Overview: How to implement the Twist Human Core Exome kit on the MIRO CANVAS

Experimental set-up

The fully automated Twist Universal Library Prep protocol has been tested using 50 ng of high molecular weight DNA. DNA should be quantified using the Qubit™ dsDNA Broad Range Quantification Assay or similar before starting.6 Fragmentation, end repair, adapter ligation, amplification and purification steps are all automated in this protocol. The Twist Fast Hybridization Target Enrichment protocol has been tested using single samples and 8-plex pools, and the volume of each sample library used depends on their respective concentrations.7 Minimal hands-on sample preparation is required at the beginning of this protocol, leaving most steps automated on the MIRO CANVAS, including hybridizing probes with pools, binding targets to beads, post-capture amplification and purification.

Downloads: App note for implementing the Twist Human Core Exome Kit on the MIRO CANVAS

Methods and results

Library preparation

The MIRO Universal Library Prep (Twist) protocol has been tested using 50 ng of NA12878* gDNA. Hybridization capture was then performed manually to assess the quality of the library preparation protocol alone on the MIRO CANVAS. Replicates of the libraries prepared on the MIRO CANVAS and manually (n=4, 8 in total) were sequenced on an Illumina NextSeq 500 or 550 platform, 75 Paired-End. Key metrics, such as depth of coverage and fold-80 scores, were comparable between the methods. Additionally, both methods yielded a similar percentage of duplicated reads and reads on target (Figure 2).

*NA12878 DNA was obtained from the NIGMS Human Genetic Cell Repository at the Coriell Institute for Medical Research.

Target enrichment

The Twist Fast Hybridization Target Enrichment protocol has been tested using inputs of 1500 ng of mixed DNA for 8-plex pools (multiplex, 187.5 ng per library) and 500 ng for individual libraries (singleplex). Samples run on the MIRO CANVAS for hybridization capture previously underwent manual library preparation. Sequencing was performed using the NextSeq High Output 75PE platform. Singleplex and 8-plex pools run on the MIRO CANVAS were compared to singleplex samples that had been enriched manually. Coverage of target bases at 20x and 30x was comparable between manual and automated protocols for single samples and 8-plex pools. Fold-80 scores were also similar between methods, with median scores varying by no more than 0.02 (Figure 3).

Additional sequencing was completed using 2 x 8-plex pools, one prepared manually and the other on the MIRO CANVAS. Targets covered at 30x and fold-80 scores for each pool were comparable (Table 1). Reads for these runs were assessed using the MIRO CANVAS Integrative Genomics Viewer (IGV). The IGV outputs displayed confident mapping of target genes across multiple exons from pools enriched using the MIRO CANVAS (Figure 4).

Table 1: Key metrics were comparable for 8-plex pools enriched manually and on the MIRO CANVAS. All samples were subsampled to 150x raw sequencing coverage (70 M reads, 5.3 GB of data per sample).

Time savings

While the overall time taken between the library preparation and hybridization capture protocols does not vary significantly between manual preparation and MIRO CANVAS automation, the hands-on time is far less for the automated applications. Manual library preparation requires about 3 hours of hands-on time, while the Twist Universal Library Prep requires about 15 minutes to set up for a fully automated run of 3 hours and 20 minutes.

The Twist Fast Hybridization Target Enrichment protocol begins with several steps that cannot be automated, such as preparing pools and hybridization mix, and therefore requires about an hour of benchtop work. The steps that follow are fully automated on the MIRO CANVAS, taking about 5.5 hours to complete. This hour of hands-on time pales in comparison to the time taken by the manual protocol, which requires as much as 7.5 hours.

References

1. Rabbani B et al. The promise of whole-exome sequencing in medical genetics. J Hum Genet 2014; 59: 5–15. https://doi.org/10.1038/ jhg.2013.114
2. Lelieveld SH et al. Comparison of exome and genome sequencing technologies for the complete capture of protein-coding regions. Hum Mutat 2015; 36: 815-822. https://doi.org/10.1002/humu.22813
3. Holland I and Davies JA. Automation in the life science research laboratory. Front Bioeng Biotechnol 2020; 8: 571777. https://doi.org/10.3389/fbioe.2020.571777
4. Twist Bioscience. Human core exome kit. Available at: Human Core Exome - Twist Bioscience Accessed Feb 2022.
5. Miroculus. New Class of Technology. Available at: https://miroculus.com/technology/. Accessed February 2022.
6. Miroculus, Inc. Miro Universal Library Prep (Twist) Protocol. Available at: https://miroculus.com/. Accessed: Feb 2022.
7. Miroculus, Inc. Miro Human Exome Hyb Capture (Twist) Protocol. Available at: https://miroculus.com/. Accessed: Feb 2022.