Library Preparation Kits
This kit is recommended for users who:
- require a short preparation time
- have limited access to laboratory equipment.
The Rapid Sequencing Kit generates sequencing libraries from extracted gDNA in 10 minutes using a simple 2-step protocol. At the heart of the kit is a transposase which simultaneously cleaves template molecules and attaches tags to the cleaved ends. Rapid Sequencing Adapters are then added to the tagged ends.
Key features:
| Prep time | 10 minutes |
| Input amount | 400 ng HMW gDNA |
| Read length | Random distribution, dependent on input fragment length |
| Typical throughput | 1-2 Gb in 6 hours, 4-8 Gb in 48 hours per flow cell on MinION/GridION |
Workflow
The Rapid Sequencing Kit generates sequencing libraries from extracted gDNA in 10 minutes using a simple 2-step protocol. At the heart of the kit is a transposase which simultaneously cleaves template molecules and attaches tags to the cleaved ends. Rapid Sequencing Adapters are then added to the tagged ends.
This kit is recommended for users who:
- Want to target specific regions of 5-20 kb to high coverage (>100x) on a single MinION Mk 1B flow cell without PCR.
- Want to sequence a larger, contiguous Region of Interest (ROI), using up to 100 target sites, in a single assay*
- Have 1-10 µg of available high quality gDNA
- Are interested in methylation patterns or other modified bases.
- Have gene targets that are highly repetitive, or wish to evaluate the number of repeats in an expansion, where traditional amplification method or sequencing-by-synthesis methods could yield a biased result
- Want to sequence long gene targets in a single pass that are not amenable to long-range PCR (>30 kb)
* Note: This is known as ‘tiling’ an ROI.
Key features:
| Prep time | 110 minutes |
| Input amount | 1–10 µg of double-stranded DNA (5 µg recommended) |
| Read length | = fragment length |
| Typical throughput | <1 Gb in 6 hours, 1-4 Gb in 48 hours per flow cell on MinION/GridION |
Workflow
The Cas9 Sequencing Kit offers a method of enriching regions of interest (ROI) from a genomic DNA sample. After DNA extraction, 5’ ends are dephosphorylated to reduce ligation of sequencing adapters to non-target strands. Cas9 ribonucleoprotein particles (RNPs), with bound crRNA and tracrRNA, are added to the genomic DNA, and bind then cleave the region of interest (ROI). dsDNA cleavage by Cas9 reveals blunt ends with ligatable 5’ phosphates. All of the DNA in the sample is dA-tailed, which prepares the blunt ends for sequencing adapter ligation. Sequencing adapters are ligated primarily to Cas9 cut sides, which are both 3’ dA-tailed and 5’ phosphorylated. The library preparation is cleaned up to remove excess adapters using AMPure XP beads and resuspended in Sequencing Buffer (Non-target molecules are not removed). The subsequent library preparation is added to the flow cell for sequencing.
This kit is highly recommended for users who:
- would like to identify and quantify full-length transcripts
- are interested in differential gene expression
- want to characterise and quantify isoforms, splice variants and fusion transcripts
- wish to avoid bias and artefacts introduced through PCR
The Direct cDNA Sequencing Kit features:
Key features:
| Prep time | 270 minutes |
| Input amount | 100 ng poly-A+ RNA |
| Read length | Enriched for full-length cDNA |
| Typical throughput | 2-3+ Gb in 6 hours, 8+ Gb in 48 hours per flow cell on MinION/GridION; 10-15+ Gb in 6 hours, 40+ Gb in 48 hours per flow cell on PromethION |
Workflow
Taking full-length messenger RNA, the complementary strand is synthesised using a kit-supplied oligo adapter. The RNA is then degraded, and the second (complementary) strand synthesised. Adapters supplied in the Direct cDNA Sequencing Kit are then ligated onto the cDNA, which introduce the components needed for the cDNA strands to enter the pore. One strand of the duplex is sequenced at a time, producing 1D reads.
This kit is recommended for users who:
- would like to process multiple samples simultaneously, either with a multichannel pipette or a liquid handling robot
- want to optimise their sequencing experiment for throughput
- require control over read length
- would like to utilise upstream processes such as size selection or whole genome amplification.
