This kit is recommended for users who

• wish to multiplex samples to reduce price per sample
• want to do 16S sequencing
• are interested in genus level bacterial identification

Barcoding or multiplexing is useful when the amount of data required per sample is less than the total amount of data that can be generated from a single flow-cell: it allows a user to pool multiple samples and sequence them together making more efficient use of the flow-cell.

 Multiplexing samples onto one flow cell can reduce the cost per sample for a user. Below are some worked examples:

The 16S Barcoding Kit offers a method of amplifying and barcoding the ~1500 bp 16S rRNA gene from multiple samples and sequencing them together. By narrowing down to a specific region of interest, a user can see all the organisms in the sample without sequencing unnecessary regions of the genome, making the identification quicker and more economical.

The 16S Barcoding Kit features:

The DNA is amplified by PCR using specific 16S primers (27F and 1492R) that contain barcodes and 5’ tags which facilitate the ligase-free attachment of Rapid Sequencing Adapters. The kit contains 12x barcoded primer pairs.

The kit is supported by the EPI2ME 16S-BLAST workflow, which can be used to analyse data from the 16S protocol. Deconvolution of barcoded sequencing data is supported by Guppy and EPI2ME which classify the barcode sequence and sort reads into corresponding folders.

Further considerations:

Oxford Nanopore Technologies deem the useful life of the product to be 3 months from receipt by the customer.

The DNA is amplified by PCR using specific 16S primers (27F and 1492R) that contain barcodes and 5’ tags which facilitate the ligase-free attachment of Rapid Sequencing Adapters. The kit contains 12x barcoded primer pairs.

The kit is supported by the EPI2ME 16S-BLAST workflow, which can be used to analyse data from the 16S protocol. Deconvolution of barcoded sequencing data is supported by Albacore and EPI2ME which classify the barcode sequence and sort reads into corresponding folders.

Example 16S Barcoding protocol - for illustration purposes

Equipment and comsumables for the Rapid Sequencing Kit family

Safety Data Sheet for 16S Barcoding Kit

Safety Data Sheet for Flow Cell Priming Kit

The 16S Barcoding Kit contains 12 unique barcodes and sufficient reagents to generate six sequencing libraries.

The Flow Cell Priming Kit Expansion Pack is also supplied.

Consumables

• Agencourt AMPure XP beads
• 1.5 ml Eppendorf DNA LoBind tubes
• 0.2 ml thin-walled PCR tubes
• Nuclease-free water (e.g. ThermoFisher, cat # AM9937)
• Freshly prepared 70% ethanol in nuclease-free water
• LongAmp Taq 2X Master Mix (e.g. NEB M0287)
• 10 mM Tris-HCl pH 8.0 with 50 mM NaCl

Equipment

• Microfuge
• Timer
• Thermal cycler
• P1000 pipette and tips
• P100 pipette and tips
• P20 pipette and tips
• P10 pipette and tips
• P2 pipette and tips

Optional Equipment

• Agilent Bioanalyzer (or equivalent)
• Qubit fluorometer (or equivalent for QC check)
• Eppendorf 5424 centrifuge (or equivalent)

The PCR Sequencing Kit can be used together with:

Kits

• RAP Top-up Kit (EXP-RAP001)
• Flow Cell Wash Kit (EXP-WSH003)

Flow cells

• FLO-MINSP6
• FLO-MIN106D

Devices

• MinION Mk 1B
• GridION X5