole-genome sequenced with an average coverage of 100x and 30x, respectively. Few patients had more than one tumour sample sequenced.

Step Method
Preparation Illumina TruSeq DNA Nano, TruSeq DNA PCR-Free or FFPE library preparation kit
Sequencing HiSeq X generating 150 bp paired-end reads
Primary WGS analysis Illumina’s North Star pipeline (version 2.6.53.23)
Read alignment against human reference genome GRCh38-Decoy+EBV with iSAAC Aligner (version iSAAC- 03.16.02.19)
Small variant calling together with tumour-normal subtraction Strelka2 (version 2.4.7)

Strelka FILTERs flag the following germline variant calls as NOT PASS, they are nonetheless included in the single vcf files and somAgg:

  • All calls with a sample depth three times higher than the chromosomal mean
  • Site genotype conflicts with proximal indel call. This is typically a heterozygous SNV call made inside of a heterozygous deletion
  • Locus read evidence displays unbalanced phasing patterns
  • Genotype call from variant caller not consistent with chromosome ploidy
  • The fraction of basecalls filtered out at a site > 0.4
  • Locus quality score < 14 for for het or hom SNP
  • Locus quality score < 6 for het, hom or het-alt indels
  • Locus quality score < 30 for other small variant types or quality score is not calculated

Single sample decomposition

The annotated small variant VCF files used as input have been decomposed. The annotated single VCF files are generated from the somatic small variant VCF files (somatic_small_variants_vcf_path in cancer_analysis) generated by the variant calling pipeline, which comprises Strelka2 and vt for the decomposition. In other words, the somatic variant VCF files are the ones decomposed, which means that no multi-allelic entries are found, because each multi-allelic is represented by two or more bi-allelic variants.

The decomposition procedure is done in three steps by vt as presented here:

  1. Decompose variants of the same length.

vt decompose_blocksub -p {vcf_input} -o {vcf_output}

  1. Split records with multiple alternate alleles into multiple bi-allelic records an e.g. 1/2 genotype will be split to 1/. and ./1. The flag -s (“smart”) option makes INFO and FORMAT fields of type A and R to be retained and decomposed appropriately.

vt decompose -s {vcf_input} -o {vcf_output}

  1. Left-align indels and trim redundant bases. The “non-ambiguous” reference genome is used. This file only contains A,T,G,C and N characters.

vt normalize -n -w {window_size} -r {reference} {vcf_input} -o {vcf_output}

Genotype-level metrics

All 16,341 samples included in somAgg have successfully passed our internal sequencing and interpretation pipeline. These sample are listed in the LabKey table cancer_analysis. Some quality control statistics for these samples are provided below.

Sample Attribute Description
Tumour Cross-Contamination less than 5%
Germline Cross-Contamination less than 3%
Median Fragment Size greater than 279bp
Excess of Chimeric Reads mean of 0.3%
Percentage of Mapped Reads mean of 93.4%
Percentage AT Dropout mean of 3.1%

Sample source and library preparation

The vast majority of the samples has been collected using surgical resection.

tissue_source number_of_samples percent_of_samples
SURGICAL RESECTION 14602 89.36
NOT SPECIFIED 521 3.19
USS GUIDED BIOPSY 490 3.00
ENDOSCOPIC BIOPSY 227 1.39
NON GUIDED BIOPSY 136 0.83
BMA TUMOUR SORTED CELLS 133 0.81
NON STANDARD BIOPSY 85 0.52
CT GUIDED BIOPSY 69 0.42
STEREOTACTICALLY GUIDED BIOPSY 49 0.30
ENDOSCOPIC ULTRASOUND GUIDED FNA 12 0.07
LAPAROSCOPIC EXCISION 8 0.05
MRI GUIDED BIOPSY 6 0.04
ENDOSCOPIC ULTRASOUND GUIDED BIOPSY 3 0.02

Also, the majority (~92%) of somAgg are from fresh-frozen (FF) and (~88%) from PCR-free.

library_type preparation_method number_of_samples percent_of_samples
PCR-Free FF 13711 83.91
PCR FF 1285 7.86
PCR FFPE 602 3.68
PCR-Free EDTA 494 3.02
PCR-Free ASPIRATE 124 0.76
PCR-Free CD128 SORTED CELLS 70 0.43
PCR CD128 SORTED CELLS 25 0.15
PCR EDTA 19 0.12
PCR ASPIRATE 8 0.05
PCR-Free FFPE 3 0.02

As expected, we see an increased AT drop-out for FFPE samples, but overall the vast majority of samples have good mapping rate.

Tumour purity and coverage