GETSET™: Help Page


Predict high-fidelity junction sets.

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 overhangs

Choose a number of overhangs in the set.

4. Required overhangs

Specify overhangs that must be present in the set. Note that palindromic overhangs are disregarded.

5. Excluded overhangs

Specify overhangs that must not be present in the set. The 'excluded' overhangs are disregarded if they are also present in the 'required' list.

6. 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):

Appendices

Most frequent four base mismatch pairs

-----------------------
O1	O2    Relative
              frequency
-----------------------
ACGG	CCGC	110
CCGG	TCGG	109
ACGC	GCGC	107
CGCC	GGCA	105
ACCG	CGGC	99
CGAC	GTCA	97
CGGC	GCCA	96
ACAG	CTGC	93
CAGG	TCTG	92
CGAG	TTCG	83
AGGT	GCCT	83
CTGT	GCAG	82
CGCA	CGCG	82
GGCT	TGCC	82
GTCT	TGAC	81
CGGG	TCCG	80
CCGT	GCGG	78
AGAC	GTCA	77
ACGG	TCGT	77
GACT	TGTC	77
-----------------------
(Full list)

Most frequent three base mismatch pairs

-----------------------
O1      O2    Relative
              frequency
-----------------------
GCC	GGT	34
GCT	GGC	34
GAT	GTC	33
CTC	GTG	29
GAC	GTT	25
ACG	CGC	22
CGT	GCG	20
ACC	GGC	19
ACT	GGT	19
CTG	CTG	19
CCT	GGG	19
ATG	CTT	18
GGG	TCC	18
GCG	TGC	18
AGT	GCT	17
GTC	GTC	15
ACG	CGG	15
AGC	GCC	14
CAT	GTG	14
ACC	GGG	14
-----------------------
(Full list)

Estimated assembly fidelity

Generally, the predicted assembly fidelity is expected to be >90% for sets of overhangs that are <20 fragments (4-base overhangs) or <10 fragments (3-base overhangs); however, the predicted maximum assembly fidelity could be significantly impacted requiring that certain junction sequences be included or excluded from the overhang set.

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