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Rapid-based kits
Oxford Nanopore has previously offered the following rapid-based sequencing kits:
- Rapid Sequencing Kit (SQK-RAD004)
- Rapid Barcoding Kit (SQK-RBK004)
- Ultra-Long DNA Sequencing Kit (SQK-ULK001)
- Field Sequencing Kit (SQK-LRK001)
- PCR Sequencing Kit (SQK-PSK004)
- PCR Barcoding Kit (SQK-PBK004):
- Rapid PCR Barcoding Kit (SQK-RPB004)
- 16S Barcoding Kit (SQK-RAB204)
- 16S Barcoding Kit 1-24 (SQK-16S024)
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Rapid Sequencing Kit
SQK-RAD004
The Rapid Sequencing Kit (SQK-RAD004) is optimised for speed and simplicity, using limited laboratory equipment. The transposase-based workflow takes approximately 10 minutes to generate sequencing libraries from extracted gDNA in two steps. We recommend an input of 400 ng of HMW gDNA (>30 kb).
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Ultra-Long DNA Sequencing Kit
As our transposase-based kits are relatively simple, very long reads can be achieved due to the minimal pipetting required during library preparation. The Ultra-Long DNA Sequencing Kit (SQK-ULK001) was developed using our rapid-based kit.
This kit requires ultra-high molecular weight (uHMW) gDNA to be extracted. In our protocol, we recommend using the NEB Monarch® HMW DNA Extraction Kit for Tissue (T3060) to extract the uHMW gDNA from either frozen cells or whole blood. After gDNA extraction, a diluted fragmentation mix containing transposases are added to the extracted gDNA to fragment and simultaneously tag the fragmented template with transposase adapter sequences. Post-transposition, sequencing adapters are then attached to the transposase adapters in an enzyme-free reaction. After an overnight elution with Elution Buffer, the library is ready for sequencing.
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Field Sequencing Kit
The Field Sequencing Kit (SQK-LRK001) is recommended for those working in the field with limited access to cold storage and laboratory equipment. It is recommended to use ~400 ng high molecular weight gDNA as input for the library preparation. This kit uses a simple two-step protocol, using a transposase to simultaneously cleave the template molecules and attach tags to the cleaved ends. The Rapid Sequencing Adapters are then added to the tagged ends in approximately 10 minutes. This kit is stable at ambient temperatures for extended periods of time and many of the reagents are lyophilised.
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PCR Sequencing Kit
The PCR Sequencing Kit (SQK-PSK004) is designed to prepare sequencing libraries when the available gDNA input is limited (<100 ng). We recommend starting with ~100 ng gDNA but less can be used. The gDNA is fragmented in a Covaris g-TUBE and the sheared ends are repaired and dA-tailed before adapters containing primer binding sites are ligated onto the prepared ends. An amplification step uses primers containing 5’ tags for the ligase-free attachment of Rapid Sequencing Adapters. Prior to PCR, this workflow takes approximately 50 minutes and post-PCR, the attachment of sequencing adapters takes about 10 minutes.
The barcoding version is the PCR Barcoding Kit (SQK-PBK004), enabling the multiplexing of up to 12 samples.