Overview: How to automate the ONT Ligation Sequencing Kit on MIRO CANVAS

Experimental set-up

The protocol was designed for fully automated use on the MIRO CANVAS, and has been tested using 1 μg of high quality, high molecular weight (HMW) input DNA. Before beginning, DNA should be quantified using a broad range Qubit quantification kit or similar. DNA repair and end prep, post repair bead clean-up, adapter ligation and library clean-up are all automated on the MIRO CANVAS (Figure 1). Downstream quantification requires additional hands-on time.

Downloads: App note for automating the Oxford Nanopore Ligation Sequencing Kit on MIRO CANVAS®

Results

For both manual library preparation and libraries prepared on the MIRO CANVAS, 1 μg of ZymoBIOMICS HMW DNA standard was used as input. For both preparation types, volumes listed in the ONT Genomic DNA by Ligation Protocol were reduced by 75 %. Each prepared library was loaded into a MinION flow cell and sequenced for up to 3 hours. 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 using the manual technique.

Table 1: Summary of the sequencing metrics for libraries prepared manually and using the MIRO CANVAS. Representative sequencing metrics for libraries prepared manually and using the MIRO CANVAS are shown below. Read length and quality statistics are comparable between MIRO CANVAS and manual preparation.

The five longest sequenced reads in the MIRO CANVAS library were all longer than 120 kb, and of similar length to the five longest ranked reads from the manually prepared library. Additionally, the longest read from the MIRO CANVAS library exceeded the length of the longest read from the manually prepared library, and had a higher mean call base quality score (Figure 3).

The ZymoBIOMICS HMW DNA standard is composed of genomic DNA from seven bacteria and one yeast species. All of the eight expected 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.

Due to its compact nature, simple set-up and minimal infrastructure requirements (a 120V adapter), the MIRO CANVAS can support scientists outside of the laboratory and facilitate collaboration between working groups. The MIRO CANVAS has been tested after air travel in carry-on baggage and in a backpack (Figure 5).

References

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