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Abbreviations

SNV:

single nucleotide variant

nt:

nucleotide

bp:

base pair

indel:

insertion or deletion

ref:

reference

alt:

alternate

Notes:

• 1 bp insertion probability = proportion of indel outcomes that are 1 bp insertions
• 1 bp frameshift frequency = proportion of indel outcomes that induce a 1 bp frameshift
• 2 bp frameshift frequency = proportion of indel outcomes that induce a 2 bp frameshift
• (Overall) frameshift frequency = proportion of indel outcomes that induce a frameshift (1 or 2 bp)

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Visualizations

Frameshift, variant load, variant impact on frameshift

These three text boxes put CROTON’s frameshift frequency predictions for a gene in the whole genome context, describe the variant load for the user’s gene query, and examine the extent to which variants on a gene impact predicted frameshift frequency. These texts are particularly helpful for users looking to induce a knockout of their gene of interest and would like more contextual information regarding their target gene’s frameshift frequency.

Frameshift predictions

This bar graph shows CROTON’s average frameshift probability predictions over the entire queried gene body. This graph provides a visualization of a gene’s overall frameshift probabilities, and it is helpful to users looking to evaluate whether their potential gene targets have higher or lower frameshift frequency relative to all potential PAM targets on their gene if interest.

Nucleotide Substitution Frequency Map

This bar graph displays the SNVs present on a particular gene. On the x-axis are the alleles present in the reference genome and along the y-axis are stacked bars representing the frequency of alternative alleles.

Histograms for alternate 1 bp insertion, 1 bp frameshift, 2 bp frameshift

This is a series of three histograms describing the distribution of CROTON’s alternate 1 bp insertion probability, 1 bp frameshift frequency, and 2 bp frameshift frequency predictions. These histograms can help users evaluate whether variants present in their potential target regions have a significant effect on CRISPR/Cas9 editing outcomes.

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Table

#	Column header	Description
1	Variant ID	unique identifier for the SNV
2	Variant Position	the genomic position of the SNV
3	Reference Allele	the nt present in the reference genome
4	Alternate Allele	the nt present in an alternate genome
5	PAM ID	unique identifier for the PAM site PAM ID is defined as [`genename`] | [`num`] where `num` identifies the PAM site of a specific gene. The `num` of the PAM site increases as the position of the first nt of the 60 bp section of the gene-of-interest increases on the positive strand.
6	PAM Range	describes the position of the 60 bp sequence CROTON uses to make predictions PAM Range is defined as [`strand`] : [`start`] – [`end`] where `strand` indicates if the PAM site is sense (+) or antisense (–), and `start` and `end` indicate the position of the first and last nt, respectively, of the CROTON input sequence.
7	Max Variant Effect	The maximum absolute difference between CROTON’s set of predictions for the reference and alternate sequence inputs. [This is the maximum between CROTON’s reference and alternate predictions for the set of statistics: 1 bp insertion probability, 1 bp frameshift frequency, and 2 bp frameshift frequency (shown in table), as well as deletion frequency, 1 bp deletion frequency, and overall frameshift frequency (not shown in table).]
8	Ref. 1 bp insertion	CROTON’s 1 bp insertion probability prediction for the reference sequence
9	Ref. 1 bp frameshift	CROTON’s 1 bp frameshift frequency prediction for the reference sequence
10	Ref. 2 bp frameshift	CROTON’s 2 bp frameshift frequency prediction for the reference sequence
11	Alt. 1 bp insertion	CROTON’s 1 bp insertion probability prediction for the alternate sequence
12	Alt. 1 bp frameshift	CROTON’s 1 bp frameshift frequency prediction for the alternate sequence
13	Alt. 2 bp frameshift	CROTON’s 2 bp frameshift frequency prediction for the alternate sequence
14	Ref_seq and alt_seq	The 60 bp PAM-containing sequence in the reference and alternate genome (the 0-index PAM site is at position 33). There is a single nucleotide difference between the reference and alternate sequences.

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