Key features:
| Prep time | 110 minutes |
| Input amount | 1000 ng high molecular weight dsDNA 100+ ng DNA if performing fragmentation or PCR |
| Read length | = fragment length |
| Typical throughput | 2-3+ Gb in 6 hours, 8+ Gb in 48 hours per flow cell on MinION/GridION; 10-15+ Gb in 6 hours, 40+ Gb in 48 hours per flow cell on PromethION |
Workflow
The Ligation Sequencing Kit offers a flexible method of preparing sequencing libraries from dsDNA (e.g. gDNA, cDNA or amplicons). The library preparation method is straightforward: DNA ends are FFPE repaired and end-prepped/dA-tailed using the NEBNext End Repair/dA-tailing module, and then sequencing adapters, supplied in the kit, are ligated onto the prepared ends.
This kit is recommended for users who:
- want to optimise their sequencing experiment for throughput
- require control over read length
- would like to utilise upstream processes such as size selection or whole genome amplification.
Key features:
| Prep time | 60 minutes minutes |
| Input amount | 1000 ng high molecular weight dsDNA 100+ ng DNA if performing fragmentation or PCR |
| Read length | = fragment length |
| Typical throughput | 2-3+ Gb in 6 hours, 8+ Gb in 48 hours per flow cell on MinION/GridION; 10-15+ Gb in 6 hours, 40+ Gb in 48 hours per flow cell on PromethION |
Workflow
The Ligation Sequencing Kit offers a flexible method of preparing sequencing libraries from dsDNA (e.g. gDNA, cDNA or amplicons). The library preparation method is straightforward: DNA ends are FFPE repaired and end-prepped/dA-tailed using the NEBNext End Repair/dA-tailing module, and then sequencing adapters, supplied in the kit, are ligated onto the prepared ends.
This kit is highly recommended for users who:
- would like to remove RT or PCR bias
- have transcripts that are difficult to reverse transcribe
- are exploring attributes of native RNA such as modified bases
Key features:
| Prep time | 115 minutes |
| Input amount | 500 ng RNA (poly-A+) |
| Read length | = RNA length |
| Typical throughput | <1 Gb in 6 hours, 1-4 Gb in 48 hours per flow cell on MinION/GridION; <5 Gb in 6 hours, 5-20 Gb in 48 hours per flow cell on PromethION |
Workflow
The Direct RNA Sequencing kit (SQK-RNA002) is used to prepare any RNA with a 3’ polyA tail for 1D sequencing on the Oxford Nanopore sequencing devices. Possible input RNA includes eukaryotic mRNA, viral RNA with a polyA tail, or any RNA prepared with a polyA-tailing kit. For RNA without a polyA tail, users can follow simple instructions to design their own custom adapter to ligate a specific terminal 3’ sequence like the 3’ of tRNA or 16S rRNA. The kit requires 500 ng of input RNA.
This kit is recommended for users who:
- are working in the field, with limited access to cold storage and laboratory equipment.
The Field Sequencing Kit generates sequencing libraries from extracted gDNA in 10 minutes using a simple 2-step protocol. At the heart of the kit is a transposase which simultaneously cleaves template molecules and attaches tags to the cleaved ends. Rapid Sequencing Adapters are then added to the tagged ends.
Key features:
| Prep time | 10 minutes |
| Input amount | 400 ng dsDNA |
| Read length | Random distribution, dependent on input fragment length |
| Typical throughput | 1-2 Gb in 6 hours, 4-8 Gb in 48 hours per flow cell on MinION/GridION |
Workflow
The Field Sequencing Kit generates sequencing libraries from extracted gDNA in 10 minutes using a simple 2-step protocol. At the heart of the kit is a transposase which simultaneously cleaves template molecules and attaches tags to the cleaved ends. Rapid Sequencing Adapters are then added to the tagged ends.
This kit is recommended for users who
- have a low starting amount of DNA
- want to optimise their sequencing experiment for throughput
- require control over read length
- are interested in targeted amplicon sequencing
The PCR Sequencing Kit is designed to prepare sequencing libraries when there is a limited amount of starting gDNA available.
