Inconsistent Methylation Levels Between Pairs in WGBS Data After Moving to NovaSeq X

[MichalM]

Subject: Inconsistent Methylation Levels Between Pairs in WGBS Data After Moving to NovaSeq X

Hi everyone,

I'm analyzing some WGBS data and have noticed a discrepancy in methylation levels between sequencing pairs since moving to the new NovaSeq X.

Previously, methylation levels were consistent between pair one and pair two at around 75%. However, with the NovaSeq X, pair two now shows only 50% methylation, while pair one remains at approximately 75%.

When examining the data in IGV, the alignment quality is high in non-CpG bases for both pairs. However, there’s a frequent issue with CpG bases where:

• Pair one consistently registers a C (indicating a methylated base) with a high quality score.
• Pair two, on the other hand, frequently calls a T at the same CpG site with a much lower quality score.

This suggests there may be a readout issue with the "dark base" G, which seems underrepresented in pair two. Could this be related to a compensation issue or an incorrect background level setting for the dark base in pair two on the NovaSeq X?

Has anyone encountered similar issues with the NovaSeq X, or does anyone have advice on troubleshooting or potential adjustments?

Thanks for any insights!