flowchart LR D[DNA] --> E[End repair + dA tail] E --> A[EM-seq adaptor ligation] A --> P[TET2 + T4-BGT protect 5mC/5hmC] P --> C[APOBEC converts unmodified C] C --> PCR[Q5U amplification + unique dual index] PCR --> L[Sequence-ready library]
Protocol 6: NEBNext EM-seq library preparation
protocol
NEBNext EM-seq
library preparation
methylation
From fragmented DNA to indexed Illumina libraries that encode modified-cytosine state
What the protocol does
NEBNext EM-seq converts a DNA sample into an indexed Illumina-compatible library while encoding cytosine modification state. End repair/dA-tailing and adaptor ligation precede enzymatic protection and deamination.
| Output | What it is |
|---|---|
| indexed library tube | amplified molecules ready for Illumina sequencing |
| fragment-analyzer trace | library-size distribution |
| concentration/molarity | pooling input |
| index/sample sheet | demultiplexing key |
| control metrics | evidence for protection/conversion performance |
The NEB v2 manual states that the assay jointly detects 5mC and 5hmC and does not distinguish them. The site must record whether the original E7120 or v2 E8015 kit was used; their ranges and workflow details differ.
QC gate
Check library yield, size, adapter dimer, cycle count, unique-dual-index assignment, positive methylated and unmethylated controls, plate/batch, and replicate agreement. Never infer sample methylation from library concentration.