Construction of mosaic reference standards

Introduction

A robust pipeline for constructing mosaic reference standards. The reference standards are consisting of 386,613 mosaic SNVs and INDELs in a wide range of variant allele frequencies, from 0.5% to 56%. Negative controls of non-variant positions (35,113,417) and germline variants (19,936 SNVs and INDELs) are accompanied with abundant positive controls. The reference standards were constructed by mixing genetic materials of six pre-genotyped normal cell lines, mimicking the cumulative aspect of mosaic variant acquisition in the early development.

1. Genotyping six cell lines

A. Germline variant calling

- Alignment and preprocessings
- Strelka2 
- DeepVariant 
- CNVkit   

B. Generating candiates

- Positive controls 
  + Mutually exclusive germline variants    

- Negative controls   
  + Common wildtype positions (Set A)   
  + MRC5 germine variants (Set B)   

2. Generating Set B

- Down-sampling of MRC5 (39 times with random seeds) 
- Extraction of reads embedding positive controls from Set A
- Replacement of extracted reads to MRC5 down-sampled data

3. Finalizing the reference standards

A. Pileup for post-filters

B. Apply post-filters

- Sequencing coverage
- Variant coverage
- High-quality alternative alleles