- Materials
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- 1 µg (or 100-200 fmol) high molecular weight genomic DNA
- OR 100+ ng high molecular weight genomic DNA if performing DNA fragmentation
- Ligation Sequencing Kit XL V14 (SQK-LSK114-XL)
- Consumables
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- NEBNext® Companion Module for Oxford Nanopore Technologies® Ligation Sequencing (NEB, E7180S or E7180L).
Alternatively, you can use the NEBNext® products below:
- NEBNext FFPE Repair Mix (NEB, M6630)
- NEBNext Ultra II End repair/dA-tailing Module (NEB, E7546)
- NEBNext Quick Ligation Module (NEB, E6056)
- Agencourt AMPure XP Beads (Beckman Coulter™, A63881)
- Nuclease-free water (e.g. ThermoFisher, AM9937)
- Freshly prepared 80% ethanol in nuclease-free water
- Bovine Serum Albumin (BSA) (50 mg/ml) (e.g Invitrogen™ UltraPure™ BSA 50 mg/ml, AM2616)
- Hard-Shell® 96-Well PCR Plates, low profile, thin wall, skirted, red/clear (Bio-Rad™, cat # HSP9611)
- Thermo Scientific™ Abgene™ 96 Well 1.2 ml Polypropylene Deepwell Storage Plate (Thermo Scientific, cat # AB1127)
- 1000 µl Disposable Conductive Tips - Liquid Handling Flexible Channel Arm - Filtered, Pure, ANSI/SLAS-format box (same as SBS) (Tecan , cat# 30057817)
- 200 ul Disposable Conductive Tips - Liquid Handling Flexible Channel Arm - Filtered, Pure, ANSI/SLAS-format box (same as SBS) (Tecan , cat# 30057815)
- 50 ul Disposable Conductive Tips - Liquid Handling Flexible Channel Arm - Filtered, Pure, ANSI/SLAS-format box (same as SBS) (Tecan , cat# 30057813)
- Small SBS Box to place conductive tips & refill, compatible with 10uL, 50uL, 200uL tips (Tecan , cat# 30058506)
- Big SBS Box to place conductive tips & refill, compatible with 1000uL tips (Tecan , cat# 30058507)
- 150 µl Disposable Tips - MultiChannel Arm™ 384/96 - Filtered, Sterile, Single Stack (Tecan, cat # 30038618)
- 50 µl Disposable Tips - MultiChannel Arm 384/96 - Filtered, Sterile, Single Stack (Tecan, cat # 30038608)
- 100 ml disposable trough (Tecan, cat # 10613049)
- 25 ml disposable trough (Tecan, cat # 30055743)
- Arched Auto-Sealing Lids with Wide Tabs for PCR Plates (Bio-Rad™, cat # MSL2032 or equivalent)
- Sarstedt Inc Screw Cap Micro tube 2 ml, sterile (Sarstedt™, cat # 72.694.321)
- 1.5 ml Eppendorf DNA LoBind tubes
- 2 ml Eppendorf DNA LoBind tubes
- 5 ml Eppendorf DNA LoBind tubes
- Qubit™ Assay Tubes (Invitrogen, Q32856)
- Qubit dsDNA HS Assay Kit (Invitrogen, Q32851)
- Low-lint scientific wipes (e.g. Kimberly-Clark™, cat # 7552 or equivalent)
- Double distilled water (ddH2O) (e.g. ThermoFisher, cat # 11983084)
- Freshly prepared ≥80% ethanol in nuclease-free or double distilled water, for cleaning
- ~10% Bleach (or equivalent): Thermo Scientific Alfa Aesar Sodium hypochlorite, 11-15% available chlorine, (e.g. ThermoFisher, cat # 15429019)
- Equipment
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- Ice bucket with ice
- Vortex mixer
- Microplate centrifuge, e.g. Fisherbrand™ Mini Plate Spinner Centrifuge (Fisher Scientific, 11766427)
- Tecan DreamPrep NGS workstation with full configuration
- Optional equipment
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- Agilent Bioanalyzer (or equivalent)
- Qubit fluorometer plate reader (or equivalent for QC check)
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For this protocol, you will need 1 µg (100-200 fmol) of gDNA.
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Input DNA
How to QC your input DNA
It is important to use a plate reader to ensure the input DNA meets the quantity and quality requirements. Using too little or too much DNA, or DNA of poor quality (e.g. highly fragmented or containing RNA or chemical contaminants) can affect your library preparation.
For instructions on how to perform quality control of your DNA sample, please read the Input DNA/RNA QC protocol.
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Tecan DreamPrep NGS
This method has been tested and validated using the Tecan DreamPrep NGS including an on deck thermal cycler (ODTC). An option to not use the ODTC is available in the method.
