Overview: How to automate PacBio SMRTbell WGS on the MIRO CANVAS

Experimental set-up

The SMRTbell Prep Kit 3.0 protocol was designed with automated systems – such as the MIRO CANVAS – in mind, and has been tested using high quality, high molecular weight 1-3 μg DNA inputs. Before beginning, DNA should be fragmented to 15-18 kb using a Megaruptor®, and quantified before and/or after fragmentation using a Q32850 Qubit™ dsDNA Quantification Assay Kit, Broad Range, or similar. Post-shearing clean-up, repair and A-tailing, adapter ligation, post-ligation clean-up, nuclease treatment and bead-based size selection for the SMRTbell Prep Kit 3.0 are all automated on the MIRO CANVAS (Figure 1), resulting in a ready-to-sequence library.

Downloads: App note for automating PacBio® SMRTbell® whole genome sequencing library prep on the MIRO CANVAS

Method and results

SMRTbell libraries were constructed with 1 μg of high quality, high molecular weight NA24385 (HG002)* DNA, manually and using the MIRO CANVAS. Final library quantities were assessed using broad range or high sensitivity Qubit kits. The MIRO CANVAS produced comparable libraries to manual preparation for each of the kits evaluated (Table 1).

* NA24385 DNA was obtained from the NIGMS Human Genetic Cell Repository at the Coriell Institute for Medical Research.

Table 1: SMRTbell Prep Kit 3.0 libraries generated with the MIRO CANVAS were comparable to manually prepared libraries. Total library quantity shown as average +/- standard deviation (n=6).

Efficient removal of small molecules from libraries using diluted bead-based size selection with the SMRTbell Prep Kit 3.0

Diluted bead-based size selection offers many advantages over traditional gel-based size selection of long-read libraries, including automatability, reduced workflow times and lower input requirements. Performing the SMRTbell Prep Kit 3.0 protocol on the MIRO CANVAS enabled construction of libraries from just 1 μg of input material, and efficiently removed small molecules from the library with automated bead-based size selection (Figure 2).

SMRTbell libraries were sequenced on a Sequel® II System using binding kit 2.2, sequencing kit 2.0 (PacBio), and 30-hour movies. This demonstrated equivalency across manual and automated library preps. Of particular note, the new SMRTbell Prep Kit 3.0 not only requires less input material and eliminates the need for cumbersome gel-based size selection methods, it also results in libraries with excellent sequencing performance (Figure 3) and structural variant detection (Figure 4).

References

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2. Logsdon GA, Vollger MR, Eichler EE. Long-read human genome sequencing and its applications. Nature reviews. Genetics. 2020 Oct;21(10):597-614. doi: 10.1038/s41576-020-0236-x. Epub 2020 Jun 5. PMID: 32504078.