Cas9 Sequencing Kit
SQK-CS9109

Targeted sequencing of genomic regions using CRISPR-Cas9

Information

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.
FeatureProperty
Preparation time110 minutes
Input requirement1–10 µg of double-stranded DNA (5 µg recommended)
PCR RequiredNo
FragmentationNot required
Read lengthEqual to fragment length
Read type produced1D
Typical throughput*•••
Associated protocols/documentationCas9 Sequencing Kit (SQK-CS9109), Targeted, amplification-free DNA sequencing using CRISPR/Cas
Associated analysis toolsCas9 targeted sequencing tutorial in EPI2ME Labs, and Evaluation of read-mapping characteristics from a Cas9 targeted sequencing experiment tutorial
Multiplexing optionsPlease contact Technical Support to discuss multiplexing
Pack size6 reactions

Further Considerations:

Cas9 Targeted sequencing is a fast, flexible method for sequencing targets of interest without amplification. SQK-CS9109 offers users our protocol with the convenience and cost reduction of a kit.

Best coverage is achieved when using the CRISPR/Cas system to target a 5–20 kb region cutting twice at either end. For applications where only one end of the region is known, a single cut and read-out approach is possible.

For users that wish to target regions longer than 20 kb, we suggest a tiling approach and/or ensuring as highly intact starting DNA material as possible. It will not be possible to span larger regions without intact starting gDNA.

We strongly recommend starting with multiple probes and working out which approach works best for your experimental aims, DNA quality etc. Detailed recommendations can be found in the Targeted, amplification-free DNA sequencing using CRISPR/Cas document.

Licenses

Oxford - Broad

Oxford - Caribou

Shipping and logistics:

Flow cells and kits are shipped together in a temperature-controlled shipping box with gel ice packs. Ice packs are stored at 2-8° C, and -20° C ice packs are added to the top of the box (but not in direct contact with product) to maintain the integrity of the shipment.

Products are shipped to customers within the USA and EU Monday to Thursday. Shipments to Canada, Norway, Korea and Japan are expedited Monday to Wednesday; with Australia and New Zealand leaving our warehouses on a Friday. Shipments to the rest of the world are made on Mondays to allow the full working week for packages to arrive.

The delivery charges are calculated when a quote is raised or during checkout. Once an order is made, the delivery ID and delivery information can be tracked in the Store.

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. Cas9 sequencing workflow

Protocols

Cas9 Sequencing Kit (SQK-CS9109) - for illustration purposes only

What's in the box

The Cas9 Targeted Sequencing Kit contains sufficient reagents to generate six sequencing libraries.

SQK-CS9109 kit contents v2

The Flow Cell Priming Kit is also supplied.

FLP

3rd party materials

  • S. pyogenes Cas9 Alt-R™ crRNAs (resuspended at 100 µM crRNA in TE pH 7.5)
  • S. pyogenes Cas9 tracrRNA (e.g., IDT Alt-R™, Cat # 1072532, 1072533 or 1072534) resuspended at 100 µM in TE pH 7.5 • Alt-R® S. pyogenes HiFi Cas9 nuclease V3, 100 µg or 500 µg (IDT, Cat # 1081060 or # 1081061)
  • Nuclease-free duplex buffer (IDT Cat # 11-01-03-01)
  • 1.5 ml Eppendorf DNA LoBind tubes
  • 0.2 ml thin-walled PCR tubes
  • Nuclease-free water (e.g. ThermoFisher, cat # AM9937)
  • Agencourt AMPure XP beads

Compatibility

The Cas9 Sequencing Kit can be used together with:

Kits

  • Flow Cell Wash Kit (EXP-WSH003)

Flow cells

  • FLO-MINSP6
  • FLO-MIN106D

Devices

  • MinION Mk1B
  • MinION Mk1C
  • GridION Mk1