Future work will focus on specific improvements to hybridization capture, primarily to minimize hybridization-based bias. For example, we have observed that lower-sequence complexity targets, such as those carrying SNPs or repeats, are more problematic for hybridization capture due to the tendency for probes to cross-hybridize with their homologs and when a homolog is present it can be counted as multiple targets. We have also observed that probes that hybridize well at higher temperatures are less likely to hybridize to their homologs, which is an intrinsic property of the hybridization capture protocol. We are working toward developing an approach to mitigate bias by restricting hybridization capture to optimal conditions. In addition, we are exploring the utility of re-designing the capture probes to enable discrimination of homologous targets using variables such as base composition, GC content, or chemical modification. Current approaches are based on structural differences, such as length, specificity and binding affinity, but recently developed machine learning approaches have shown promise for analyzing extrinsic sequences such as DNA methylation (65). We are currently exploring this strategy for discrimination of homologous targets and its potential to reduce bias.
Future work will also concentrate on continued development of this technology and a transition to commercialization, both of which will require the close collaboration of Proto-Nano with commercial partners in the industry. While the current version of multiplex hybridization capture is largely designed for low-level detection, we believe that an optimized version could be used to enrich targeted molecules to much higher levels such that low-level detection directly from the enriched molecules would be robust to PCR inhibitors. Alternatively, for pathogen detection purposes, one could envision hybridization capture purification followed by PCR or other amplification techniques, such as digital PCR to enhance detection (66). We are currently exploring these scenarios. d2c66b5586