We have found that Single Cell 3’ v2 libraries sequenced on NextSeq 500/550 can show significant variability in sequencing metrics across different flow cell lots. For SC3' v1 libraries sequencing variability will primarily affect i7 Read (barcode read) quality, whereas for SC3 v2 libraries the Read 2 (transcript read) quality is affected. In both cases the root cause is believed to be an interaction between the long poly-T stretch and the flow cell surface chemistry on Illumina NextSeq flow cells. This is based on the observation that if we create libraries with shorter or no poly-T tails but otherwise follow the same protocol, the Q30 quality scores goes back up to normal.
We encourage users to carefully analyze their data with Cell Ranger and Loupe Cell Browser to determine the impact of variable primary sequencing performance on the secondary analysis.
For more information about sequencing variability on the NextSeq for SC libraries please review the Technical Note Chromium™ Single Cell 3’ v2 Libraries – Sequencing Performance on Illumina® NextSeq® 500 Flow Cells (https://support.10xgenomics.com/single-cell-gene-expression/index/doc/technical-note-chromium-single-cell-3-v2-libraries-sequencing-performance-on-illumina-nextseq-500-flow-cells)