-
Ligation-based barcoding kits
Native Barcoding Expansions 1-12, 13-24 and 96
For the Kit 9 Ligation Sequencing Kit (SQK-LSK109), there are three native barcoding expansions to enable barcoding:
- Native Barcoding Expansion 1-12 (EXP-NBD104)
- Native Barcoding Expansion 13-24 (EXP-NBD114)
- Native Barcoding Expansion 96 (EXP-NBD196)
These native barcoding expansions are recommended for users who want to multiplex their samples with a PCR-free method to preserve base modifications. There are up to 96 unique barcodes which can be used with the Ligation Sequencing Kits (SQK-LSK109) to sequence gDNA or cDNA with the Direct cDNA Sequencing Kit (SQK-DCS109). For more information about sequencing cDNA, please see the RNA and cDNA sequencing and kits section.
The barcode expansion packs are optimised to generate maximum output without the need for PCR. For highest data yields, we recommend the following inputs:
Native Barcoding Expansion 1-12 (EXP-NBD104) and 13-24 (EXP-NBD114) for barcoding up to 24 samples:
- 1 µg or 1.5-3 µg pure input DNA for R9.4.1 or R10.3 flow cells.
Native Barcoding Expansion 96 (EXP-NBD196) for barcoding up to 96 samples:
- ~400 ng DNA per barcode for R9.4.1 or R10.3 flow cells.
Multiplex Ligation Sequencing Kit XL
The Multiplex Ligation Sequencing Kit XL is a standalone kit providing 96 unique barcodes to enable PCR-free multiplexing of dsDNA samples such as gDNA and amplicons. This kit provides an easier workflow to enable Whole Genome Sequencing (WGS) by enabling low-plex sequencing of 2-3 samples, allowing users to identify regions more common and others more prone to mutations.
The library preparation 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.
We recommend starting with 1 μg gDNA per sample and our manual protocol recommends sequencing two samples per flow cell enabling the sequencing of 96 samples across 48 flow cells. There is also an automated protocol on the Hamilton NGS STAR 96 (NGS STAR with Multi-Probe Head 96) to sequence either two samples per flow cell or three samples across two flow cells which is fully supported by Oxford Nanopore Technologies.
-
Rapid-based barcoding kits
Rapid Barcoding Kit
The Rapid Barcoding Kit (SQK-RBK004) can be used to barcode up to 12 samples quickly with limited laboratory equipment. It is recommended to use ~400 ng high molecular weight gDNA to generate barcoded libraries in 15 minutes using the simple two-step protocol.
This kit uses the transposase simultaneously cleaves template molecules and attaches barcoded tags to the cleaved ends. Barcoded samples are pooled and Rapid Sequencing Adapters are then added to the tagged ends.
-
Rapid-based PCR Barcoding Kits
PCR Barcoding Kit (SQK-PBK004):
The PCR Barcoding Kit (SQK-PBK004) is the multiplexing version of the PCR Sequencing Kit (SQK-PSK004), allowing up to 12 samples to be multiplexed on one flow cell when starting with low input. When the quantity of input DNA is low or users want to maximise output, we recommend using PCR to increase the amount of template molecules. This kit implements PCR and it is recommended to start with ~100 ng gDNA. However, less input can be used but it may be necessary to increase the number of PCR cycles.
The gDNA samples are fragmented in a Covaris g-TUBE and the sheared ends are repaired and dA-tailed before the adapters containing primer binding sites are ligated onto the prepared ends. This kit contains 12 primer pairs which are used to amplify each sample during PCR. After amplification, the Rapid Sequencing Adapters are attached to the pooled barcoded samples. Each primer pair contains a barcode and 5’ tags which facilitates the ligase-free attachment of the Rapid Sequencing Adapter. Prior to PCR, this workflow takes approximately 50 minutes and post-PCR, the attachment of sequencing adapters takes about 10 minutes.
Locus-specific protocols:
We also offer methods to enrich for loci of interest using the PCR Barcoding Kit (SQK-PBK004) and PCR Sequencing Kit (SQK-PSK004) where users tag their own specific amplicons with the same 5’ group before barcoding to simplify post-PCR processing. This is done via a four-primer PCR outlined in the Four-Primer PCR (SQK-PSK004 or SQK-PBK004) protocol with a recommended input of 30 ng DNA. Users add a 5’ tail to their own primer sequences and this acts as a priming site for a set of barcoded “outer” primers which are supplied in the PCR Barcoding Kit. These primers contain the 5’ tags which facilitate the ligase-free attachment of Rapid Sequencing Adapters.
