In this minimal, reproducible example of wellDA-seq, we will address the following questions/tasks:

1. how to preprocess the wellDA-seq data to create analysis-ready data objects?
2. how to identify subclones and cell types (or states)?
3. how to investigate the interplay of CNA events and open/closed chromatin regions (e.g., the GtoE and EbyG scores, and the plasticity/heritability score)?

This tutorial contains all the analysis that we performed on each of the samples in the manuscript.

In brief, the following key tools are required:

• preprocessing the DNA data by single-cell CNV pipeline.
• preprocessing the ATAC data by scATAC-pro and ArchR.
• creating an analysis-ready DNA object by copykit.
• creating an analysis-ready ATAC object by Signac.

See the folder wellDA-seq/install for detailed instruction of installing software requirement.

Here, we use the real sample P8 (DCIS66T_chip2) in the manuscript for demonstration.

See the detailed instruction in 01.preprocessing.md

Input: FASTQ files of the DNA and ATAC modality data.

Expected result:

• a preliminary Signac object for ATAC
• a preliminary Copykit object for DNA

item	ATAC	DNA
Cell Dispensing
Basic Quality Control
Basic Clustering

See the detailed instruction in 02.wellDA_initiation.md

Expected result:

• a folder of wellDA data

├── metadata.csv      <--- data frame of single-cell metadata
├── metadata.df.rds   <--- data frame of single-cell metadata
├── obja.rds          <--- Signac object
├── objd.rds          <--- Copykit object

See the detailed instruction in 03.wellDA_scATAC_annotation.md

Expected result:

• a Signac object with cell types / states annotated.

See the detailed instruction in 04.wellDA_scCNA_annotation.md

Expected result:

• a Copykit object with the diploid and low-quality cells removed and the tentative subclones determined.

Before data cleaning the aneuploid cells	After data cleaning the aneuploid cells

See the detailed instruction in 05.wellDA_refine.md

Expected result:

• an analysis-ready folder of wellDA-seq data for each sample

See the detailed instruction in 06.GtoE_EbyG.md

Expected result:

• GtoE and EbyG scores for each sample

See the detailed instruction in 07.global_concordance.md

Expected result:

• a global concordance score for each sample

See the detailed instruction in 08.plasticity_heritability.md

Expected result:

• Heritable/plastic score of tumorigenesis-related gene signatures for each sample