This is a web tool to rapidly assess genome editing by CRISPR-Cas9 of a target locus determined by a guide RNA (sgRNA). Based on the quantitative sequence trace data from two standard capillary sequencing reactions the TIDE software quantifies the editing efficacy and identifies the predominant types of insertions and deletions (indels) in the DNA of a targeted cell pool. See Brinkman et al, Nucl. Acids Res. (2014) for a detailed explanation and examples.
Enter a 20nt ('5-'3) DNA character string representing the used sgRNA guide sequence immediately upstream of the PAM sequence (PAM not included). Numbers and other invalid (non-IUPAC) DNA characters will be automatically removed. TIDE assumes that a dsDNA break is induced between nucleotides 17 and 18 in this sequence.
Next, upload the chromatogram sequence files of respectively the control sample (e.g. transfected without Cas9 or without the sgRNA) and the test sample (e.g. DNA of pool of cells treated with both Cas9 and the sgRNA).
We advise to sequence a stretch of DNA ~700bp enclosing the designed editing site. The projected break site should be located preferably ~200bp downstream from the sequencing start site. This region upstream of the break site is used to align the sequencing data of the test sample with that of the control sample.
Currently, ABIF (.ab1) and SCF (.scf) files are supported. SCF is an open standard and several tools exist to convert other formats to SCF files.
The following parameters have default settings but can be adjusted if necessary in the panel to the left by checking the 'advance settings' box.
These settings determine the window in which the control and test sequences are aligned to determine any offset between the two reads. There is usually no need to deviate from the default settings, except when long repetitive sequences are present.
By default this is set to 100, because base-calling at the start of a Sanger sequence read is often of poor quality.
right boundary: This is automatically set at break site - 10bp
These settings determine the sequence segment used for decomposition. The default setting is the largest window possible for the uploaded sequences:
max indel size + 5bp downstream of the break site.
right boundary: max indel size + 5bp before the end of the shortest sequence read.
Set the maximum size of deletions and insertions to be modeled. The default value is 10.
Significance cutoff. Any value between 0 and 1 is accepted. Default is p<0.001
Once the data are uploaded and parameters are set, submit the data by clicking on the "update view" button and the plots will appear in the three tabs: "Quality", "Decomposition, "+1 insertion". If the settings are incorrect or too stringent, warnings or remarks will be dispayed in the "Quality and/or Decomposition" tab.
Quality measures: Results depend on the quality of the sequence reads. As a rule of thumb, we recommend to aim for an average aberrant sequence signal strength before the breaksite <10% (both control and test sample), and R2>0.9 for the decomposition result. Sequencing of the opposite strand is recommended to confirm the results.
All copyright is exclusively owned by Stichting het Nederlands Kanker Insituut - Antoni van Leeuwenhoek ziekenhuis (The Netherlands Cancere Institute). The availability and use of this software is subject to a license from the copyright holder. If you use this software for data analysis in a publication, please cite Brinkman et al, Nucleic Acids Res. 2014 Dec 16;42(22):e168. doi:10.1093/nar/gku936.
R Core Team (2013). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. www.R-project.org . R version 3.1.1.
H. Pages, P. Aboyoun, R. Gentleman and S. DebRoy. Biostrings: String objects representing biological sequences, and matching algorithms. R package version 2.32.1.
J.T. Hill and B. Demarest (2014). sangerseqR: Tools for Sanger Sequencing Data in R. R package version 1.3.1. http://www.bioconductor.org/packages/devel/bioc/html/sangerseqR.html
K. M. Mullen and I. H. M. van Stokkum. The Lawson-Hanson algorithm for non-negative least squares (NNLS). R package version 1.4.
RStudio and Inc. (2013). shiny: Web Application Framework for R. R package version 0.11.1. http://CRAN.R-project.org/package=shiny
A plot will be shown here when the valid sequencing files and guide string have been uploaded.
Check the following criteria to determine the quality of your data (use the Aberrant sequence signal plot)
The prediction of the +1 inserted nucleotide will be shown here when the valid sequencing files and guide string have been uploaded.
In some instances the webtool appears not to respond due to firewall problems. Please contact us at email@example.com and let us know the nature of the problem.
Some .ab1 sequence files are not compatible with the TIDE webtool. Various software programs that process the raw sequence data can cause this problem. We recommend that you export the data as a .scf file and then uploaded in TIDE. The .ab1 format can also be converted to .scf using software such as 4peaks (Mac) or FinchTV (Windows & Mac).
TIDE should work with shorter sequences if the quality of the sequence reads is good. In that case the start of sequence read (alignment window) might have to be set lower than the default setting of 100. Often with shorter sequences the break site is too close to the start of the sequence read in the default setting (see figure). The alignment window can be changed under Advanced settings.
The overall efficiency doesn't need to add up to 100%, because there is also noise in the data.
For example if the R2 value is 0.95, it tells that 95% of the variance can be explained by the model; the remainder 5% is noise or very large indels.
A low R2 can be caused when the settings are not optimal or when the sequence quality is not good.
By default, the decomposition window is set to its maximum size and the Indel size range is set to 10. The settings can be adjusted in advanced settings.
If the forward and reverse indel spectra don't give the same indel spectra, one of the results is not reliable. Often there is a misannotation in one of the sequence files (see explanation Wrongly annotated nucleotides).
Sometimes a mismatch occurs in the control sequence at the location of the sgRNA. This will stop the TIDE analysis. In this case, change the guide into identical IUPAC nucleotides as the control sequence.
With the used settings the decomposition cannot be performed. This can be caused when the settings are not optimal or when the breaksite is too close to the sequence start or end. Try if possible to set decomposition boundaries further apart or use smaller indel size or use lower the alignment window. If that doesn't help you might have to resequence to perform the TIDE analysis. It can also help to sequence the opposite strand. We advise to sequence a stretch of DNA ~700bp enclosing the designed editing site. The projected break site should be located preferably ~200bp downstream from the sequencing start site.
When the beginning of the sequence is of poor quality, the alignment function can make a mistake. This can be observed in the quality plot that has high aberrant sequence signal over the whole length of the sequence trace (see figure). The aberrant sequence signal should only increase around the expected cut site (blue dotted line).
In case of poor alignment, try to shift the start of sequence read (alignment window) higher or lower. The alignment window can be changed in advance settings.