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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. Specify a list of overhangs

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

• Overhangs should be written in 5' to 3' direction.
• Overhangs should be four bases in length using only canonical A, C, G, T bases; degenerate and non-standard bases are not supported.
• Complementary (reverse) overhangs are automatically added to the query.
• Incorrect or duplicate overhangs are automatically discarded; spaces (if any) are excluded; overhangs may be entered in upper or lower case.

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. 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.

Results

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.

Legend

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

• Watson-Crick pairs are indicated in green:
• High ligation frequency is expected for WC pairs
• (
  ) Strong WC pairs are shown in dark green.
• such pairs ligate frequently: >100 ligations (expected maximum 300-400 ligation events).
• (
  ) Weak WC pairs are shown in light green.
• such pairs ligate less efficiently: <100 ligations (users may wish to avoid these overhangs).


• Mismatch ligations are indicated in orange:
• Three orange color gradations are pre-defined:
• (
  ) trace mismatch ligations: <10 ligations; low frequency events have only a modest impact on multiplexed assembly accuracy.
• (
  ) modest mismatch ligations: 10 - 100 ligations; these mismatch ligation events occur with significant frequency and should be avoided.
• (
  ) high-count mismatch ligations: >100 ligations; these events are observed on a frequency similar to that of proper Watson-Crick ligations.

Ligation preferences

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

• Overhangs in rows correspond to top strand
• Overhangs in columns correspond to bottom strand
• Overhangs are always written in 5' to 3' direction
• For example, ATTC overhang in a row and GAAT overhang in a column corresponds to the WC pair

  5'-ATTC-3'
  3'-TAAG-5'

  This is a strongly ligating WC pair as indicated by the dark green color.
• TACC overhang in a row and GGTT overhang in a column corresponds to a pair

  5'-TACC-3'
  3'-TTGG-5'

  with a A/T mismatch. This is a mismatch ligation that is modestly frequent as indicated by the light orange color.

References

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, https://doi.org/10.1093/nar/gky303
2. Comprehensive profiling of four base overhang ligation fidelity by T4 DNA Ligase and application to DNA assembly (2018). ACS Synthetic Biology, https://doi.org/10.1021/acssynbio.8b00333i

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