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Native Barcoding Expansion 13-24 (PCR-free)

EXP-NBD114

$288.00

A versatile method of preparing barcoded sequencing libraries for applications where high throughput is required

gDNA / amplicon / cDNA ≤ 1 µg ~80 mins No PCR Throughput / read-length control
  • Information

    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.

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

     No barcodes6 barcodes12 barcodes

    Flow cell price

    $500

    $500

    $500

    Library price

    $99

    $99

    $99

    Barcode price

    -

    $25

    $50

    Price per sample

    $599

    $104

    $54

     

    The Native Barcoding Expansion 13-24 is an expansion pack, which provides 12 unique barcodes to enable PCR-free multiplexing of dsDNA samples such as:

    • gDNA – use in combination with Ligation Sequencing Kit
    • cDNA – use in combination with Direct cDNA Sequencing Kit.

    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.

    The Native Barcoding Kit features:

    Feature Property
    Preparation time 80 minutes
    Input requirement 1000 ng of double stranded DNA
    PCR Required No
    Fragmentation Optional
    Read length Equal to fragment length
    Read type produced 1D
    Typical throughput* •••
    Associated protocols

    Native Barcoding Kit

    Flow Cell Wash Kit

    Pack size 6 reactions
    Stability

    Shipped at  2–8° C

    Long-term storage  -20° C

     

    The kit is optimised to generate maximum throughput, without the need for PCR. For highest data yields, we recommend starting with a total of 200 fmol of pure input DNA (for two samples, this means starting with 100 fmol of each). Starting with lower amounts of input material or impure samples can affect library preparation efficiency and lower sequencing throughput.

    In order to ensure that 200 fmol of total DNA is used, it is possible to generate the required number of molecules by shearing samples using a Covaris g-TUBE: 1 μg of gDNA sheared to ~8 kb corresponds to 200 fmol. However, if your experiment requires long reads, fragmentation/shearing is neither advised nor required. Note, if long reads are required, then long fragments must be present in the starting material. To determine purity, we suggest using the Nanodrop to measure the A260/280 and A260/230 ratios and we recommend that the sample should meet the following criteria:

    • A260/280 = 1.8
    • A260/230 = 2.0-2.2.

    The Native Barcoding Expansion 12-24 is compatible with upstream processes such as whole genome amplification (for applications where under 1 ng of sample is available) and size selection (for enrichment of specified fragment lengths, using the BluePippin, for example). When size selecting, we recommend increasing the amount of input used, as size selection can be a wasteful process.

    Deconvolution of barcoded sequencing data is supported by Albacore and EPI2ME, which classify the barcode sequence and sort reads into corresponding folders.

    Further considerations:

    Starting with lower amounts of input material, or impure samples, can affect library preparation efficiency and lower sequencing throughput. PCR can be used to generate more input material in cases where sample amount is limiting. Also, where sample purity is compromised and library preparation efficiency may be reduced, PCR will select for molecules which have been successfully adapted, and generate more of them. The original impurity is lost or diluted. It is often observed that a sample which produces poor sequencing results can be improved by inclusion of PCR (for example, using the PCR Barcoding Kit).

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

  • 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.

  • Protocols

    Example experiment using the Native Barcoding Expansions with the Ligation Sequencing Kit (EXP-NBD104 or EXP-NBD114 & SQK-LSK108) - for illustration purposes only

  • Safety and legal
  • What's in the box

    The Native Barcoding Expansion 13-24 contains 12 unique barcodes and sufficient reagents for six sequencing libraries.

  • Compatibility

    The Native Barcoding Expansion 13-24 can be used together with:

    Kits

    • Ligation Sequencing Kit (SQK-LSK109)
    • Direct cDNA Sequencing Kit (SQK-DCS109)
    • Flow Cell Wash Kit (EXP-WSH003)

    Flow cells

    • FLO-MINSP6
    • FLO-MIN106D
    • FLO-MIN111
    • FLO-PRO002
    • FLO-FLG001

    Devices

    • MinION Mk1B
    • MinION Mk1C
    • Flongle
    • GridION Mk1
    • PromethION P24/P48
Information

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.

