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Multiplex Ligation Sequencing Kit XL

A sequencing kit optimised for output and PCR-free sequencing of 2-3 samples for Whole Genome Sequencing (WGS).

This is an Early Access product

This uses our Kit 11 chemistry

SKU SQK-MLK111.96-XL Categorías ,
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Descripción

Information

This kit is recommended for users who:

  • Want to optimise their sequencing experiment for output
  • Wish to sequence 2-3 samples for Whole Genome Sequencing (WGS)
  • Need a PCR-free method of multiplexing to preserve additional information, such as base modifications
  • Require control over read length
  • Would like to utilise upstream processes, such as size selection or whole genome amplification
  • Would like to process multiple samples simultaneously, either with a multichannel pipette or a liquid handling robot

This is an Early Access product

For more information about our Early Access programmes, please see this article on product release phases.

Please note that to use this kit, you will need to purchase 3rd party reagents: see the «3rd Party Materials» tab for more detail.

Feature Property
Preparation time 135 mins manual library prep, 245 mins automated library prep
Input requirement 1 µg gDNA per sample
PCR required No
Fragmentation Optional; recommended for input 100 – 500 ng
Kit chemistry Kit 11
Read length Equal to fragment length
Associated protocols • Ligation sequencing gDNA – Multiplex Ligation Sequencing Kit XL (SQK-MLK111.96-XL)

• Ligation sequencing gDNA – automated Hamilton NGS STAR 96 with Multiplex Ligation Sequencing Kit XL (SQK-MLK111.96-XL)

Pack size 48 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

The Multiplex Ligation Sequencing Kit XL is a standalone kit providing 96 unique barcodes to enable PCR-free multiplexing of dsDNA samples such as gDNA and amplicons. This kit provides an easier workflow to enable Whole Genome Sequencing (WGS) by enabling low-plex sequencing of 2-3 samples, allowing users to identify regions more common and others more prone to mutations.

The library preparation is similar to the Ligation Sequencing Kit protocol; DNA ends are repaired and dA-tailed using the NEBNext End Repair/dA-tailing module, and then a unique dT-tailed barcode adapter is ligated on the dA-tailed template. Barcoded samples are then pooled together. Each barcode adapter also has a cohesive end and this is used as a hook to ligate to the supplied sequencing adapters.

We recommend starting with 1 μg gDNA per sample and our manual protocol recommends sequencing two samples per flow cell enabling the sequencing of 96 samples across 48 flow cells. There is also an automated protocol on the Hamilton NGS STAR 96 (NGS STAR with Multi-Probe Head 96) to sequence either two samples per flow cell or three samples across two flow cells which is fully supported by Oxford Nanopore Technologies.

The Multiplex Ligation Sequencing Kit XL (Kit 11) includes the Adapter Mix II T (AMII T), with two key features: Firstly, the adapter is higher capture, enabling lower flow cell loading amounts. Secondly, the adapter contains the fuel fix technology, enabling users to run long experiments without the need for fuel addition during the run.

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 library preparation method is similar to the Ligation Sequencing Kit protocol; DNA ends are repaired and dA-tailed using the NEBNext End Repair/dA-tailing module, and then a unique dT-tailed barcode adapter is ligated on the dA-tailed template. Barcoded samples are then pooled together. Each barcode adapter also has a cohesive end and this is used as a hook to ligate to the supplied sequencing adapters.

Native barcoding workflow1

What’s in the box

The Multiplex Ligation Sequencing Kit XL contains enough reagents for 48 reactions.

SQK-MLK111.96-xl tubes

Name Acronym Cap colour Number of vials Fill volume per vial (µl)
Adapter Mix II T AMII T Green 1 320
Sequencing Buffer II SBII Red 4 1,500
Loading Beads II LBII Pink 4 1,500
Loading Solution LS White cap, pink sticker 4 1,500
EDTA EDTA Clear 1 700
Elution Buffer EB White cap, black label 1 10,000
Long Fragment Buffer LFB White cap, orange label 1 20,000
Flush Buffer FB White cap 6 15,500
Flush Tether FLT White cap, purple sticker 2 1,600
Native Barcodes NB01-96 N/a 1 plate 8 µl per well

3rd party materials

Consumables

  • NEB Blunt/TA Ligase Master Mix (M0367)
  • NEBNext® Quick Ligation Reaction Buffer (NEB B6058)
  • NEBNext® Companion Module for Oxford Nanopore Technologies® Ligation Sequencing (cat # E7180S)
    • Alternatively to the NEBNext® Companion Module and the NEBNext® Quick Ligation Reaction Buffer, you can use the three NEBNext® products below:
    • NEBNext FFPE Repair Mix (M6630)
    • NEBNext Ultra II End repair/dA-tailing Module (E7546)
    • NEBNext Quick Ligation Module (E6056)
  • 1.5 ml Eppendorf DNA LoBind tubes
  • 0.2 ml thin-walled PCR tubes
  • Qubit™ Assay Tubes (ThermoFisher Q32856)
  • Nuclease-free water (e.g. ThermoFisher, cat # AM9937)
  • Freshly prepared 70% ethanol in nuclease-free water
  • Agencourt AMPure XP beads
  • Qubit dsDNA HS Assay Kit

Equipment

  • Hula mixer (gentle rotator mixer)
  • Microfuge
  • Magnetic rack
  • Vortex mixer
  • Thermal cycler
  • P1000 pipette and tips
  • P200 pipette and tips
  • P100 pipette and tips
  • P20 pipette and tips
  • P10 pipette and tips
  • P2 pipette and tips
  • Ice bucket with ice
  • Timer
  • Qubit fluorometer (or equivalent)

Optional equipment

  • Agilent Bioanalyzer (or equivalent)
  • Eppendorf 5424 centrifuge (or equivalent)

Compatibility

The Multiplex Ligation Sequencing Kit XL can be used together with:

Kits

  • Flow Cell Wash Kit (EXP-WSH004)
  • Sequencing Auxiliary Vials (EXP-AUX002)

Flow cells

  • FLO-PRO002

Devices

  • PromethION

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