---
title: "How do I confirm the quality of my CUT&Tag sequencing library?"
slug: "confirm-quality-of-cut-and-tag-library"
updated: 2025-09-24T13:43:02Z
published: 2025-09-24T13:43:02Z
canonical: "support.epicypher.com/confirm-quality-of-cut-and-tag-library"
---

> ## Documentation Index
> Fetch the complete documentation index at: https://support.epicypher.com/llms.txt
> Use this file to discover all available pages before exploring further.

# How do I confirm the quality of my CUT&Tag sequencing library?

There are two general approaches to confirm the quality of CUT&Tag sequencing libraries:

## **Fragment distribution of purified library**

- Fragment distribution analysis of purified sequencing libraries is the single BEST method to confirm CUT&Tag success. Libraries should show enrichment of mononucleosome-sized DNA fragments (~300 bp, including CUT&Tag DNA + sequencing adapters).
- Use the H3K4me3 and H3K27me3 positive control reactions and IgG negative control reactions (**Figure 1**) to help confirm CUT&Tag success.
- The "trident pattern" is observed for many histone PTM CUT&Tag libraries, including the H3K27me3 and H3K4me3 libraries in **Figure 1B & C**. These fragments are generated during tagmentation and subsequent indexing PCR. Peaks represent:
  - Subnucleosome fragments: ~200 bp, orange text in **Figure 1**. Note that these fragments are **NOT** adapter dimer/primer dimer.
  - Mononucleosome fragments: ~300 bp, blue text in **Figure 1**. Predominant enrichment at ~300 bp is the best metric for CUT&Tag success.
  - Dinucleosome fragments: ~500 bp, green text in **Figure 1**.
  - Primer dimer fragments: ~25-100 bp, red text in **Figure 1**. If primer dimers comprise >5% of the library, additional cleanup should be performed; see our recommendations for bead:DNA ratios to use in the Application Notes for our CUTANA™ DNA Purification Beads ([EpiCypher 21-1407](http://www.epicypher.com/product/cutana-dna-purification-beads/)).
- The trident peak pattern is not cause for concern, as it typically reflects on-target enrichment. They do not impact sequencing and we do **NOT** recommend removing them, as it risks loss of the mononucleosome peak (~300 bp).

## **Library yields**

- There is no typical yield for CUT&Tag. Library yields vary widely by cell type, number of cells, target abundance, and antibody quality.
- In general, aim for 2 ng/μL or ~30 ng total DNA, which will allow accurate library quantification and minimize PCR duplicates. Library molarity >0.5 nM for the 200-700 bp region will allow pooling at standard concentrations for sequencing.
- For library concentrations below 0.5 nM, see the [CUT&Tag Optimization and Troubleshooting](/v1/docs/assay-optimization-and-troubleshooting-cut-and-tag) section of this website for guidance.

![](https://cdn.document360.io/1fc69f91-cf1e-45ab-bc51-4063eb80e37c/Images/Documentation/TapeStation CUT&Tag Libraries Representative@2x.png)

**Figure 1.** Typical TapeStation traces from CUTANA CUT&Tag libraries targeting IgG (negative control), H3K27me3 (EpiCypher 13-0055), and H3K4me3 (EpiCypher 13-0041 shown; replaced by 13-0060). All libraries are predominantly enriched for mononucleosome-sized library fragments, indicated by the peak at ~300 bp (blue text). Subnucleosome (~200 bp; orange text) and dinucleosome (~500 bp; green text) peaks are present, forming the trident patten discussed above. Minimal primer dimers are observed in the H3K4me3 library (~75 bp; red text).