SPLITSET™: Help Page


Split DNA sequence for scarless high-fidelity assembly.

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. Number of fragments

Specify a number of fragments to divide the provided sequence into.

4. Nucleotide sequence

Provide a nucleotide sequence to divide into fragments. Only canonical A/C/G/T/ bases are allowed.

5. Define split regions

Provide non-overlapping regions in the nucleotide sequence where it can be divided.

6. Excluded overhangs

Specify overhangs that must not be present in the resulting split set. Overhangs must be provided as a comma-separated list. Only canonical A/C/G/T/ bases are allowed.

7. Request ID

Specify Request ID for the previously run analysis to retrieve the results.

Results

Overhangs

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.

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:

Ligation preferences

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

Overhang coordinates

Location of the proposed junctions in the DNA sequence.

Sequence fragments

The resulting split up DNA sequence.

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