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Multiplex Ligation Sequencing Kit XL V14
SQK-MLK114.96-XL

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

This uses our latest Kit 14 chemistry

Product lead time: 1 week

US$4,080.00
3Released
  • Products from Oxford Nanopore Technologies that have completed their Early Access
  • Users can continue to provide feedback through feature request pinboard
  • Warranty periods extended to 1 – 3 months
  • Product continues to iterate with upgrade notifications of up to 3 months
  • Lead time visible in store
  • Product subject to availability

Information

This kit is recommended for users who:

  • Want to optimise their sequencing experiment for output
  • Wish to low-plex 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

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

FeatureProperty
Preparation time150 mins manual library prep, 245 mins automated library prep
Input requirement1 µg gDNA per sample
PCR requiredNo
FragmentationOptional; recommended for input 100 - 500 ng
Kit chemistryKit 14 (V14)
Read lengthEqual to fragment length
Associated protocolsLigation sequencing gDNA - Multiplex Ligation Sequencing Kit V14 XL (SQK-MLK114.96-XL)
Pack sizeUp to 48 reactions

Options:

• For preparations of 2 barcoded samples on one flow cell, up to 48 reactions across 48 flow cells.

• For preparations of 3 barcoded samples on two flow cells, up to 32 reactions across 64 flow cells.
StabilityShipped 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 V14 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, allowing users to identify regions more common and others more prone to mutations.

This kit has been upgraded to use our Kit 14 chemistry, which includes improved modal raw read sequencing accuracies (Q20+) with high output on our latest nanopore: R10.4.1. The flow cell priming and sequencing reagents included as part of the sequencing kit have been reformulated to be compatible with the Kit 14 chemistry upgrade and R10.4.1 nanopore. Other upgrades include higher capture rate of gDNA to enable lower flow cell loading amounts and fuel fix technology, allowing users to run longer experiments without the need for fuel addition during the run.

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 ug gDNA per sample and our manual protocol recommends sequencing two samples per flow cell to sequence up to 96 samples across 48 flow cells. Three samples across two flow cells can also be used to sequence up to 96 samples across 64 flow cells to maximise data output.

Deconvolution of barcoded sequencing data is supported by the MinKNOW software, the stand-alone Guppy basecaller, and EPI2ME Labs, all of which classify the barcode sequence and sort reads into corresponding folders.

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.

MLK114.96 genomic DNA workflow

What's in the box

The Multiplex Ligation Sequencing Kit XL V14 contains enough reagents for:

  • 48 reactions when using 2 barcodes per run, across 48 flow cells.
  • 32 reactions when using 3 barcodes per run, across 64 flow cells.

MLK114.96-XL tubes

NameAcronymCap colourNumber of vialsFill volume per vial (µl)
Native AdapterNAGreen1320
Sequencing BufferSBRed41,700
Library BeadsLIBPink41,800
Library SolutionLISWhite cap, pink sticker41,800
EDTAEDTABlue1700
Elution BufferEBClear cap, black label110,000
Long Fragment BufferLFBClear cap, orange label120,000
Flow Cell FlushFCFClear cap, light blue label615,500
Flow Cell TetherFCTPurple21,600
AMPure XP BeadsAXPClear cap, light teal label16,000
Native BarcodesNB01-96N/a1 plate8 µl per well

Note: This product contains AMPure XP Reagent Manufactured by Beckman Coulter, Inc. and can be stored at -20°C with the kit without detriment to reagent stability.

3rd party materials

Consumables:

  • NEB Blunt/TA Ligase Master Mix (NEB, cat # M0367)
  • NEBNext® FFPE Repair Mix (NEB, M6630)
  • NEBNext Ultra II End repair/dA-tailing Module (NEB, cat # E7546)
  • NEBNext Quick Ligation Module (NEB, cat # E6056)
  • Eppendorf twin.tec® PCR plate 96 LoBind, semi-skirted (Eppendorf™, cat # 0030129504) with heat seals
  • 0.2 ml thin-walled PCR tubes
  • 1.5 ml Eppendorf DNA LoBind tubes
  • 2 ml Eppendorf DNA LoBind tubes
  • Nuclease-free water (e.g. ThermoFisher, AM9937)
  • Freshly prepared 80% ethanol in nuclease-free water
  • Qubit™ Assay Tubes (Invitrogen, Q32856)
  • Qubit dsDNA HS Assay Kit (Invitrogen, Q32851)

Equipment:

  • Hula mixer (gentle rotator mixer)
  • Microfuge
  • Magnetic rack
  • Magnetic rack
  • Vortex mixer
  • Thermal cycler
  • Multichannel pipette
  • 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)

Compatibility

The Multiplex Ligation Sequencing Kit XL V14 can be used together with:

Kits:

  • Flow Cell Wash Kit (EXP-WSH004)
  • Flow Cell Wash Kit XL (EXP-WSH004-XL)
  • Sequencing Auxiliary Vials V14 (EXP-AUX003)
  • Native Barcoding Auxiliary Kit V14 (EXP-NBA114)

Flow cells:

  • FLO-PRO114M

Devices:

  • PromethION