• Automated library preparation for a nanopore DNA barcoding protocol on the MIRO CANVAS

    Enabling automation of multiplex library preparation using the Native Barcoding Kit from Oxford Nanopore Technologies

    Long read sequencing allows the in-depth characterization of formerly unreadable regions, supporting de novo genome assembly and haplotype phasing.1

    The Native Barcoding Kit (SQK-NBD114.24) uses DNA barcoding and multiplex nanopore sequencing to enable the identification of DNA molecules in a single sample that is being sequenced in parallel with other samples.2 It can use PCR amplicons or non-amplified DNA.3 This is important for applications where native DNA structure preservation is key – such as the analysis of base modifications, metagenomic and epigenetic studies – or when detecting species of interest, such as pathogens and their evasion mechanisms.4

    The MIRO CANVAS NGS prep system is a digital microfluidics platform that automates NGS library preparation. It is compact (20.2x40.6x17.6 cm, WxDxH), requires minimal infrastructure (a standard 120V adapter), and has been shown to work after air travel in carry-on baggage and in a backpack.

    This application note describes results from the Native Barcoding Kit partially automated on the MIRO CANVAS, which can handle up to 8 native barcoded samples or
    >60 bead purified amplicon samples.

  • Table of contents

    Long read sequencing allows the in-depth characterization of formerly unreadable regions, supporting de novo genome assembly and haplotype phasing.1

    The Native Barcoding Kit (SQK-NBD114.24) uses DNA barcoding and multiplex nanopore sequencing to enable the identification of DNA molecules in a single sample that is being sequenced in parallel with other samples.2 It can use PCR amplicons or non-amplified DNA.3 This is important for applications where native DNA structure preservation is key – such as the analysis of base modifications, metagenomic and epigenetic studies – or when detecting species of interest, such as pathogens and their evasion mechanisms.4

    The MIRO CANVAS NGS prep system is a digital microfluidics platform that automates NGS library preparation. It is compact (20.2x40.6x17.6 cm, WxDxH), requires minimal infrastructure (a standard 120V adapter), and has been shown to work after air travel in carry-on baggage and in a backpack.

    This application note describes results from the Native Barcoding Kit partially automated on the MIRO CANVAS, which can handle up to 8 native barcoded samples or
    >60 bead purified amplicon samples.

Key benefits

  • Library preparation using the Native Barcoding Kit is partially automated on the MIRO CANVAS, which can process a barcoded pool of 8 end-prepped samples.
  • This protocol uses ~3 μg of high quality, high molecular weight input DNA in the form of an 8-plex pool.
  • The MIRO CANVAS enables a 50 % reduction in reaction volumes compared to manual library preparation.
  • The automated protocol has a total run time of 2 hours and 40 minutes.
  • N50 read lengths are comparable to manual library preparation.

Overview: How to automate the Native Barcoding Kit on the MIRO CANVAS

Experimental set-up

This experiment used 3.2 μg of short read eliminated (PacBio PN: SKU 102-208-300) ZymoBIOMICS HMW DNA Standard (Zymo research PN: D6322) as input for both manual library preparation and libraries prepared on the MIRO CANVAS. 400 ng of each ZymoBIOMICS HWM DNA Standard was quantified using a broad range Qubit™ quantification kit, followed by manual end-prep, unique barcode ligation and pooling to create an 8-plex pool. Reagent volumes listed in the Native Barcoding Kit protocol were reduced by 50 % after the barcoding step. The MIRO CANVAS protocol was then used to automate bead clean-up, adapter ligation and final library bead clean-up, which was also performed manually in parallel.

Overview of the experimental set-up for the ONT Native Barcoding Kit on the MIRO CANVAS.
Figure 1: Experimental set-up for the Native Barcoding Kit workflow. Following manual pooling of end-prepped and barcoded DNA samples, the MIRO CANVAS automates pooled sample bead clean-up, adapter ligation and library clean-up.

Downloads: App note for fully automated DNA barcoding workflow

Results

Each prepared library pool was loaded into a R10.4.1 MinION flow cell and sequenced until ~5 Gb of data was obtained. Libraries prepared using the automated workflow on the MIRO CANVAS produced comparable read length distributions (Figure 2) and N50 read lengths (Table 1) to those prepared manually.

Comparison of read length distributions between manual and automated NGS library preparation after multiplex sequencing.
Figure 2: Representative read length histograms show similar distributions for libraries prepared manually or using the       MIRO CANVAS.

Representative sequencing metrics for libraries prepared manually and using the MIRO CANVAS are shown below. Read length (bp) and quality statistics are comparable between the MIRO CANVAS and manual preparation.