Rapid PCR Barcoding Kit (SQK-RPB004):
The Rapid PCR Barcoding Kit (SQK-RPB004) is a fast and simple method of preparing barcoded libraries for low quantities of gDNA (1‒5 ng), with only ~15 minutes of hands-on preparation time. The transposase simultaneously cleaves the 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.
16S Barcoding Kit (SQK-RAB204)
The 16S Barcoding Kit (SQK-RAB204) 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. There are up to 12 unique barcodes, allowing the user to pool up to 12 different samples in one sequencing experiment using 10 ng of high molecular weight gDNA per sample as input. The DNA is amplified by PCR using the specific 16S barcode primers (27F and 1492R) and 5’ tags which facilitate the ligase-free attachment of Rapid Sequencing Adapters.
16S Barcoding Kit 1-24 (SQK-16S024):
The 16S Barcoding Kit 1-24 (SQK-16S024) 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. There are up to 24 unique barcodes, allowing the user to pool up to 24 different samples in one sequencing experiment with 10 ng of high molecular weight gDNA per sample as input. The DNA is amplified by PCR using the specific 16S barcode primers (27F and 1492R) and 5’ tags which facilitate the ligase-free attachment of Rapid Sequencing Adapters.
-
cDNA barcoding kits
PCR-cDNA Barcoding Kit (SQK-PCB109)
This kit is the barcoding version of the cDNA-PCR Sequencing Kit (SQK-PCS109) and is recommended for users who want to multiplex cDNA samples with limited input material, whilst optimising for output. This kit may also be used to identify and quantify full-length transcripts or characterise and quantify isoforms, splice variants and fusion transcripts. This is also appropriate for users who are interested in differential gene expression or differential transcript usage. Up to 12 samples of cDNA can be prepared from an input as low as 1 ng of poly(A) RNA. Users who do not have poly(A)+ enriched RNA can use 50 ng of total RNA but additional optimisation may be required, as further explained below.
Complementary strand synthesis and strand switching are performed on full-length poly(A) RNA using the kit supplied oligonucleotides. There are 12 primer pairs that are used to generate and then amplify double-stranded cDNA by PCR amplification. Each primer pair contains a barcode and a 5’ tag which facilitates the ligase-free attachment of Rapid Sequencing Adapters. Amplified and barcoded samples are then pooled together and sequencing adapters are added to the pooled mix.
PCR-cDNA Barcoding Kit (SQK-PCB111.24):
This kit is the barcoding version of the cDNA-PCR Sequencing Kit (SQK-PCS111) and is recommended for users who want to multiplex cDNA samples with limited input material, whilst optimising for output. This kit may also be used to identify and quantify full-length transcripts or characterise and quantify isoforms, splice variants and fusion transcripts. This is also appropriate for users who are interested in differential gene expression or differential transcript usage. Up to 12 samples of cDNA can be prepared from an input as low as 1 ng of poly(A) RNA. Users who do not have poly(A)+ enriched RNA can use 50 ng of total RNA but additional optimisation may be required, as further explained below.
We have recently upgraded this kit to Kit 11 chemistry (SQK-PCB111.24) and increased the number of barcodes available to 24. This upgrade requires starting inputs of 4 ng poly(A) RNA or 200 ng of total RNA. Further the Kit 11 upgrades includes a new Rapid Adapter T (RAP T), which has a higher capture rate and fuel fix technology for longer experiments without the need for fuel addition during the run.
We have also included a new cDNA RT adapter (CRTA), Annealing Buffer (AB) and RT primer (RTP) to prime cDNA synthesis from the end of a transcript to reduce overlaps during the reverse transcription step. This also enables users to measure poly(A) tail lengths.
The kit workflow includes complementary strand synthesis and strand switching of full-length poly(A) RNA using the kit supplied oligonucleotides. There are 24 primer pairs that are used to generate and then amplify double-stranded cDNA by PCR amplification. Each primer pair contains a barcode and a 5’ tag which facilitates the ligase-free attachment of Rapid Sequencing Adapters. Amplified and barcoded samples are then pooled together and sequencing adapters are added to the pooled mix.