This kit is recommended for users who:

• Wish to multiplex samples to reduce price per sample
• Need a PCR-free method of multiplexing to preserve additional information such as base modifications
• Require a short preparation time
• Have limited access to laboratory equipment

This protocol describes how to carry out rapid barcoding of genomic DNA using the Rapid Barcoding Kit 96 (Q-SQK-RBK110.96).

Steps in the sequencing workflow:

Prepare for your experiment

You will need to:

• Extract your DNA and check its length, quantity, and purity. The quality checks performed on input samples are essential in ensuring experimental success.
• Ensure you have the Q-Line Rapid Barcoding Kit 96, the correct equipment, and third-party reagents

Library preparation

You will need to:

• Tagment your DNA using the Rapid Barcodes in the kit; this simultaneously attaches a pair of barcodes to the fragments
• Pool the barcoded samples
• Attach sequencing adapters supplied in the kit to the DNA ends
• Prime the flow cell and load your DNA library into the flow cell

Sequencing and analysis

You will need to:

• Start an assay using the sequencing software, which will collect raw data from the device and convert it into basecalled reads. There is also an option to demultiplex reads into barcode-specific folders.

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.

This product contains AMPure XP reagent manufactured by Beckman Coulter, Inc.

The GridION device contains all the hardware required to control up to five sequencing experiments simultaneously and acquire the data. The device is further enhanced with high performance GPU technology for real-time basecalling. Read more in the GridION Q IT Requirements document in the Q system channel.

The sequencing software controls the GridION, collects sequencing data in real-time and processes it into basecalls. The software can also demultiplex reads by barcode, and basecall/demultiplex data after a sequencing run has completed. You will be using the sequencing software for every assay you run.

For instructions on how to run the sequencing software on the GridION, please refer to the Q-Line sequencing software user guide.

We recommend using the Loading Beads II (LBII) for loading your library onto the flow cell for most sequencing experiments. However, if you have previously used water to load your library, you must use Loading Solution (LS) instead of water. Note: some customers have noticed that viscous libraries can be loaded more easily when not using Loading Beads II.

Follow the instructions in the Q-Line sequencing software user guide beginning from the "Set up and run an assay" section.