• 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

app-note-miro canvas-nanopore native barcoding.pdf ( )

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

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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