Question: What sequencing configuration is required for antibody libraries derived from using Proteintech Group (PTG) antibodies mixed with Biolegend antibodies or antibodies conjugated with an alternative feature barcode structure, generated from multiplexing experiments using Flex Gene Expression, so that the data can be analyzed properly using Cell Ranger?
Answer: You will need to sequence the Multiplexed Protein Expression (Ab) library with an alternative sequencing configuration. This configuration is found in the “Alternate Sequencing Parameters” section of the Multiplexed User Guide.
Alternative Sequencing Configuration:
Read 1: 48 cycles
i7 index: 10 cycles
i5 index: 10 cycles
Read 2: 50 cycles
This read scheme, which we refer to as MFRP-R1 chemistry, allows the Antibody Multiplexing Barcode to be extracted in the same positions of Read 1 as opposed to the default chemistry, which extracts the Antibody Multiplexing Barcode in the same positions of Read 2. The alignment of the Multiplexing Barcodes is required by Cell Ranger v8.0 to properly demultiplex sample barcodes. Proteintech Group (PTG) antibodies and Biolegend antibodies contain differently structured conjugated oligos, which is why the constructed library sequences preclude aligned extraction in the same positions of Read 2. By extension, the use of any pool of oligo-conjugated antibodies that would result in misaligned Antibody Multiplexing Barcodes within Read 2 would require the alternative sequencing configuration noted above.
In the Alternate Sequencing Configuration, the following information is extracted from Read 1:
Cycles 1 - 16: GEM barcode
Cycles 17 - 28: UMI
Cycles 29 - 40: pCS1 constant sequence (discarded)
Cycles 41 - 48: GEX Probe and Ab Multiplexing Barcodes
This read configuration also requires a 10% PhiX spike-in when sequencing on the NovaSeq 6000 or a 5% PhiX spike-in when sequencing on the MiSeq, NextSeq 500/550, or NextSeq 2000.
For details on how to perform intracellular protein labeling, see the following article: Is the Fixed RNA Profiling assay compatible with intracellular protein labeling?
Please Note: For Singleplexed Ab libraries, there is no need to utilize the Antibody Multiplexing Barcodes during analysis, so the default sequencing configuration of Read 1 (28 cycles), i7 index (10 cycles), i5 index (10 cycles), and Read 2 (90 cycles) can be used.
Products: Single Cell Gene Expression Flex