The gDNA is fragmented in a Covaris g-TUBE. The sheared ends are repaired and dA-tailed using the NEBNext End Repair/dA-tailing module, and then adapters which contain primer binding sites are ligated onto the prepared ends. An amplification step follows using primers that contain 5’ tags which facilitate the ligase-free attachment of Rapid Sequencing Adapters.
The kit involves ~50 mins pre-PCR work. The PCR step is variable (depends on the number of cycles, polymerase speed and template length). The standard protocol suggests starting with 100 ng of gDNA: less can be used, but it may be necessary to increase the number of PCR cycles. The post-PCR work is about 10 mins.
Key features:
| Prep time | PCR + 60 minutes |
| Input amount | 100 ng dsDNA |
| Read length | = fragment length post-PCR |
| Typical throughput | 2-3+ Gb in 6 hours, 8+ Gb in 48 hours per flow cell on MinION/GridION |
Workflow
The PCR Sequencing Kit is designed to prepare sequencing libraries when there is a limited amount of starting gDNA available.
The gDNA is fragmented in a Covaris g-TUBE. The sheared ends are repaired and dA-tailed using the NEBNext End Repair/dA-tailing module, and then adapters which contain primer binding sites are ligated onto the prepared ends. An amplification step follows using primers that contain 5’ tags which facilitate the ligase-free attachment of Rapid Sequencing Adapters.
The kit involves ~50 mins pre-PCR work. The PCR step is variable (depends on the number of cycles, polymerase speed and template length). The standard protocol suggests starting with 100 ng of gDNA: less can be used, but it may be necessary to increase the number of PCR cycles. The post-PCR work is about 10 mins.
This kit is highly recommended for users who:
- have a limited amount of input material
- want to optimise their sequencing experiment for throughput
- would like to identify and quantify full-length transcripts
- are interested in differential gene expression
- want to characterise and quantify isoforms, splice variants and fusion transcripts
Key features:
| Prep time | 165 minutes |
| Input amount | 1 ng poly-A+ or 50 ng total RNA |
| Read length | Enriched for full-length cDNA |
| Typical throughput | 7-12+ million full-length mapped reads in 48 hours per flow cell on MinION/GridION; 50+ million mapped reads in 48 hours per flow cell on PromethION |
Workflow
Taking full-length PolyA+ RNA, complementary strand synthesis and strand switching are performed using kit-supplied oligonucleotides. dscDNA is then generated by PCR amplification using primers that contain 5’ tags which facilitate the ligase-free attachment of Rapid Sequencing Adapters.
Barcoding Kits
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.
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.
Key features:
| Prep time | 10 minutes |
| Input amount | 400 ng HMW gDNA |
| Read length | Random distribution, dependent on input fragment length |
| Typical throughput | 1-2 Gb in 6 hours, 4-8 Gb in 48 hours per flow cell on MinION/GridION |
Workflow
The Rapid Barcoding Kit generates barcoded sequencing libraries from extracted gDNA in 10 minutes using a simple 2-step protocol. At the heart of the kit is a transposase which simultaneously cleaves template molecules and attaches barcoded tags to the cleaved ends: there are 12 unique barcoded tags in the kit. Barcoded samples are pooled and Rapid Sequencing Adapters are then added to the tagged ends.
This kit is recommended for users who:
- wish to multiplex samples to reduce price per sample
- have a low starting amount of DNA
- require a simple library preparation procedure
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.
Key features:
| Prep time | PCR + 15 minutes |
| Input amount | 1-5 ng gDNA |
| Read length | ~2 kb |
| Typical throughput | 1-2 Gb in 6 hours, 4-8 Gb in 48 hours per flow cell on MinION/GridION |
Workflow
The Rapid PCR Barcoding Kit offers the fastest and simplest method of preparation of barcoded libraries for low quantities of gDNA (1-5 ng), with only ~15 mins of hands-on preparation time.
At the heart of the kit is a transposase which simultaneously cleaves template molecules in each sample and attaches tags, which contain primer binding sites, to the cleaved ends. The kit contains 12 primers which are then used to amplify each sample: each primer contains a barcode and 5’ tag which facilitates the ligase-free attachment of Rapid Sequencing Adapters. Amplified and barcoded samples are then pooled together and Rapid Sequencing Adapters are added to the pooled mix.
