Insider is a program that identifies genomic positions where several aligned reads have the same start or end positions. The tool can be used to identify genomic positions for viral integration sites, or to find on- and off-target sites in CRISPR-Cas9 digested genomic DNA. In both cases, the sites are detected with high sensitivity and specificity by rapid processing of read alignments.
./insider.pl [options] --help -i -o -ots -offset -minp -mins -minc --silent
-i Insider input file (SAM format).
-o Output file for Insider results.
-ots Set this flag for analysis of CRISPR-Cas9 cleavage sites in
off-target sequencing (OTS) data.
-offset Offset between left-end and right-end soft clipped alignments in
host genome, used when searching for insertion sites (default=10).
-minp Minimum number of soft clipped reads starting at the exact same
position in the insertion site peak. This number of reads is
required for both the left-end and right-end alignments (default=3).
-mins Minimum number soft clipped reads starting at the exact same
position in the insertion site peak, when taking both left-end
and right-end alignments into account (default=10).
-minc Minimum number of soft clipped bases required for a read to be
considered in the Insider analysis (default=20).
--silent Do not print status to stdout.
In this example, we will use Insider for detection of CRISPR-Cas9 off-target sites in the human genome. The data was generated using the Nano-OTS protocol presented in this study. The nanopore reads were aligned to the human reference genome (GRCh38) using Minimap2.
First, download the Insider git repository using the following command:
git clone https://github.com/UppsalaGenomeCenter/insider.git
In the repository you will find both the executable file and an example SAM file.
To execute Insider, simply run the following command:
`./insider.pl -i Nano-OTS.example.minimap2.chr22.sam -o out.txt -ots`
The example SAM file contains reads for human chromosome 22, resulting from applying the Nano-OTS protocol for three gRNAs (ATXN10, MMP14 and NEK1) to DNA from the human cell line HEK293. The insider software identifies Cas9 cleavage sites for these gRNAs. For this example, the following results are obtained:
chr22 11211032 11211032 5
chr22 11215498 11215498 7
chr22 16937226 16937226 20
chr22 45689924 45689924 5
chr22 45794862 45794862 51
chr22 16937206 16937206 5
chr22 47094773 47094773 8
The three first result columns display the chromosome, start and end positions for the Cas9 cleavage site (GRCh38 coordinates). The fourth column displays the number of reads in support of a Cas9 cleavage site. The coordinate chr22:45794862 corresponds to the on-target site for ATXN10 and all other coordinates are potential off-target sites.
Copyright (C) 2015-2020 Ignas Bunikis, Adam Ameur
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