This protocol will require installation by a Tecan Filed Application Specialist, please contact your Tecan representative for further details. -
Tecan DreamPrep NGS worktable layout
After having the system installed according to specifications, users will need to load the automated workstation before running the protocol.
Please always refer to the latest instructions on how to load the worktable, which are displayed and explained in the touchscreen display (with TouchTools™).
Examples of how to load the system are displayed from steps 11 to 20, which can be found in the library preparation section of the protocol.
Please also find below reference images for the workbench layouts:
Trough mounting sites:
Hotel sites:
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NEBNext® Companion Module for Oxford Nanopore Technologies® Ligation Sequencing
For customers new to nanopore sequencing, we recommend buying the NEBNext® Companion Module for Oxford Nanopore Technologies® Ligation Sequencing (catalogue number E7180S or E7180L), which contains all the NEB reagents needed for use with the Ligation Sequencing Kit.
Please note, for our amplicon protocols, NEBNext FFPE DNA Repair Mix and NEBNext FFPE DNA Repair Buffer are not required.
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Third-party reagents
We have validated and recommend the use of all the third-party reagents used in this protocol. Alternatives have not been tested by Oxford Nanopore Technologies.
For all third-party reagents, we recommend following the manufacturer's instructions to prepare the reagents for use.
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Consumables and reagent quantities required:
Consumables X8 samples X24 samples X48 samples X96 samples Tecan 1000 µl Flexible Channel Arm filter tips 49 tips 50 tips 51 tips 53 tips Tecan 200 µl Flexible Channel Arm filter tips 33 tips 32 tips 32 tips 32 tips Tecan 50 µl Flexible Channel Arm filter tips 16 tips 16 tips 16 tips 16 tips Tecan 150 µl MultiChannel Arm 384/96 filter tips 1 box 3 boxes 5 boxes 10 boxes Tecan 50 µl MultiChannel Arm 384/96 filter tips 1 box 2 boxes 3 boxes 5 boxes Bio-Rad Hard-Shell® 96-Well PCR Plate 8 8 8 8 Abgene™ 96 Well 1.2mL Polypropylene Deepwell Storage Plate 1 1 1 1 Tecan 25 ml disposable trough 4 4 4 3 Tecan 100 ml disposable trough 1 1 1 2 ODTC plate lid 1 1 1 1 Sarstedt Inc Screw Cap Micro tube 2 ml 2 2 3 5 Reagents/kits X8 samples X24 samples X48 samples X96 samples 80% ethanol 10 ml 16 ml 24 ml 42 ml Nuclease-free water 3.5 ml 4.6 ml 6.3 ml 9.5 ml AMPure XP Beads 3 ml 4.9 ml 7.8 ml 13.5 ml Ligation Sequencing Kit XL V14 (SQK-LSK114-XL) 1 kit 1 kit 1 kit 2 kits NEBNext Companion Module for Oxford Nanopore Technologies Ligation Sequencing (Cat# E7180L) 1 kit 1 kit 1 kit 1 kit NEBNext Companion Module for Oxford Nanopore Technologies Ligation Sequencing (Cat# E7180S) - - - 1 kit Alternative to the NEBNext Companion Module, individual reagents can be bought as seen below: - - - - NEBNext FFPE DNA Repair Mix (Cat# M6630L) 1 kit 1 kit 1 kit 2 kits NEBNext Ultra II End Repair/dA-Tailing Module (Cat# E7546L) 1 kit 1 kit 1 kits 2 kits NEBNext Quick Ligation Module (Cat# E6056L) 1 kit 1 kit 1 kit 2 kits NEBNext Quick Ligation Module (Cat# E6056S) - - 1 kit 1 kit Note: These are the number of kits required for one run through for the selected number of samples.
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Ligation Sequencing Kit XL V14 (SQK-LSK114-XL) contents
Name Acronym Vial colour Number of vials Fill volume per vial (µl) DNA Control Strand DCS Yellow 1 100 Ligation Adapter LA Green 1 320 Ligation Buffer LNB White 1 1,500 Elution Buffer EB White cap, black strip label 1 10,000 Long Fragment Buffer LFB White cap, orange strip label 2 20,000 Short Fragment Buffer SFB White cap, blue strip label 2 20,000 Library Beads LIB Pink 2 1,800 Library Solution LIS White cap, pink label 2 1,800 Sequencing Buffer SB Red 3 1,700 Flow Cell Flush FCF Clear 4 15,500 Flow Cell Tether FCT Purple 1 1,600 Note: The DNA Control Sample (DCS) is a 3.6 kb standard amplicon mapping the 3' end of the Lambda genome.