Ligase Fidelity Viewer™ (v2): Help Page

This tool allows the user to determine whether a set of four base overhangs is expected to show mismatch ligation between any members of the set. The data used comes from recent publications on ligation fidelity using T4 DNA Ligase (1, 2) and represents subsets of the data sets discussed in those papers.

Basic usage

1. Overhang length

Choose an overhang length. This choice determines which published data set will be used to judge mismatch potential (2).

2. Choose experimental ligation conditions

Choose an experimental condition for which to determine ligation fidelity. This choice determines which published data set will be used to judge mismatch potential (2).

The default conditions are ligation at 25°C for 18 hours; these conditions have been shown to well predict the results of Golden Gate assembly using typical cycled conditions (16°C 5 min/37°C 5 min, 30 cycles).

3. Specify a list of overhangs

Specify the overhangs to be checked as a comma-separated list:

4. Show normalized ligation counts (optional)

By default, the tool provides ligation data in a graphical output, indicating the general efficiency of each connection.

Toggle the checkbox to display normalized ligation counts. In multiplex ligation assay, the relative ligation frequency) was experimentally determined for all 256 four-base overhangs in a single experiment. Total ligation events for each experiment were normalized to 100,000; in this case, a typical frequency for any single Watson-Crick pair was 300-400 observations per 100,000 ligation events. Please see publications for further details.


Estimated fidelity

Given a set of overhangs, the tool estimates the total fraction of correct ligation events out of all ligation events. This value represents the approximate expected fidelity when using all overhangs in the set in a Golden Gate assembly reaction or similar method.


Relative frequency of ligation events for various overhang pairs is indicated by colors:

Ligation preferences

The ligation preferences for all specified overhangs are provided in a tabular format (see example below):


  1. A single-molecule sequencing assay for the comprehensive profiling of T4 DNA ligase fidelity and bias during DNA end-joining (2018). Nucleic Acids Research, gky303,
  2. Comprehensive profiling of four base overhang ligation fidelity by T4 DNA Ligase and application to DNA assembly (2018). ACS Synthetic Biology,