Question: Why are my Q30 quality scores low on NextSeq 500/550?
Answer: While sequencing metrics do differ across sequencing platforms (e.g. MiSeq versus HiSeq 4000), they are generally consistent across runs on the same platform. However, Single Cell 3’ v2 libraries sequenced on NextSeq 500/550 v2 sequencing kits can show significant variability in sequencing metrics (especially for Read 2) across different flow cell lots compared to other sequencers.
We have tested running the same library on different flow cell lots and observe Read 2 quality scores ranging from the upper 70s for one flow cell lot and between 20-40% for the worst performing flow cell lot. We consider a Q30 quality score less than 50% is to be poor. Overall, the majority of sequenced libraries we tested or our customers tested (55 out of 75) showed Read 2 Q30 scores greater than 50.
Low Q30 quality score may impact application performance. However, key metrics such as the number of “Valid Barcodes”, “Estimated Number of Cells”, “Fraction Reads in Cells”, and “Total Genes Detected” are largely unaffected by variation in Read 2 Q30 scores. Cluster analysis with Loupe Cell Browser indicates that individual PBMC subtypes, for instance, can be identified from even low-quality sequencing runs. Application performance, however, will be sample dependent.
There have been recent reports of improved sequencing quality with NextSeq v2.5 sequencing kits.
Products: Single Cell Gene Expression