Considerations for identifying somatic variants in GEI
| Sample sequencing . |
|---|
| Genome sequencing (∼30×): Identifies coding, non-coding, and structural variants |
| Exome sequencing (∼200×): Focuses on coding regions |
| Targeted sequencing (>500×): Allows for deep sequencing of genes of interest for disease pathogenesis |
| Single-cell sequencing: Used to pair variant identification with additional data (i.e., scRNA-seq, ATAC-seq) |
| Variant caller considerations |
| Controls: Does the caller require a matched normal (are there other tissues from a patient that could be used)? What samples are being used as a “panel of normals?” |
| Types of variants: What does the caller perform best with (e.g., SNVs, insertion/deletions, structural variants)? |
| Consensus calling: What algorithms complement each other the best? How many callers need to agree on a variant? |
| Sequencing method: Are there specific callers designed for the method of sequencing used that can help extend the limit of detection? |
| Limit of detection: What is the lowest VAF that a caller can reliably call? |
| Variant validation methods |
| Sanger sequencing of cell type enriched for variant |
| ddPCR |
| Resequencing with an independent library |
| Alternative NGS method (e.g., long-read sequencing) |
| Evaluate other sequencing data |
| Sample sequencing . |
|---|
| Genome sequencing (∼30×): Identifies coding, non-coding, and structural variants |
| Exome sequencing (∼200×): Focuses on coding regions |
| Targeted sequencing (>500×): Allows for deep sequencing of genes of interest for disease pathogenesis |
| Single-cell sequencing: Used to pair variant identification with additional data (i.e., scRNA-seq, ATAC-seq) |
| Variant caller considerations |
| Controls: Does the caller require a matched normal (are there other tissues from a patient that could be used)? What samples are being used as a “panel of normals?” |
| Types of variants: What does the caller perform best with (e.g., SNVs, insertion/deletions, structural variants)? |
| Consensus calling: What algorithms complement each other the best? How many callers need to agree on a variant? |
| Sequencing method: Are there specific callers designed for the method of sequencing used that can help extend the limit of detection? |
| Limit of detection: What is the lowest VAF that a caller can reliably call? |
| Variant validation methods |
| Sanger sequencing of cell type enriched for variant |
| ddPCR |
| Resequencing with an independent library |
| Alternative NGS method (e.g., long-read sequencing) |
| Evaluate other sequencing data |