Two probes are better than one

Scientists from David Zhang's group in the NABlab at Rice University, Texas, and nanoString technologies had their work featured on the front cover of Decembers Nature Methods: Continuously tunable nucleic acid hybridization probes. The paper describes their work with X-probes and/or ToeHold probes to tune the performance of oligos in a hybridisation reaction, and demonstrate this with almost perfect yield across 31 probes designed to Staphylococcus aureus in a Human genomic DNA background.

Uniformity of capture: Capture uniforminty has an impact on the amount of sequencing required to reach a specified minimum coverage. Most commercial kits are quite variable, especially in regions of high and low GC content. The X-probes used in the paper went through two rounds of  stoichiometric tunig to get the results shown in figure 2a in the paper (reproduced above). 

The work is exciting as it works on sequences irrespective of their AT:GC content from 0-100%! For SNV discrimination the group showed that their tuning method allows uniform reaction conditions across multiple probes. They compared X-probes to molecular beacons, which did not have a single temperature that generated high sensitivity (high signal for SNV targets) and high specificity (low signal for the wild type); whereas the X-probes achieved a uniform yield across the three temperatures tested.

Mutant allele enrichment from a normal background: The methods offers the ability to tune probes to enrich specifically for mutant alleles (somewhat in the way Boreal Genomics enrich). This may well increase detection sensitivity for mutant alleles making screening for low-abundance tumour DNA in plasma easier. Although detection of mutant alleles is not as immediately useful as quantitation; better to have something than nothing.

Some of the work was done in conjunction with nanoString and the group present a multiplexed RNA panel for detecting variants from FFPE samples on the nCounter. See also their paper in Nature Chemistry which describes the X-probes in more detail.

I am sure Illumina's new venture Grail, Guardant, Inivata, Foundation Medicine, and all the other ctDNA companies will be looking carefully at this, and similar technologies.