The appropriate size of single-cell VDJ sequencing data depends on various factors, such as sample complexity, sequencing depth, and experimental design. The considerations may differ when comparing mixed library building and individual library building.

 

In the case of mixed library building, the inter-sample competition can impact the choice of data volume. If there is a large number of samples with similar clonotypes, deeper sequencing depths may be necessary to ensure sufficient coverage and resolution. Based on experience, for mixed library construction, a minimum of 2000 reads per single cell is typically recommended to ensure reliable VDJ rearrangement and accurate clonotype identification.

 

When it comes to individual library building, the amount of sequencing data can be chosen more flexibly since each single cell is processed and sequenced individually. Generally, in single-cell V(D)J sequencing, the goal is to obtain comprehensive clonotype information, requiring sufficient sequencing depth to support high-quality rearrangement and accurate clonotype identification. Based on experience, sequencing each single cell to at least 4000 reads is typically recommended to ensure reliable VDJ analysis results.

 

However, it is important to note that the appropriate amount of sequencing data should be selected based on the specific experimental design and research questions. Some research questions may require greater sequencing depth, while others may require less. Therefore, when designing experiments and conducting data analysis, it is crucial to consider multiple factors comprehensively and select an appropriate amount of sequencing data based on the specific needs of the study.