Table 4. CRISPResso2 common batch parameters.

Careful analysis is required to ensure accurate assessment of editing and indels. As a starting point, we recommend the following parameters. Below the parameter descriptions, we have provided example setups for standard mode (to be used for SNPs) and HDR mode (to be used for insertions, deletions, or multiple base changes).

Description of CRISPResso2 parameters important for prime editing analysis
r1 (fastq_r1)	Specifies the name of the fastq file to be analyzed (a second r2 entry is required for analyzing paired end reads).
a (amplicon_seq)	Specifies the nucleotide sequence of the unedited amplicon
n (name)	Specifies the desired output filename
g (guide_seq)	Specifies the nucleotide sequence of the protospacer targeted for editing
q (min_average_read_quality)	Specifies the minimum average phred quality score needed for a read to be included in the analysis. The recommended value is 30.
qwc (quantification_window_coordinates)	Specifies the region of the unedited reference amplicon that CRISPResso2 will analyze for indels. The specified range is inclusive and zero-indexed, meaning that the first nucleotide of the amplicon is position 0. We recommend setting a range spanning from 10 bp 5′ upstream of the pegRNA-guided nick to 10 bp 3′ downstream of the 3′ flap generated by the RT or any secondary nick, whichever is longer, such that the entire inter-nick distance, flanked by 10 bp on either side, is analyzed for indels.
e (expected_amplicon_seq)	Specifies the nucleotide sequence of the edited amplicon. Only include this parameter when running CRISPResso2 in HDR mode to quantify insertions, deletions, or multiple-base pair substitutions.
discard_indel_reads	When set to TRUE, CRISPResso2 will discard reads containing an indel and count the number of discarded reads with respect to the reference amplicon (and also the expected amplicon in HDR mode). Doing so streamlines quantification of PE indels, as discarded reads can be easily counted after analysis.
Example batch parameter file for CRISPResso2 standard mode
r1	a	n	g	q	qwc	discard_indel_reads
SampleX_filename	Unedited reference amplicon sequence	SampleX_outputname	Protospacer sequence	30	StartingBP-EndingBP	TRUE
Example batch parameter file for CRISPResso2 HDR mode
r1	a	n	g	q	qwc	Discard_indel_reads	e
SampleX_filename	Unedited reference amplicon sequence	SampleX_outputname	Protospacer sequence	30	StartingBP-EndingBP	TRUE	Edited reference amplicon sequence