TIDE provides rapid and reliable assessment of genome editing experiments of a target locus. 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.
The input to TIDE is Sanger sequencing data.
The output of TIDE is a comprehensive profile of all insertions and deletions (indels) in the edited sample.
This web tool was developed by Eva Brinkman, Tao Chen and Bas van Steensel from the Bas van Steensel lab.
For more information and to report bugs, please contact email@example.com
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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
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 decomposition window is set on a sequence segment downstream of the break site. The default setting is the largest window possible for the uploaded sequences. In general, the larger the decomposition window is chosen, the more robust the estimation of mutations is.
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 uploaded samples appear in the ???Uploads??? tab. Here you can check if the guide sequence, control file and test files are correctly matched. Also potential errors if particular files will be noted. The plots can be downloaded in the: "Download" tab. If the settings are incorrect or too stringent, warnings or remarks will be displayed in the file.
A depiction of the combination of trace models (indels) that can best explain the composite sequence trace in the experimental sample as determined by non-negative linear modelling. An R2 value is calculated as a measure of the goodness of fit, and the statistical significance of the detection of each indel is calculated.
The signals of all nucleotides: A, G, T, C at each position in the sequence file are used. In general, each position in the sequence trace is represented by one predominant nucleotide signal indicative of the actual nucleotide. The minor signals from the other three nucleotides are normally considered as background. The percentage of these aberrant nucleotides is plotted along the sequence trace of the control and the experimental sample.
Thus, a value of 0% at a position indicates that the detected nucleotide does not differ from the control sequence while a value of 100% indicates that the expected nucleotide was not detected at all (and instead only one or more of the other three nucleotides). The percentages of aberrant nucleotides in the control should be low along the whole sequence trace. However, the experimental sample consists of a mixture of multiple sequences due to the presence of indels and possible point mutations. Around the break site the sequences start to deviate from the control, which is visible with consistently elevated signal of the aberrant sequence signal. Note that there is a 25% chance that an identical nucleotide in a mutated sequence is found as is present in the control sequence at the same position, because there are only 4 different nucleotides available.
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.
An estimate of the base composition of this insertion.
Data can be downloaded when the valid sequencing files and guide string have been uploaded.
The requirements of sequence length are flexible. This region upstream of the break site is used to align the sequencing data of the test sample with that of the control sample. The region behind the break site is used for a decomposition to determine the various indels in the pool of cells. In general, with a bigger stretch of sequence trace a better estimation can be performed by TIDE. 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.
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.
TIDE should work with sequences with a break site closer or further than 200 bp to the start of the sequence trace if the quality of the sequence reads is good. In that case of break site closer to beginning, the start of sequence read (alignment window) have to be set lower than the default setting of 100. The break site is too close to the start of the sequence read in the default setting to perform an alignment (see figure). The alignment window can be changed under Advanced settings. In case of break site further from the beginning, the decomposition window become smaller. Note, a larger stretch of nucleotides selected often makes the estimation of the indels more reliable. The minimal decomposition window spans from 2x n bp indel size & it has to be at least 5bp from the sgRNA target site or the end of the sequence.
The overall efficiency doesn't need to add up to 100%, because there is also noise in the data.
The overall efficiency is calculated as R2 - % wildtype (zero indels). 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.
The different indel bars represent the different insertion and deletions in the population. You can’t tell for an individual cell what the specific indels of each allel is. To determine allel specific information you have to isolate a cell clone and perform TIDE analysis.
The different indel bars represent the different insertion and deletions in the allels in a cell clone. With a diploid cell you should get a percentage of ~50% per indel.
The sequences of the specific indels cannot be deduced from the sequence trace. We tested this option, but Sanger sequening is not sensitive enough to give reliable results on this. To know the precise sequence of the mutations you can use next generation sequencing or sequencing the DNA of individual mutated clones.
You can use TIDE for other RNA guided nucleases by entering in the TIDE webtool the DNA sequence around the expected cut site. TIDE assumes that a dsDNA break is induced between nucleotides 17 and 18 in the input sequence. You can estimate which sequence this will be for the other nucleases. Note that if you don’t know the exact breakpoint, TIDE should work fine to estimate the efficiency, but the +1 estimation is not be reliable anymore.
At the moment 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 are working on this issue. For now, 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).
Sometimes the quality of the peaks in chromatogram looks fine, but the file has some wrongly unannotated or wrongly additional annotated nucleotides. These will interfere with the indel spectra (see figure wrongly unannotated nucleotide). TIDE gives a warning when the spacing between the nucleotides in the chromatogram of the sequence trace are not consistent, which is often an indication for wrongly unannotated or wrongly additional annotated nucleotides. Then the sequence file cannot be used for a reliable TIDE analysis. Try if possible to set the right boundary of the decomposition window lower. In case this warning stays, carefully investigate your chromatogram.
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.