Question: Why was the percentage of PhiX increased for Multiplexed Fixed RNA Profiling libraries sequenced on NovaSeq 6000?
Answer: To improve the flow cell diversity when sequencing through the constant region before the multiplexing barcode (i.e., boosting the Q30s in the multiplexing barcode), we recommend increasing the percentage of PhiX used for Multiplexed libraries sequenced on NovaSeq 6000. In most cases, sequencing Multiplexed libraries with 5% Phi X on NovaSeq 6000 results in > 90% valid barcodes. On rare occasions, there may be lower sequencing quality in the multiplexing barcode region. To reduce this risk, we recommend increasing the concentration of PhiX to 10% when sequencing on the NovaSeq 6000 for multiplexed FRP libraries. For other Illumina sequencers (e.g., NextSeq, etc.), we still recommend using 5% PhiX. Please refer to the updated Multiplex User Guide.
Products: Fixed RNA Profiling, Single Cell Gene Expression Flex