Leave this at the default setting of 50 bp. The Variants of Interest section of the setup dialog allows you to specify how far upstream and downstream of the CRISPR cleavage site to look for variants. If your reference sequence does not have a CDS annotation, you can set which translation frame to use under the Variant Analysis section. This allows variants to be analyzed for their protein effect using the correct frame. Our reference sequence is a portion of the APC gene and is annotated with a partial CDS annotation. Apc Reference should automatically be set as the Reference sequence. Then open Analyze CRISPR Editing Results from the Annotate and Predict menu. Select the reference file Apc Reference along with the Sample Reads (trimmed) (merged) file you created in Step 1 (hold down control/command to select both files). The reference sequence can be set in the operation dialog, or selected along with the sample reads prior to opening the operation. This will normally be the unedited amplicon sequence. The reference sequence should be a short sequence spanning the CRISPR editing site, of similar length to the reads. The Analyze CRISPR Editing Results tool maps the merged reads to a reference sequence, trims them to the region of interest around the CRISPR editing site, and then collapses the reads into identical clusters and outputs the number of reads in each cluster as a percentage of the total. Step 2: Running “Analyze CRISPR Editing Results” Your BBDuk settings should look as in the screenshot below: To discard reads that are too short to be useful after trimming, select Discard Short Reads and set the minimum length to 20 bp. This will trim poor quality bases with phred scores of less than 20 from the ends of the reads. Then select Trim Low Quality and set the Minimum Quality to 20. Select Trim Adapters and leave the settings as they are. First click the Reset to Defaults option under the grey settings cog in the bottom left of the window to clear any previous settings you have been using. Select Sample Reads and go to Annotate and Predict → Trim using BBDuk. We will first trim this dataset using the BBDuk plugin. The Sample Reads dataset provided for this tutorial contains already paired reads. If you have forward and reverse reads spanning your amplicon, you should assemble these into a single sequence prior to the analysis, using the de novo assembly tool (as described at this link). Note that Analyze CRISPR Editing Results can also be run on Sanger sequences. We also recommend performing basic quality trimming prior to merging to ensure the accuracy of the merging step. Paired reads must then be merged prior to running the analysis. If your reads are paired, you should select the appropriate pairing settings upon importing your file. ![]() Analyze CRISPR Editing Results is designed to be run on unaligned NGS reads imported from fastq files.
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