DNA sequencing made easy
Developed in 1977, Sanger DNA sequencing mimics the natural process of DNA synthesis to read the sequences of small DNA regions. The past 10 years have seen the emergence of superfast technologies – collectively termed next-generation sequencing (NGS) – that enable the sequencing of multiple DNA fragments at higher speeds and a fraction of the cost. In essence, NGS is like running multiple small Sanger sequencing reactions in parallel. Despite these advances, it remains difficult to meaningfully sequence many genomic regions of medical and agricultural importance.
Microfluidics in genomic analysis
The EU-funded IMPACT initiative aimed to overcome key challenges associated with established genomics workflows. “Our goal was to enable the analysis of genomic regions that are inaccessible with other technologies,” explains Marie Just Mikkelsen, CTO and co-founder of Samplix. The IMPACT team developed a microfluidics-based approach (Xdrop®) that can encapsulate biological material, such as DNA, in highly stable double-emulsion droplets for downstream genomic analysis. The workflow begins with the capture of DNA molecules in droplets, and then the detection sequence that relates to the region of interest is amplified using droplet polymerase chain reaction (droplet PCR). The amplified sequence has a fluorescent signal that enables sorting of the droplets with the desired target, which is then further enriched using droplet multiple displacement amplification (dMDA). Xdrop® supports the analysis of samples of low DNA concentration, delivering material suitable for both long and short sequence reads. Importantly, it can capture long DNA regions enabling the identification and investigation of insertions, deletions, repeats and gaps in the genomic region of interest. This is of particular importance for genomic regions implicated in disease. It can also retrieve single DNA molecules from a minute amount of biological sample, such as tumour biopsies.
IMPACT applications and prospects
“Understanding the needs of users is paramount in the development of new technologies,” emphasises Mikkelsen. IMPACT partners invested time and effort in user interaction, visiting user facilities to obtain invaluable feedback that led to the optimisation of Xdrop® for various applications. The technology has been successfully employed in genome-editing projects, where it has demonstrated the capacity to identify genomic modifications as well as unintended edits. Moreover, interesting results have emerged from projects using Xdrop® to study cancer genes and biosynthetic gene clusters in the genome of barley. The technology is also suitable for resolving unknown genomic regions in plants and animals. “Overall, Xdrop® has shown significant versatility, and it is expected to have a lasting impact in the fields of genomics and cell biology,” concludes Mikkelsen. The instrument has the capacity to encapsulate single molecules, organelles, or cells in double- or single-emulsion droplets, opening new downstream analytics possibilities. Ongoing efforts focus on developing additional Xdrop®-based services and supporting users who are investigating new applications. The team is also working on new possibilities to further improve the workflow while maintaining the user-friendly nature of the technology.
Keywords
IMPACT, Xdrop®, DNA sequencing, microfluidics, genomic analysis, healthcare, droplet polymerase chain reaction, droplet multiple displacement amplification