Validation of CRISPR/Cas Experiments

Output Description

Analysis was done with Crispresso2 (Clement et al. 2019, see download). Note that depending on the analysis settings, the output files may vary. For example, if only indels are considered for analysis, output files for substitutions will not be generated.

Summary HTML report

• Location: bfx*.crispresso.html
• Summary of the results of all samples

Per-sample results

HTML report

• Location: CRISPRessoPooled_on_*.html
• Summary report that can be viewed in a web browser containing all of the output plots and summary statistics

Overall preprocessing statistics

• Location: CRISPRessoPooled_on_*/MAPPING_STATISTICS.txt
• Text file showing the number of reads in the input, reads after preprocessing (filtering, trimming, and merging) and reads aligned

Overall amplicon statistics

• Location: CRISPRessoPooled_on_*/REPORT_READS_ALIGNED_TO_AMPLICONS.txt
• Tab-separated text file showing for each amplicon its name, sequence, sgRNA, expected alternative allele (in case of homology-directed repair), coding sequence as well as the name of the corresponding fastq file after preprocessing, the number of reads aligned and the percentage of reads aligned

Overall quantification summary

• Location: CRISPRessoPooled_on_*/SAMPLES_QUANTIFICATION_SUMMARY.txt
• Tab-delimited text file showing for each amplicon the percentage of unmodified and modified reads, numbers for total, aligned, unmodified, modified and discarded reads per amplicon as well as numbers for reads with insertions, deletions, substitutions, and combinations of these modifications

Per-amplicon results

The following files are generated for each amplicon provided enough reads are aligned to the amplicon.

Mapping statistics

• Location: CRISPRessoPooled_on_*/CRISPResso_on_*/Crispresso_mapping_statistics.txt
• Tab-delimited text file showing the number of reads in the input, reads after preprocessing (filtering, trimming, and merging), reads aligned, reads for which alignments were computed, reads for which alignments were used from cache (i.e. have already been computed), reads for which no alignments could be computed and reads for which no alignments could be computed from cache

Quantification summary

• Location: CRISPRessoPooled_on_*/CRISPResso_on_*/Quantification_of_editing_frequency.txt
• Tab-delimited text file showing the percentage of unmodified and modified reads, numbers for total, aligned, unmodified, modified and discarded reads as well as numbers for reads with insertions, deletions, substitutions, and combinations of these modifications

Alleles

• Location: CRISPRessoPooled_on_*/CRISPResso_on_*/Alleles_frequency_table.zip
• Tab-delimited text file showing for each allele the sequence, the corresponding amplicon sequence, amplicon name, whether the allele sequence was modified, how many deletions, insertions, substitutions the allele sequence has, the number of reads for that allele sequence, and the percentage of reads for that allele sequence

Alleles per amplicon at CRISPR/Cas9 cut site

• Location: CRISPRessoPooled_on_*/CRISPResso_on_*/Alleles_frequency_table_around_sgRNA_NNNNN.txt
• Tab-delimited text file showing for each allele at CRISPR/Cas9 cut site the sequence, the corresponding amplicon sequence, whether the allele is unmodified (hence the reference), the number of deletions, insertions and substitutions as well as the number and percentage of reads accounting for this allele

Frameshift analysis

• Location: CRISPRessoPooled_on_*/CRISPResso_on_*/Frameshift_analysis.txt
• Text file with the number of noncoding mutations, in-frame mutations and frameshift mutations

For a more detailed description of the output files, please refer to the CRISPResso2 documentation.

References

Clement, Kendell, Holly Rees, Matthew C Canver, Jason M Gehrke, Rick Farouni, Jonathan Y Hsu, Mitchel A Cole, et al. 2019. “CRISPResso2 Provides Accurate and Rapid Genome Editing Sequence Analysis.” Nature Biotechnology 37 (3): 224–26. https://www.nature.com/articles/s41587-019-0032-3.