This kit is recommended for users who:
- wish to multiplex samples to reduce price per sample
- have a limited amount of input material
- want to optimise their sequencing experiment for throughput
- would like to identify and quantify full-length transcripts
- are interested in differential gene expression or differential transcript usage
- want to characterise and quantify isoforms, splice variants and fusion transcripts
Key features:
| Prep time | 165 minutes |
| Input amount | 1 ng poly-A+ RNA, or 50 ng total RNA |
| Read length | Enriched for full-length cDNA during PCR |
| Typical throughput | 7-12+ million full-length mapped reads in 48 hours per flow cell on MinION/GridION; 50+ million mapped reads in 48 hours per flow cell on PromethION |
Workflow
Taking full-length poly-A+ RNA, complementary strand synthesis and strand switching are performed using kit-supplied oligonucleotides. The kit contains 12 primer pairs which are used to generate and then amplify double-stranded cDNA by PCR amplification: each primer pair contains a barcode and 5’ tag which facilitates the ligase-free attachment of Rapid Sequencing Adapters. Amplified and barcoded samples are then pooled together, and Rapid Sequencing Adapters are added to the pooled mix.
This kit is recommended for users who:
- wish to multiplex samples to reduce price per sample
- have a low starting amount of DNA
- want to optimise their sequencing experiment for throughput
- require control over read length
- are interested in cDNA or targeted amplicon sequencing.
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.
Key features:
| Prep time | PCR + 60 minutes |
| Input amount | 100 ng dsDNA |
| Read length | = fragment length post-PCR |
| Typical throughput | 2-3+ Gb in 6 hours, 8+ Gb in 48 hours per flow cell on MinION/GridION |
Workflow
The PCR Barcoding Kit is designed to prepare barcoded sequencing libraries when there is a limited amount of starting gDNA available from multiple samples.
The gDNA samples can be fragmented in Covaris g-TUBEs. Sheared ends are repaired and dA-tailed using the NEBNext End Repair/dA-tailing module, and then adapters which contain primer binding sites are ligated onto the prepared ends. The kit contains 12x primer pairs which are then used to amplify each sample: each primer pair contains a barcode and 5’ tags which facilitate the ligase-free attachment of Rapid Sequencing Adapters. Amplified and barcoded samples are then pooled together and Rapid Sequencing Adapters are added to the pooled mix
The kit involves ~50 mins pre-PCR work. The PCR step is variable (depends on the number of cycles, polymerase speed and template length). The standard protocol suggests starting with 100 ng of gDNA: less can be used, but it may be necessary to increase the number of PCR cycles. The post-PCR work is about 10 mins.
This kit also offers a method whereby users are able to barcode and tag their own specific amplicons with the same 5’ group, simplifying downstream post-PCR processing. This is done via a four-primer PCR. Users add a 5’ tail to their own primer sequences and this acts a priming site for a set of barcoded “outer” primers (supplied in the PCR Barcoding Kit) and these primers contain the 5’ tags which facilitate the ligase-free attachment of Rapid Sequencing Adapters.
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.
Key features:
| Prep time | PCR + 10 minutes |
| Input amount | 10 ng gDNA |
| Read length | = full-length 16S gene (~1.5kb) |
| Typical throughput | 1-2 Gb in 6 hours, 4-8 Gb in 48 hours per flow cell on MinION/GridION |
Workflow
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.
This kit is recommended for users who:
- 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.
Key features:
| Prep time | PCR + 10 minutes |
| Input amount | 10 ng gDNA |
| Read length | = full-length 16S gene (~1.5kb) |
| Typical throughput | 1-2 Gb in 6 hours, 4-8 Gb in 48 hours per flow cell on MinION/GridION |
Workflow
The DNA is amplified by PCR using specific 16S primers (27F and 1492R) that contain 5’ tags which facilitate the ligase-free attachment of Rapid Sequencing Adapters. The kit contains 24x 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.
