Does the methylation + transcriptome workflow include Lambda DNA spike-in controls for conversion efficiency estimation?

Answer:
No. The SeekOne DD Single-Cell Multiome Methylation + RNA workflow does not use Lambda DNA spike-in controls for methylation conversion efficiency estimation.

Instead, overall conversion efficiency is calculated directly from endogenous library sequences using internal non-methylated cytosine-containing regions incorporated into the library structure.

Principle of C-to-T conversion efficiency estimation

In the methylation workflow, enzymatic methyl-sequencing chemistry (EM-seq) is used to distinguish methylated and unmethylated cytosines.

During enzymatic conversion:

• Unmethylated cytosines (C) are converted into uracil (U) 
• During sequencing, uracil is subsequently read as thymine (T) 
• As a result: Unmethylated C detected as T; Methylated C remains as C 

The overall C-to-T conversion rate therefore reflects the proportion of unmethylated cytosines successfully converted during library preparation.

Library structure overview

• SP1 / SP2: Sequencing adapter sequences
• Barcode: 17 bp cell barcode
• UMI: 12 bp unique molecular identifier sequence
• TSO: 13 bp template switch oligo sequence (TTTCTTATATGGG)
• 17L: 17 bp fixed sequence (CgtCCgtCgttgCtCgt)
• ME: 19 bp fixed sequence (AGATGTGTATAAGAGACAG)
• 9 bp region: Transposase insertion-derived extension sequence

Conversion-efficiency calculation strategy

To prevent incorrect conversion of sequencing adapters during enzymatic conversion:

• Cytosines within the SP1 and SP2 adapter regions are methylation-protected 
• Cell barcode (CB) and UMI regions do not contain cytosines 
• Cytosines within the TSO, 17L, and ME regions are intentionally left unmethylated. These cytosines are expected to undergo C-to-T conversion during EM-seq conversion. 

The fixed 17L and ME sequence regions are therefore used as internal references for estimating overall conversion efficiency.

Advantages of the internal conversion-efficiency strategy

Compared with external spike-in approaches, this strategy:

• Avoids introducing additional exogenous DNA molecules 
• Enables conversion-rate estimation directly from sequencing reads 
• Reduces variability associated with spike-in quantification 
• Simplifies library composition and downstream QC workflows 

Important notes

• Conversion efficiency is an important QC metric for methylation library quality assessment. 
• Insufficient C-to-T conversion may affect downstream methylation calling accuracy. 
• Internal fixed-sequence conversion analysis is performed during downstream bioinformatic processing.