Scripts to analyze and model DNA double-strand break repair kinetics, see https://www.biorxiv.org/content/early/2017/05/26/142802.
Eva Brinkman 20180328
We uploaded 1 perl script, 3 R/Markdown .Rmd scripts and 2 zipfolders with data necessary for the scripts.
- script fastqForCRISPR_20160627_LBR2.pl process the raw sequence files
Input data: fastq.files (GEO) Output data: each read annotated with type (wt, insertion, deletion, point mutation or not clear) and size of the mutations Note: This is annotated for LBR2, but script can be adjusted for other guide according to the instructions in script.
- script EB20180323_data_kinetics.Rmd processes the sequence files of the timeseries at 4 different loci
We performed a series of PCR experiments to quantify uncut dsDNA and repaired dsDNA induced by CRISPR-Cas9 and a single guide RNA, using NGS as readout. We do this in timeseries with 12 measurements. The fastq data files contain reads from which we can infer whether they originate from intact or from repaired DNA. We simply count those to infer abundances. We repeated this experiment and also PCR amplified 3 other cut sites in the genome in a similar way.
Input data: output.txt files and sum_output.txt files (GEO) Output data: - abundances of mutations at each timepoint in each timeseries saved in txt files similar as in the folder timeseries.zip - plots of the intact/indel fraction in time - plots of abundances +1/-7 indels generated by sgRNA LBR2
- script TC20170313_ZLWB2_EB20180413.Rmd
The abundances of intact and repaired DNA data after Cas9 cut is uploaded per time series. We use this data to estimate the rates at which the dsDNA is cut and is repaired by different DNA repair pathways by mathematelical modelling. Validation of the model by measuring the broken ends and parameter sweeps are included in the script.
Inputdata: - abundances of mutations at each timepoint in each timeseries saved in txt files similar as in the folders timeseries.zip - quantification of the broken fraction detecion assay, txt files in folder LM-PCR.zip (raw image files and normalization in Mendeley: https://data.mendeley.com/datasets/wg4ssg7pfw/draft?a=536407f5-2248-4cef-a949-bf7e709ebf17)
- script EB20180319_doublecut.Rmd
We performed a series of PCR experiments to quantify uncut dsDNA and repaired dsDNA induced by CRISPR-Cas9 and two guide RNAs, using NGS as readout. 5 combinations of guide RNAs are tested 1 combination is tested in timeseries experiments. The fastq data files contain reads from which we can infer whether they originate from intact or from repaired DNA. We simply count those to infer abundances.
Input data: output.txt files and sum_output.txt files (GEO) Output data: - plots of abundances mutations products after transfection of 2 sgRNAs - plots of the intact/indel/excised fraction after transfection of sgRNA LBR2/LBR8 in time