 

Table 1: Summary of the sequencing metrics for libraries prepared manually and using the           MIRO CANVAS.

Summary of the sequencing metrics for libraries prepared manually and using the MIRO CANVAS demonstrating key metrics are comparable between the MIRO CANVAS and manual preparation.

The 5 longest sequenced reads in the MIRO CANVAS library were all >150 kb, and greater in length than the 5 longest ranked reads from the manually prepared library pool (Figure 3). Additionally, the MIRO CANVAS had the higher mean call base quality score.

Comparison of read length and mean call base quality score for the 5 longest reads sequenced using manual and automated NGS library preparation methods. MIRO CANVAS produced longer reads of higher quality than manual processing in multiplex DNA sequencing experiments.
Figure 3: Read length and mean call base quality score for the 5 longest reads sequenced.

The ZymoBIOMICS HMW DNA Standard is composed of genomic DNA from 7 bacteria and 1 yeast species. All 8 species were identified through nanopore sequencing of libraries prepared both manually and using the MIRO CANVAS. Figure 4 shows how the cumulative reads of each species compare between manual and MIRO CANVAS runs.

Comparison of cumulative read numbers for representative mock community per genus using libraries prepared manually and with automated NGS library preparation. All mock community members were identifiable in both the MIRO CANVAS and manual nanopore multiplexing results.
Figure 4: Cumulative read numbers for representative mock community per genus using libraries prepared manually and with the MIRO CANVAS.

The number of sequenced reads for each of the clinical samples was similar between pooled libraries for both the MIRO CANVAS and manual experiments. This reflected a balanced pool, where each barcode was +/-30 % read count on average, with no more than 20 % of barcodes falling outside the 30 % median bracket.

Similar results were obtained when the input DNA pool was comprised of pooled SARS-CoV-2 amplicons generated using the nanopore protocol pcr-tiling-SARS-CoV-2-nbd-PTCN_9103_v109_revR_13Jul2020-minion (data not shown, poster available upon request).

Comparison of the number of sequenced reads for each one of the 8 uniquely barcoded samples in the manually prepared pool and the pool processed using automated NGS library preparation. DNA barcoding of each of the individual samples is equally represented in both the MIRO CANVAS and manual libraries.
Figure 5: The number of sequenced reads for each one of the 8 uniquely barcoded samples in both the manually prepared and MIRO CANVAS processed pools.

References

  1. Bayliss SC, Hunt VL, Yokohama M, Thorpe HA, Feil EJ. The use of Oxford Nanopore native barcoding for complete genome assembly. GigaScience. 2017 Mar; 6(3): doi: 10.1093/gigascience/gix001. PMID: 28327913.
  2. Florian T, Karen L, Emily F, Alon S, Hilary GM, Luis B, Bana J, Murat E. High molecular weight DNA extraction strategies for long-read sequencing of complex metagenomes. Microbiology Spectrum. 2022 Jul;22(5):1786-1802. doi: 10.1111/1755- 0998.13588. PMID: 35068060.
  3. Robert P, Kathleen V, Andrea S, Ellen F, Amanda E, Mihir SJ, Rebecca D, David R, Sarah G, Bruce G, Shanmuga S. Optimization of Oxford Nanopore Technology Sequencing Workflow for Detection of Amplicons in Real Time Using ONT-DART Tool. Genes (Basel). 2022 Oct 3;13(10):1785. doi: 10.3390/genes13101785. PMID: 36292670.
  4. Mojnu M, Mohammad EH, Rashedul H, Md Shaheen A, Joynob AP, Md Mahmudul H, Ariful I, Sukanta C, Mohammed ZR. Culture-Independent Workflow for Nanopore MinION-Based Sequencing of Influenza A Virus. Genes (Basel). 2022 Oct. 3;13(10):1785. doi: 10.3390/genes13101785. PMID: 37212605.

Conclusion

  • The MIRO CANVAS protocol uses the Native Barcoding Kit to automate the preparation of up to 8 samples for multiplex sequencing on a single flow cell.
  • Following pooling, the entire process – from pooled sample bead clean-up to elution – is fully automated, reducing reagent requirements by 50 % and yielding results comparable to manual library preparation.
  • For higher throughput sequencing, pools greater than
    8-plex and/or amplicon DNA pools can be prepared on the MIRO CANVAS, as long as the pool volume is adjusted for loading compatibility.
  • The system is portable, so can accompany highly portable ONT sequencers, offering multiplex library preparation and sequencing applicable to outbreak scenarios.

