Purpose

AmpliCan is an analysis tool for genome editing that unites highly precise quantification and visualization of genuine genome editing events. ampliCan features nuclease-optimized alignments, filtering of experimental artifacts, event-specific normalization, and off-target read detection and quantifies insertions, deletions, HDR repair, as well as targeted base editing. It is scalable to thousands of amplicon sequencing-based experiments from any genome editing experiment, including CRISPR. It enables automated integration of controls and accounts for biases at every step of the analysis.

Config file details

Columns of the config file:

• ID - should be a unique identifier of your experiments.
• Barcode - use this to group experiments with the same barcode.
• Forward_Reads, Reverse_Reads - put names of your files in these fields, you can put here files compressed to .gz, we will unpack them for you.
• Group - use this field if you want to group ID by other criteria, here for instance we will group by person that performed experiment, on later stage it is possible to generate report with breakdown using this field.
• guideRNA - put your guideRNA in 5’ to 3’ fashion here, if he has to be reverse complemented to be found in the amplicon put “1” into Direction field, we will use this field to make sure your guideRNA is inside the amplicon and during plotting of the results.
• Forward_Primer, Reverse_Primer - make sure these primers are correct for each of your IDs, Forward_Primer should be found in the amplicon as is, on the left side of the guide, Reverse_Primer reverse complement should be found on the amplicon on the right side of the guide.
• Direction - here “0” means guide does not requires to be reverse complemented to be matched to the amplicon, “1” means it should be reverse complemented, this field is also used when plotting deletions, mismatches and insertions.
• Amplicon - insert amplicon sequence as lower case letters, by putting UPPER CASE letters you can specify expected cut site, you should specify at least one cut site, here for example we specify in uppercase letter PAM sequence of the corresponding guideRNA.
• Donor - insert donor template if you have designed HDR experiments or used base editors, otherwise leave empty, but keep the column. amplican will estimate HDR efficiency based on the events from aligning donor and amplicon sequences, donor and reads and reads and amplicon.

If you have only forward primers leave column Reverse_Primer empty, leave empty also the Reverse_Reads column. You can still use amplican like normal.