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.
Please note that to use this kit, you will need to purchase additional 3rd party reagents: see the "3rd Party Materials" tab for more detail.
Please also note that the Cas9 Sequencing Kit is compatible with R9.4.1 flow cells (FLO-MIN106D or FLO-MINSP6) only.
The Cas9 Sequencing Kit features:
| Feature | Property |
|---|
| Preparation time | 110 minutes |
| Input requirement | 1–10 µg gDNA (5 µg recommended) |
| PCR Required | No |
| Fragmentation | Not required |
| Read length | Equal to fragment length |
| Kit chemistry | Kit 9 |
| Associated protocols/documentation | Ligation sequencing gDNA - Cas9 enrichment (SQK-CS9109) |
| Associated analysis tools | Cas9 Targeted Sequencing tutorial in EPI2ME Labs |
| Multiplexing options | Please contact Technical Support to discuss multiplexing |
| Pack size | 6 reactions |
| Stability | Shipped at 2–8°C
Long-term storage -20°C
Oxford Nanopore Technologies deem the useful life of the product to be 3 months from receipt by the customer. |
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 an environmentally friendly temperature-controlled shipping box.
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.
Protocols
Ligation sequencing gDNA - Cas9 enrichment (SQK-CS9109)
To access the protocol, you will need to register for a Nanopore Community account.
What's in the box
The Cas9 Targeted Sequencing Kit contains sufficient reagents to generate six sequencing libraries.
| Name | Acronym | Cap colour | No. of vials | Fill volume per vial (μl) |
|---|
| Adapter Mix | AMX | Green | 2 | 40 |
| Ligation Buffer | LNB | Clear | 2 | 200 |
| Elution Buffer | EB | Black | 1 | 200 |
| Sequencing Buffer | SQB | Red | 2 | 300 |
| L Fragment Buffer | LFB | Orange | 2 | 1,800 |
| S Fragment Buffer | SFB | Grey | 2 | 1,800 |
| Loading Beads | LB | Pink | 1 | 360 |
| Sequencing Tether | SQT | Purple | 1 | 10 |
| Phosphatase | PHOS | Brown tube, yellow label | 1 | 50 |
| TAQ Polymerase | TAQ | Brown tube, green label | 1 | 15 |
| SPRI Dilution Buffer | SDB | Brown tube, red label | 1 | 1,200 |
| T4 DNA Ligase | LIG | Brown tube, blue label | 1 | 140 |
| dATP | dATP | Brown tube, grey label | 1 | 15 |
| Reaction Buffer | RB | Brown tube, orange label | 1 | 180 |
The Flow Cell Priming Kit is also supplied.
| Name | Acronym | Cap colour | No. of vials | Fill volume per vial (μl) |
|---|
| Flush Buffer | FB | Blue | 6 | 1,170 |
| Flush Tether | FLT | Purple | 1 | 200 |
Compatibility
The Cas9 Sequencing Kit can be used together with:
Kits
- Flow Cell Wash Kit (EXP-WSH004)
Flow cells
Devices
- MinION Mk1B
- MinION Mk1C
- GridION