EvercodeTM BCR will result in two sets of sequencing libraries: one that corresponds to molecules containing the single cell whole transcriptome (gene expression) and one that corresponds to molecules containing single cell BCR information, such as V(D)J sequences of heavy and light chains.
BCR Sequencing Libraries
For Evercode BCR v3 or Evercode BCR v4 libraries, we recommend sequencing at 2,000-5,000 reads per cell. Parse BCR libraries require a 300 cycle kit minimum (which will get you up to 318 total cycles as Illumina includes extra reagents as a standard practice). BCR sequencing libraries should be diluted and denatured according to the instruction for the relevant sequencing instrument. We recommended adding 5% PhiX for optimal sequencing quality. BCR libraries contain Truseq read 1 and read 2 sequences.
The run configuration requirements for BCR sequencing libraries are as follows and paired end sequencing should be used:
- Read: Read 1; Cycles: 242
- Read: i7 Index (Index 1); Cycles: 8
- Read: i5 Index (Index 2); Cycles: 8
- Read: Read 2; Cycles: 58
Note: This run configuration includes a longer Read 1 and is therefore different from whole transcriptome libraries.
WT Sequencing Libraries
For WT libraries generated from an Evercode BCR v3 kit, we recommend a minimum sequencing depth of 20,000 reads per cell. Due to improved library efficiency, for WT libraries generated from an Evercode BCR v4 kit, we recommend a minimum of 10,000 reads/cell. However, ideal sequencing depth is dependent on the sample type and experimental goals. A 100 cycle kit minimum is required (which will get you up to 138 total cycles as Illumina includes extra reagents as a standard practice). WT sequencing libraries should be diluted and denatured according to the instruction for the relevant sequencing instrument. We recommended adding 5% PhiX for optimal sequencing quality.
The run configuration requirements for WT sequencing libraries are as follows and paired end sequencing should be used:
- Read: Read 1; Cycles: 64
- Read: i7 Index (Index 1); Cycles: 8
- Read: i5 Index (Index 2); Cycles: 8
- Read: Read 2; Cycles: 58
Sequencing Amplicon
The diagram below illustrates the composition of the sequencing libraries generated. This
library structure applies to both the whole transcriptome library and the BCR-enriched library.
Note: Sequencing both the whole transcriptome libraries and BCR libraries together is possible if different lanes are used for WT and BCR libraries. Different lanes are required because WT and BCR sublibraries contain the same index sequences and need to be kept separate during sequencing. In addition, a 300 cycle kit would be required if running all libraries on the same sequencing run. In practice, it may be more cost effective to sequence whole transcriptome libraries separately because you can use an Illumina sequencing kit with fewer cycles, but this may be dependent on your timing, resources, and your pricing for sequencing.