Expansion Packs
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
- want to optimise their sequencing experiment for throughput
- require control over read length
- are interested in utilising upstream processes such as size selection or whole genome amplification.
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.
Workflow
The library preparation method is similar to the Ligation Sequencing Kit protocol; DNA ends are repaired and dA-tailed using the NEBNext End Repair/dA-tailing module, and then a unique dT-tailed barcode adapter is ligated on the dA-tailed template. Barcoded samples are then pooled together. Each barcode adapter also has a cohesive end and this is used as a hook to ligate to the supplied sequencing adapters.
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
- want to optimise their sequencing experiment for throughput
- require control over read length
- are interested in utilising upstream processes such as size selection or whole genome amplification.
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.
Workflow
The library preparation method is similar to the Ligation Sequencing Kit protocol; DNA ends are repaired and dA-tailed using the NEBNext End Repair/dA-tailing module, and then a unique dT-tailed barcode adapter is ligated on the dA-tailed template. Barcoded samples are then pooled together. Each barcode adapter also has a cohesive end and this is used as a hook to ligate to the supplied sequencing adapters.
This kit is recommended for users who:
- wish to multiplex a high number of samples to reduce price per sample
- need a PCR-free method of multiplexing to preserve additional information such as base modifications
- require control over read length
- are interested in utlising upstream processes such as size selection or whole genome amplifcation
- want to optimise their sequencing experiement or throughput
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.
Workflow
The PCR Barcoding Expansion 1-12 is designed to allow the pooling and running together of multiple sequencing libraries on the Oxford Nanopore MinION. The kit includes adapters and Oxford Nanopore forward and reverse PCR primers with the same barcode in both forward and reverse primers. There are 12 unique barcodes and enough of the required adapters to make 6 libraries per barcode. The barcodes have been carefully designed and extensively purified to minimize the opportunity for cross-talk. There are currently three protocols for this kit, which are provided as examples of how the PCR Barcoding Expansion 1-12 for genomic, amplicon, and cDNA libraries work. Please note that if you are barcoding cDNA or amplicons, these have to be pre-made in advance. These protocols are open for adaptation and optimization for specific experimental requirements. This kit is used with the Ligation Sequencing Kit for library preparation. Store at -20 °C.
Oxford Nanopore Technologies deem the useful life of the product to be 3 months from receipt by the customer.
Workflow
The PCR Barcoding Expansion 1-12 is designed to allow the pooling and running together of multiple sequencing libraries on the Oxford Nanopore MinION. The kit includes adapters and Oxford Nanopore forward and reverse PCR primers with the same barcode in both forward and reverse primers. There are 12 unique barcodes and enough of the required adapters to make 6 libraries per barcode. The barcodes have been carefully designed and extensively purified to minimize the opportunity for cross-talk. There are currently three protocols for this kit, which are provided as examples of how the PCR Barcoding Expansion 1-12 for genomic, amplicon, and cDNA libraries work. Please note that if you are barcoding cDNA or amplicons, these have to be pre-made in advance. These protocols are open for adaptation and optimization for specific experimental requirements. This kit is used with the Ligation Sequencing Kit for library preparation. Store at -20 °C.
Oxford Nanopore Technologies deem the useful life of the product to be 3 months from receipt by the customer.
Workflow
The PCR Barcoding Expansion 1-96 (EXP-PBC096) is designed to allow the pooling and running together of multiple sequencing libraries. The kit includes adapters and forward and reverse PCR primers with the same barcode in both forward and reverse primers. There are 96 unique barcodes in the kit. There are 96 unique barcodes and enough of the required adapters to make 10 libraries per barcode. The barcodes have been carefully designed and extensively purified to minimize the opportunity for cross-talk.
There are currently three protocols for this kit which are provided as examples of how the PCR Barcoding Expansion 1-96 could be applied for genomic, amplicon, or cDNA libraries. Please note that if you are barcoding cDNA or amplicons, these have to be pre-made in advance. These protocols are open for adaptation and optimization for specific experimental requirements.
This kit is used with the Ligation Sequencing Kit for library preparation.
Oxford Nanopore Technologies deem the useful life of the product to be 3 months from receipt by the customer.
Workflow
