Copy Number Variation Calling from circulating tumore DNA (ctDNA)

This project utilises the new version of GATK4 - The command lines are different from GATK3

Pre-processing data

Raw data

• The sequencing of ctDNA of all patients were designed under the gene panel protocol
• The targeted genes are not provided, then, interval detection will be analysed through the coverage at every base of the whole sequence after finishing the alignment

Pre-processed data

Quality control with QC

• Each samples are sequenced at multiple lanes
• QC will process each lane separately
• Output from QC can be used to check the quality of sequencing
• Parameters for QC check and filter

Alignment

• Alignment tool: BWA-MEM
• Alignment first will mapped reads from each lane separately
• Then after alignment, data from many lanes of one samples will be merged, deduplicated, and reindexed to provide the final bam output files

Variant Calling

Reference data

• Hg38

Germline variation calling

• Germline mutation calling tool: GATK 3.7
• Provide BAF value

CNV Calling

Coverage calculation

Constructing Panel of Normal (PoN)

Bias correction

Build up PoN

Copy Number Variation Calling

• Tool: PureCN
• Segmentation algorithm: CBS

Single sample process

Multiple samples process