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  • Fay Christodoulou, PhD

    Chief Scientific Officer Microfluidics

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Instruments and accessories

MIRO CANVAS, NGS prep system MIRO Cartridge VIAFLO single channel, 1250 µl, electronic pipette GRIPTIPS®, 125 µl, Sterile, Filter - 10 ECO racks of 384 tips Oxford Nanopore Technologies: MIN-101B - MinION Oxford Nanopore Technologies: SQK-NBD114-24 - Native Barcoding Kit 24 V14 Oxford Nanopore Technologies: FLO-MIN114 - Flow Cell (R10.4.1) New England Biolabs: B6058S - NEBNext® Quick Ligation Reaction Buffer New England Biolabs: M0367L - Blunt/TA Ligase Master Mix Thermo Fisher Scientific: 15568025 - UltraPure 1M Tris-HCl, pH 8.0 Thermo Fisher Scientific: AM9760G - NaCl (5 M), RNase-free Thermo Fisher Scientific: Q32850 - Qubit™ dsDNA BR Assay Kit Thermo Fisher Scientific: Q33238 - Qubit 4 Fluorometer Sigma-Aldrich: E7023 - Ethyl alcohol, Pure VWR: NUPW-0050 - Nuclease-free water Eppendorf: 0030108051 - DNA LoBind® Tubes USA Scientific: 1402-4700 - TempAssure PCR Flex-Free 8-Tube Strips, natural color 0.2mL

MIRO CANVAS, NGS prep system

A revolutionary microfluidics platform which enables full automation of NGS prep protocols.

  • Fully automated - Simple, walk-away automation for on-demand NGS sample preparation

  • Efficient - Minimize costs with up to 75 % reduction in reagent use and only 15 minutes of hands-on time

  • Flexible - Established NGS sample prep protocols for both short- and long-read sequencing platforms

Part No. M-01-0001-001-01

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MIRO-CANVAS

MIRO Cartridge

The MIRO Cartridge is a single use consumable suitable for running library prep workflows on the MIRO CANVAS instrument (for Research Use Only)

Part No. M-02-0001-001-03

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Miro Cartridge

VIAFLO single channel, 1250 µl, electronic pipette

VIAFLO pipettes feature 10 preset pipetting programs, for which only a few parameters such as volume and speed need to be defined.

For more elaborate pipetting protocols, up to 40 user defined custom programs can be created. The custom program is a step-based setup queuing liquid handling steps such as aspirate, dispense, mix and more.

Part No. 4014

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INTEGRA’s range of VIAFLO electronic pipettes.

GRIPTIPS®, 125 µl, Sterile, Filter - 10 ECO racks of 384 tips

GRIPTIPS® pipette tips perfectly match the multi-lobe tip fitting, snapping firmly on during loading. This guarantees a perfect seal on every tip, preventing them from loosening, leaking or completely falling off. All tips are precisely aligned horizontally, enabling accurate touch-offs, even when pipetting with 384 tips.

ECO racks contain 60% less plastic than alternative racks, while maintaining the high standards that you expect from INTEGRA products. Once empty, ECO racks can be stacked and compressed to minimize the space needed in your lab's recycling, keeping your workspace clutter-free and supporting a positive working environment.

Part No. 3725

Learn more about GRIPTIPS® pipette tips for Benchtop Pipetting Systems
Six ECO Racks with pipette tips standing one on top of the other

Oxford Nanopore Technologies: MIN-101B - MinION

MinION is a powerful, portable sequencing device that delivers cost-effective and real-time access to gigabases of long-read sequencing data. Small enough to fit in a pocket and powerful enough to deliver up to 30 Gb data, the USB-powered MinION allows researchers to rapidly generate actionable biological insights across a wide range of application areas.

Part No. MIN-101B

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Oxford Nanopore Technologies: SQK-NBD114-24 - Native Barcoding Kit 24 V14

A versatile method of preparing barcoded sequencing libraries optimised for modal raw read accuracy of Q20+ (99%+) and long read multiplexed samples.

This uses our latest Kit 14 chemistry

Part No. SQK-NBD114.24

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Oxford Nanopore Technologies: FLO-MIN114 - Flow Cell (R10.4.1)

R10 is our nanopore chemistry designed to deliver highest consensus accuracy. Paired with the Kit 14 chemistry, R10.4.1 generates data at a modal accuracy above 99%. 

Note: R10.4.1 flow cells currently require Kit 14 chemistry.

Part No. FLO-MIN114

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New England Biolabs: B6058S - NEBNext® Quick Ligation Reaction Buffer

  • The NEBNext(r) Quick Ligation Reaction Buffer is optimized for use with Quick T4 DNA Ligase, in standard NEBNext protocols.
  • This buffer is not compatible with NEBNext Ultra™ protocols.

