Workflow Cas9

Introduction

The workflow aids with the quantification of the non-target depletion and provides information on mapping characteristics that highlight the cas9 targeted sequencing protocol performance. The figures plotted include depth-of-coverage over the target regions and strand bias over these regions. The location and peaks of coverage and local biases in strandedness may be used to assess the performance of guide-RNA sequences and may highlight guide RNAs that are not performing. A review of likely off-target regions over-represented within the sequence collection may inform of strategies to refine guide-RNA design.

Read summaries

Summary of on-target and off-target reads

On target reads are defined here as any read that contains at least 1pb overlap with a target region. Off target reads contain no target-overlapping bases.

Targets

Target coverage plots

Each of the following plots show the amount of coverage per target, for each strand in discretized bins of 100 bp.

Target summaries

• chr, start, end: target location.
• target: the target name.
• nreads: number of reads aligning.
• coverage_frac: fraction of bases within target with non-zero coverage.
• tsize: length of target (in bases).
• median_cov: average read depth across target.
• mean_read_length: average read length of reads aligning.
• strand_bias: proportional difference of reads aligning to each strand.
A value or +1 or -1 indicates complete bias to the forward or reverse strand respectively.
• kbases: number of bases in reads overlapping target.

Coverage distribution

These plots show the on-target / off-target coverage distribution of genomic regions binned by 100bp. Off-target regions are defined as any region not within 1kb of a target. The background histogram should naturally be be skewed heavily to the left, this noise being expected when many regions in the genome have a single read mapping. If the targeted sequencing approach has performed well, the on_target histogram should be skewed towards the right indicating a depletion of non-target reads.

Off-target hotspots

Off target regions are again defined here as all regions of the genome not within 1kb of a target region. An off-target hotspot is a off-target region with contiguous overlapping reads. These hotspots may indicate incorrectly- performing primers. Only regions with 10 reads or more are included

Software versions

Workflow parameters