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!

[talonaxiomatic]

From what you’ve described, it seems like there could be something going on with the readout for CpG sites, especially in pair two. The lower quality score for the T calls on CpGs is definitely odd and might point to a compensation problem or an issue with how the dark base is being handled. I’d suggest double-checking the background settings in your alignment software to see if there's any adjustment needed for the dark base compensation. It’s also worth reviewing the overall sequencing quality and perhaps even rerunning the analysis with adjusted parameters for methylation calling, just to see if that helps smooth things out. It might be a good idea to reach out to the NovaSeq team as well, especially if this is a new issue with the platform.



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