Evercode TCR products 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 TCR information, such as CDR3 sequences of alpha and beta chains. For whole transcriptome libraries, please see this article for more information.
For TCR libraries, we recommend sequencing at 2,000-5,000 reads per cell. Parse TCR 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). TCR 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. TCR libraries contain 5’ Nextera and 3’ Truseq adapters, and the UDI Plate - EC adds Nextera read 1 and Truseq read 2.
The run configuration requirements for TCR sequencing libraries are as follows and paired end sequencing should be used:
|i7 Index (Index 1)||8|
|i5 Index (Index 2)||8|
Note: This run configuration is different from whole transcriptome libraries.
Sequencing both the whole transcriptome libraries and TCR libraries on the same sequencing run is possible, and the above run configuration would still be required if running all libraries on the same sequencing run. In practice, it may be more cost effective to sequencing whole transcriptome libraries separately because you can use a Illumina sequencing kit with fewer cycles, but this may be dependent on your timing and resource considerations.
TCR Sequencing Amplicon
The CDR3 is depicted in purple (between R1 and BC1). A final PCR amplifies the TCR Amplification 1 product and appends the fourth DNA barcodes, UDIs from the UDI Plate- EC, as well as the P5 and P7 adaptors.
For UDI sequences see: Unique Dual Indices (UDI) Sequences
For WT sequencing requirements see: What are the run configuration and sequencing requirements for WT libraries?