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

 No barcodes6 barcodes12 barcodes

Flow cell price

$500

$500

$500

Library price

$99

$99

$99

Barcode price

-

$25

$50

Price per sample

$599

$104

$54

 

The Native Barcoding Expansion 13-24 is an expansion pack, which provides 12 unique barcodes to enable PCR-free multiplexing of dsDNA samples such as:

  • gDNA – use in combination with Ligation Sequencing Kit
  • cDNA – use in combination with Direct cDNA Sequencing Kit.

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.

The Native Barcoding Kit features:

Feature Property
Preparation time 80 minutes
Input requirement 1000 ng of double stranded DNA
PCR Required No
Fragmentation Optional
Read length Equal to fragment length
Read type produced 1D
Typical throughput* •••
Associated protocols

Native Barcoding Kit

Flow Cell Wash Kit

Pack size 6 reactions
Stability

Shipped at  2–8° C

Long-term storage  -20° C

 

The kit is optimised to generate maximum throughput, without the need for PCR. For highest data yields, we recommend starting with a total of 200 fmol of pure input DNA (for two samples, this means starting with 100 fmol of each). Starting with lower amounts of input material or impure samples can affect library preparation efficiency and lower sequencing throughput.

In order to ensure that 200 fmol of total DNA is used, it is possible to generate the required number of molecules by shearing samples using a Covaris g-TUBE: 1 μg of gDNA sheared to ~8 kb corresponds to 200 fmol. However, if your experiment requires long reads, fragmentation/shearing is neither advised nor required. Note, if long reads are required, then long fragments must be present in the starting material. To determine purity, we suggest using the Nanodrop to measure the A260/280 and A260/230 ratios and we recommend that the sample should meet the following criteria:

  • A260/280 = 1.8
  • A260/230 = 2.0-2.2.

The Native Barcoding Expansion 12-24 is compatible with upstream processes such as whole genome amplification (for applications where under 1 ng of sample is available) and size selection (for enrichment of specified fragment lengths, using the BluePippin, for example). When size selecting, we recommend increasing the amount of input used, as size selection can be a wasteful process.

Deconvolution of barcoded sequencing data is supported by Albacore and EPI2ME, which classify the barcode sequence and sort reads into corresponding folders.

Further considerations:

Starting with lower amounts of input material, or impure samples, can affect library preparation efficiency and lower sequencing throughput. PCR can be used to generate more input material in cases where sample amount is limiting. Also, where sample purity is compromised and library preparation efficiency may be reduced, PCR will select for molecules which have been successfully adapted, and generate more of them. The original impurity is lost or diluted. It is often observed that a sample which produces poor sequencing results can be improved by inclusion of PCR (for example, using the PCR Barcoding Kit).

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

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.

Protocols

Example experiment using the Native Barcoding Expansions with the Ligation Sequencing Kit (EXP-NBD104 or EXP-NBD114 & SQK-LSK108) - for illustration purposes only

What's in the box

The Native Barcoding Expansion 13-24 contains 12 unique barcodes and sufficient reagents for six sequencing libraries.

Compatibility

The Native Barcoding Expansion 13-24 can be used together with:

Kits

  • Ligation Sequencing Kit (SQK-LSK109)
  • Direct cDNA Sequencing Kit (SQK-DCS109)
  • Flow Cell Wash Kit (EXP-WSH003)

Flow cells

  • FLO-MINSP6
  • FLO-MIN106D
  • FLO-MIN111
  • FLO-PRO002
  • FLO-FLG001

Devices

  • MinION Mk1B
  • MinION Mk1C
  • Flongle
  • GridION Mk1
  • PromethION P24/P48

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