This kit is recommended for users who:

• Want to target specific regions of 5-20 kb to high coverage (>100x) on a single MinION Mk 1B flow cell without PCR.
• Want to sequence a larger, contiguous Region of Interest (ROI), using up to 100 target sites, in a single assay*
• Have 1-10 µg of available high quality gDNA
• Are interested in methylation patterns or other modified bases.
• Have gene targets that are highly repetitive, or wish to evaluate the number of repeats in an expansion, where traditional amplification method or sequencing-by-synthesis methods could yield a biased result
• Want to sequence long gene targets in a single pass that are not amenable to long-range PCR (>30 kb)

* Note: This is known as ‘tiling’ an ROI.

Key features:

Workflow

The Cas9 Sequencing Kit offers a method of enriching regions of interest (ROI) from a genomic DNA sample. After DNA extraction, 5’ ends are dephosphorylated to reduce ligation of sequencing adapters to non-target strands. Cas9 ribonucleoprotein particles (RNPs), with bound crRNA and tracrRNA, are added to the genomic DNA, and bind then cleave the region of interest (ROI). dsDNA cleavage by Cas9 reveals blunt ends with ligatable 5’ phosphates. All of the DNA in the sample is dA-tailed, which prepares the blunt ends for sequencing adapter ligation. Sequencing adapters are ligated primarily to Cas9 cut sides, which are both 3’ dA-tailed and 5’ phosphorylated. The library preparation is cleaned up to remove excess adapters using AMPure XP beads and resuspended in Sequencing Buffer (Non-target molecules are not removed). The subsequent library preparation is added to the flow cell for sequencing.