- Materials
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- 100–200 fmol of each DNA sample to be barcoded in 45 µl
- PCR Barcoding Expansion 1-96 (EXP-PBC096)
- Native Barcoding Expansion 1-12 (EXP-NBD104) and 13-24 (EXP-NBD114) if multiplexing more than 12 samples
- Ligation Sequencing Kit (SQK-LSK109)
- Flow Cell Priming Kit (EXP-FLP002)
- Adapter Mix II Expansion (EXP-AMII001)
- Consumables
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- Agencourt AMPure XP beads (Beckman Coulter™ cat # A63881)
- NEB Blunt/TA Ligase Master Mix (NEB, M0367)
- NEBNext Ultra II End repair/dA-tailing Module (NEB, E7546)
- NEBNext Quick Ligation Module (NEB, E6056)
- 1.5 ml Eppendorf DNA LoBind tubes
- 0.2 ml thin-walled PCR tubes
- Nuclease-free water (e.g. ThermoFisher, AM9937)
- Freshly prepared 70% ethanol in nuclease-free water
- LongAmp Taq 2X Master Mix (e.g. NEB, cat # M0287)
- Equipment
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- Hula mixer (gentle rotator mixer)
- Magnetic rack suitable for 96-well PCR plates, e.g. DynaMag™-96 Side Skirted Magnet (Thermo Fisher, cat # 12027)
- Magnetic rack
- Microplate centrifuge, e.g. Fisherbrand™ Mini Plate Spinner Centrifuge (Fisher Scientific, 11766427)
- Microfuge
- 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
- Multichannel pipette and tips
- Ice bucket with ice
- Timer
- Optional equipment
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- Agilent Bioanalyzer (or equivalent)
- Qubit fluorometer (or equivalent for QC check)
- Eppendorf 5424 centrifuge (or equivalent)
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For this protocol, you will need the following amounts of each DNA sample to be barcoded in 45 µl:
- 1 µg (or 100-200 fmol) gDNA is required for R9.4.1 flow cells
- 1.5-3 µg (or 150-300 fmol) gDNA is required for R10.3 flow cells
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Input DNA
How to QC your input DNA
It is important that 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.
Chemical contaminants
Depending on how the DNA is extracted from the raw sample, certain chemical contaminants may remain in the purified DNA, which can affect library preparation efficiency and sequencing quality. Read more about contaminants on the Contaminants page of the Community.
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PCR Barcoding Expansion Pack 1-96 (EXP-PBC096)
The kit allows up to 96 different libraries to be combined; each of these libraries will be barcoded with one of the 96 PCR barcodes (BC1-BC96). These libraries can then be barcoded for the second time with a native barcode from the Native Barcoding Expansions.
The reagents are divided between two 96 tube plates. One plate (blue caps) contains barcode adapters that are ligated to the DNA. All tubes in this plate have identical content. The other plate (white caps) contains the barcodes, one barcode per tube.
Name No. of plates Fill volume per well (µl) PCR Primer mix 1 24 Barcode adapter plate 1 240 -
Layout of barcodes in the 96 tube plate
The wells of the 96 tube plate correspond to the barcodes in the following way. All barcodes are supplied at 10 µM concentration and to be used at a final concentration of 0.2 µM.
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Native Barcoding Expansion 1-12 (EXP-NBD104) and 13-24 (EXP-NBD114) contents
EXP-NBD104 kit contents
Name Acronym Cap colour No. of vials Fill volume per vial (μl) Native Barcode 01-12 NB01-12 White 12 20 Adapter Mix II AMII Green 1 40
EXP-NBD114 kit contents
Name Acronym Cap colour No. of vials Fill volume per vial (μl) Native Barcode 13-24 NB13-24 White 12 20 Adapter Mix II AMII Green 1 40 -
Ligation Sequencing Kit contents (SQK-LSK109)
Name Acronym Cap colour No. of vials Fill volume per vial (µl) DNA CS DCS Yellow 1 50 Adapter Mix AMX Green 1 40 Ligation Buffer LNB Clear 1 200 L Fragment Buffer LFB White cap, orange stripe on label 2 1,800 S Fragment Buffer SFB Grey 2 1,800 Sequencing Buffer SQB Red 2 300 Elution Buffer EB Black 1 200 Loading Beads LB Pink 1 360 -
Flow Cell Priming Kit contents (EXP-FLP002)
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 -
Adapter Mix II Expansion contents (EXP-AMII001)
Name Acronym Cap colour No. of tubes Fill volume per vial (μl) Adapter Mix II AMII Green 2 40 -
Adapter Mix II Expansion use
Protocols that use the Native Barcoding Expansions require 5 μl of AMII per reaction. Native Barcoding Expansions EXP-NBD104/NBD114 and EXP-NBD196 contain sufficient AMII for 6 and 12 reactions, respectively (or 12 and 24 reactions when sequencing on Flongle). This assumes that all barcodes are used in one sequencing run.
The Adapter Mix II expansion provides additional AMII for customers who are running subsets of barcodes, and allows a further 12 reactions (24 on Flongle).