Pipeline version v3.0

Summary
Major overhaul of all somatic analyses (SNVs, INDELs, implied purity, BAF, copy numbers and structural variation (SVs)).

Improvements to somatic SNV / Indel calling

• Consensus method has been replaced by Strelka only calling with custom post processing. Mutect, Freebayes and Varscan callers have been removed from the pipeline.
• BQSR is now run prior to somatic calling (to improve Strelka precision).
• Strelka REPEAT filter is switched off to improve INDEL sensitivity.
• A new post calling filter is added to Strelka output: variants in the low confidence regions are hard filtered unless they have > 10% AF and Strelka Somatic score > 20.
• A soft PON (pool of normals) filter is applied to the final Strelka output to improve precision.

Improvements to somatic SV calling

• ‘Alfredi' (BPI) is run on Manta output which performs the following functions:
  • Applies a set of 8 filters to Manta output to remove obvious false positives / improve precision.
  • Determines the accurate break point of each variant.
  • Calculates a AF for each breakpoint end on each variant.

Tumor purity / BAF / copy numbers

• ‘PURPLE’ (PURity & PLoidy Estimator) replaces FreeC as the primary copy number tool
  Key features of PURPLE:
  • ‘COBALT’ (COunt BAm Lines of Tumor) counts the # of reads per kb window for both normal and tumor.
  • GC bias is fit for both normal and tumor.
  • ‘AMBER’ (A Minipileup Baf EstimatoR) calculates BAF for a set of HC common heterozygous SNPs.
  • A set of candidate copy number breakpoints is determined using a PCF (piecewise constant fitting) algorithm on tumor, normal and BAF.
  • Sample ploidy and purity is jointly fit by minimising a penalty function using a integer ploidy and minor allele ploidy model.
  • Absolute copy number is determined for each segment.
  • Candidate breakpoints are smoothed into a set of final copy number breakpoints.
  • CIRCOS and QC plots are produced.

Other changes

• Introduce damage estimator tool to estimate DNA damage.
• BQSR no longer produces a QC report, cutting out 40% of total runtime.
• BQSR writes its BAM using lower zip compression leading to faster compute time but bigger recalibrated BAM files.
• A 26 SNP filter is added for changes to SNP check design.
• Health checker is now run as part of the pipeline.
• CPCT Slicing is removed as it is no longer used.

Changes to versions

• dx_tracks updated for KG from v1 to v1.2.1

Quality

• For assessing the quality of the pipeline we do the following checks:
  • Determine germline precision & sensitivity on an internally sequenced NA12878
  • Determine somatic precision & sensitivity on an internally sequenced GIAB-mix of 70% NA24385 and 30% NA12878, against 100% of NA24385 as reference sample.

Germline precision & sensitivity

Type	Config	Algo	TP	FP	FN	Prec	Sens	Δ Prec	Δ Sens
SNV	KG	GATK	3115316	10421	38943	99,7%	98,8%	0.0%	0,0%

Somatic precision & sentivity

Type	Algo	TP	FP	FN	Prec	Sens	Δ Prec	Δ Sens
INDEL	Strelka	74432	577	22184	99,2%	77,0%	-0,4%	7,1%
SNV	Strelka	969786	1247	38058	99,9%	96,2%	0,1%	0,0%

Note: Soft PON filter has not been applied on the GIAB mixin results as that is not possible due to the nature of the sample.