Question: Can I sequence my V(D)J libraries in a different sequencing configuration than the ones recommended in the user guide?
Answer: Yes, V(D)J libraries can be sequenced in other configurations. For 5' v2 Dual Index V(D)J libraries, we have successfully tested read lengths of 26 x 10 x 10 x 77 bp and 26 x 10 x 10 x 150. With a 77 bp read length for Read 2, VDJ libraries may see a 5 - 10% reduction in sensitivity (measured in the number of cells with productive pairs) depending on sample type. V(D)J libraries will also require deeper sequencing in this configuration. Please see the table below for recommended sequencing depths at different configurations.
Alternate Read Length Configurations for 5' v2 V(D)J Dual Index Libraries
|
Sequencing configuration |
Suggested read depth |
|
26 x 10 x 10 x 150 |
2,000 read pairs per T or B cell |
|
150 x 10 x 10 x150* |
2,000 read pairs per T or B cell |
|
26 x 10 x 10 x 90** |
5,000 read pairs per T or B cell |
|
26 x 10 x 10 x 77*** |
7,500 read pairs per T or B cell |
For 5' v1/v1.1 V(D)J Libraries, similar recommendations apply for Read 1 and Read 2 lengths and sequencing depth recommendations. The most significant change is that these libraries are single-indexed. Please see the table below for more details.
Alternate Read Length Configurations for 5' v1/v1.1 V(D)J Single Index Libraries
|
Sequencing configuration |
Suggested read depth |
|
26 x 8 x 0 x 150 |
2,000 read pairs per T or B cell |
|
150 x 8 x 0 x150* |
2,000 read pairs per T or B cell |
|
26 x 8 x 0 x 91** |
5,000 read pairs per T or B cell |
|
26 x 8 x 0 x 77*** |
7,500 read pairs per T or B cell |
*Even though the sequences from Read 1 are utilized for assembly when sequencing 150bp, the overall sequencing depth recommendations are the same if the Read 1 is 26 bp or 150 bp. Additional information from Read 1 (150bp) may not necessarily increase the number of productive contigs detected.
**User guide recommended
***Libraries sequenced in this configuration may see a 5 - 10 % decrease in assay performance (measured by the number of cells with productive pairs). This decrease is sample-dependent.
Products: Single Cell Immune Profiling