Input
Result
Data: Potapov et al. 2018, T4 DNA ligase, 37 °C, 1 h, all 256 × 256 four-base overhang pairs. Limit: the score describes ligation fidelity under that one condition. Other ligases, temperatures and incubation times have their own matrices and give different numbers.
METHOD
How it is calculated
Potapov and colleagues sequenced single ligation events between every pair of four-base overhangs and published the counts as a 256 × 256 matrix. For a planned assembly, the overhangs present in the reaction are your set plus the reverse complement of each member. A junction is correct when an overhang ligates to its Watson-Crick partner, and any other ligation among the overhangs present is a mismatch.
Following Pryor et al. (2020), the fidelity of one junction is the share of its observed ligation events that are correct, and the fidelity of the whole set is the product over junctions:
Ncorrect counts events between Oi and its partner, in both directions. Ntotal counts events between Oi and any overhang present, in both directions. A palindromic overhang (one that equals its own reverse complement) is its own partner and can join two copies of the same fragment, so the page flags it. A repeated overhang is one junction species and is scored once, also with a warning.
Which calculation this page matches
Set fidelity and per-junction fidelity are identical to Nucleora's own Golden Gate fidelity module on 44 test sets of 1 to 31 overhangs, including palindromes, duplicates and reverse-complement pairs. They are also identical to an independent implementation of Pryor et al. (2020) equations 1 and 2 on the published matrix. All 65,536 matrix cells on this page equal the published file.
References
- Potapov V, Ong JL, Kucera RB, et al. (2018) Comprehensive profiling of four base overhang ligation fidelity by T4 DNA ligase and application to DNA assembly. ACS Synth Biol 7:2665-2674. doi:10.1021/acssynbio.8b00333
- Pryor JM, Potapov V, Kucera RB, et al. (2020) Enabling one-pot Golden Gate assemblies of unprecedented complexity using data-optimized assembly design. PLoS ONE 15:e0238592. doi:10.1371/journal.pone.0238592
WORKED EXAMPLE
A worked example, computed by this page
A five-junction set, entered as GGAG AATG AGGT GCTT CGCT. With their reverse complements, 10 overhang species are in the reaction.
| Overhang | Partner | Junction fidelity |
|---|---|---|
| GGAG | CTCC | 99.97 % |
| AATG | CATT | 100.00 % |
| AGGT | ACCT | 100.00 % |
| GCTT | AAGC | 99.97 % |
| CGCT | AGCG | 99.97 % |
The product is a set fidelity of 99.92 %. The lowest junction is GGAG at 99.97 %, and the most frequent mismatch among the overhangs present is ACCT : ACCT with 2 observed events. Swapping the limiting overhang is usually the quickest way to raise the score.
WHERE THIS RUNS
The calculation runs in your browser
The arithmetic on this page is JavaScript executing in your browser. The sequence you enter is not sent anywhere: the calculator makes no network request with it, and there is no account, upload or server-side step. You can disconnect from the network after the page has loaded and the calculator keeps working.