Introduction of the Hybcell-Based Compact Sequencing Technology and Comparison to State-of-the-Art Methodologies for KRAS Mutation Detection

Abstract

The detection of KRAS mutations in codons 12 and 13 is critical for anti-EGFR therapy strategies; however, only those methodologies with high sensitivity, specificity, and accuracy as well as the best cost and turnaround balance are suitable for routine daily testing. Here we compared the performance of compact sequencing using the novel hybcell technology with 454 next-generation sequencing (454-NGS), Sanger sequencing, and pyrosequencing, using an evaluation panel of 35 specimens. A total of 32 mutations and 10 wild-type cases were reported using 454-NGS as the reference method. Specificity ranged from 100% for Sanger sequencing to 80% for pyrosequencing. Sanger sequencing and hybcell-based compact sequencing achieved a sensitivity of 96%, whereas pyrosequencing had a sensitivity of 88%. Accuracy was 97% for Sanger sequencing, 85% for pyrosequencing, and 94% for hybcell-based compact sequencing. Quantitative results were obtained for 454-NGS and hybcell-based compact sequencing data, resulting in a significant correlation (r = 0.914). Whereas pyrosequencing and Sanger sequencing were not able to detect multiple mutated cell clones within one tumor specimen, 454-NGS and the hybcell-based compact sequencing detected multiple mutations in two specimens. Our comparison shows that the hybcell-based compact sequencing is a valuable alternative to state-of-the-art methodologies used for detection of clinically relevant point mutations.

Photolithographic Synthesis of High-Density DNA and RNA Arrays on Flexible, Transparent, and Easily Subdivided Plastic Substrates

“…14,15 This technical requirement has prevented high-resolution DNA patterning on surfaces with the diverse formats or non-planar geometries often found in miniaturized hybridization devices. 16,17 A conceivable solution to this problem would be to synthesize DNA arrays on flexible substrates that could later be physically manipulated or cut into desired shapes or sizes. Oligonucleotides patterned upon these substrates could, in turn, template the programmable attachment of other species, such as small molecules, proteins, or even cells, and thereby serve as a universal scaffold for high-resolution biomolecule patterning on arbitrary surfaces.…”

“…In contrast to “spotted” or “top-down” techniques of array production, where presynthesized biomolecules are coupled to a surface, in situ or “bottom-up” methods are advantageous in terms of their affordability and versatility. Nonetheless, all in situ approaches for DNA synthesis are dependent upon precision optics or highly targeted reagent delivery and have conventionally employed rigid and planar substrates. , This technical requirement has prevented high-resolution DNA patterning on surfaces with the diverse formats or nonplanar geometries often found in miniaturized hybridization devices. , A conceivable solution to this problem would be to synthesize DNA arrays on flexible substrates that could later be physically manipulated or cut into desired shapes or sizes. Oligonucleotides patterned upon these substrates could, in turn, template the programmable attachment of other species, such as small molecules, proteins, or even cells, and thereby serve as a universal scaffold for high-resolution biomolecule patterning on arbitrary surfaces …”

Disruptive innovations in the clinical laboratory: catching the wave of precision diagnostics

“…The evolution of NGS is very interesting, showing the power of disruptive innovation. Starting with two or three large manufacturers producing high-throughput instruments, many manufacturers are now moving to compact Personal Genome Machine (PGM) sequencers that are small in size, much cheaper, and have fast turnover rates but limited data throughput [40][41][42]. New technologies, such as those developed by Oxford Nanopore Technologies (Oxford, United Kingdom), offer much smaller and cheaper sequencing instruments that are also quick and portable [43].…”