Part No. B6058S

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New England Biolabs: M0367L - Blunt/TA Ligase Master Mix

Blunt/TA Ligase Master Mix is a ready-to-use solution of T4 DNA Ligase, proprietary ligation enhancer, and optimized reaction buffer. This master mix is specifically formulated to improve ligation and transformation of both blunt-end and single-base overhang substrates.

  • Simplifies reaction set-up, ensures an optimized ratio of enzyme and buffer components
  • No thawing is necessary as it remains liquid during storage at -20°C
  • Ligations for subcloning can be carried out in small volumes with low DNA concentrations, allowing users to conserve precious DNA samples and directly transform many strains of chemically competent E. coli without dilution
  • Suitable for Adaptor Ligation

Part No. M0367L 

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Thermo Fisher Scientific: 15568025 - UltraPure 1M Tris-HCl, pH 8.0

UltraPure™ 1 M Tris-HCl Buffers are pre-mixed, pH-adjusted, sterile-filtered solutions. Prepared as 1 M concentrates, these buffers can be diluted to the desired concentration and used in molecular biology or general biochemistry applications.

Part No. 15568025

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Thermo Fisher Scientific: AM9760G - NaCl (5 M), RNase-free

Ambion Molecular biology grade, 5 M NaCl solution is supplied in one bottle containing 100 mL. The solution is certified RNase-free, economical, and ready-to-use. Due to the ubiquitous presence of RNases, manufacturing products for use with RNA is especially challenging. Ambion's nuclease-free reagents and buffers are manufactured in facilities specifically designed to prevent the introduction of nucleases. Highly sensitive RNase assays are performed at several different stages of the manufacturing process to ensure the highest quality. These reagents are rigorously tested for contaminating nonspecific endonuclease, exonuclease, and RNase activity.

Part No. AM9760G

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Thermo Fisher Scientific: Q32850 - Qubit™ dsDNA BR Assay Kit

Achieve accurate and precise quantification of double-stranded DNA with Qubit dsDNA HS and BR Assay Kits. These dsDNA quantification kits enable quick and selective detection of low and high abundance DNA samples, and can distinguish dsDNA from ssDNA, RNA, protein, and free nucleotides.

Part No. Q32850

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Thermo Fisher Scientific: Q33238 - Qubit 4 Fluorometer

The Qubit 4 Fluorometer with WiFi is the latest version of the popular Qubit fluorometer designed to accurately measure DNA, RNA, and protein quantity, and now also RNA integrity and quality, using the highly sensitive Qubit assays. The Qubit 4 Fluorometer was re-engineered to enable data transfer via WiFi as well as to run the Qubit RNA IQ (integrity & quality) assay. The Qubit 4 Fluorometer and RNA IQ Assay Kit work together to accurately distinguish intact from degraded RNA in just two easy steps.

Part No. Q33238

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Sigma-Aldrich: E7023 - Ethyl alcohol, Pure

≥99.45%, for molecular biology, 200 proof, suitable for RNA extraction and DNA extraction

Part No. E7023 

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VWR: NUPW-0050 - Nuclease-free water

Sterile molecular biology grade nuclease free water. Prepared with 18.2 megOhm, 0.1 micron sterile filtered water and autoclaved in final packaging.

Part No. NUPW-0050

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Eppendorf: 0030108051 - DNA LoBind® Tubes

  • Eppendorf LoBind material ensures optimized sample recovery for improved assay results
  • Free of surface coating (e.g., silicone) to minimize the risk of sample interference
  • Lot-certified PCR clean purity grade: free of human DNA, DNase, RNase and PCR inhibitors
  • Available in tube, microplate, and deepwell plate formats for easy-up scaling
  • Precise lid sealing for minimal evaporation rates in tube format
  • Rated up to 30,000_x_g (25,000 × g for 2,0 mL) centrifugation speed for molecular biology applications

Part No. 0030108051 

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USA Scientific: 1402-4700 - TempAssure PCR Flex-Free 8-Tube Strips, natural color 0.2mL

Flex-free strips are reinforced to resist bending while handling. Individually attached flat caps are optically clear for use in real time procedures, are easy to handle, and help avoid cross-contamination. Polypropylene thin wall tubes provide rapid, even heat transfer for reliable amplifications. Manufactured in clean room conditions and certified free of detectable levels of DNase, RNase, DNA, and PCR inhibitors. Tested pyrogen-free and non-autofluorescent. Made in the USA.

Part No. 1402-4700 

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Downloads

Download App Note as PDF

DownloadAutomated library preparation for a nanopore DNA barcoding protocol on the MIRO CANVAS

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