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Stanford University

Stanford Microfluidics Laboratory

Fast, multiplex target enrichment for next-generation sequencing (NGS)

PI: Juan G. Santiago

 

NGS is poised to revolutionize disease diagnosis by offering hypothesis-free molecular diagnostics, and is continuously becoming cheaper, simpler, and faster. Target enrichment has and will continue to play a major role in decreasing NGS cost and analysis time, since NGS costs scale with the number of reads (and depth of read) required. We are developing techniques which couple isotachophoresis (ITP) with affinity chromatography for rapid and selective purification of target DNA from blood and plasma (see Figure 1). We created microfluidic systems with customized porous polymer monoliths functionalized with synthetic cDNA probes against targets in these samples. We showed 10,000-fold highly selective enrichment of target DNA sequences from up to 200 microliter samples in <10 min using a 1 mm length of our affinity column (Shkolnikov and Santiago, 2014).



Figure 1: Porous polymer monoliths for highly selective and fast target enrichment. (a) Schematic of porous polymer monolith in a channel funtionalized with synthetic cDNA capture probes to selectively capture target RNA or DNA sequences. (b) Example experimental spatiotemporal diagram showing approach of ITP zone (diagonal yellow stripe on the right) and capture of target near edge (vertical line) of functionalized porous polymer monolith.



Figure 2: Experimentally measured spatiotemporal plots for ITP-affinity chromatography purification and extraction of rare target DNA from 13,000-fold more abundant endogenous blood DNA from 13 ┬Ál whole blood sample. Plot shows target and endogenous DNA migrating from free solution into the porous polymer. Endogenous DNA (from lysed blood cells, visualized with SYBR Green I - green color) is not captured by the immobilized probe on the custom, functionalized porous monolith and continues to migrate along the channel. The Cy5-labeled, low-abundance target DNA (red color) of the correct sequence is selectively captured. The red vertical streak in time vs. distance plot shows the capture of the target sequence. This 13,000-fold targeted enrichment by sequence takes less than 5 min and less than 1 mm length of column (Shkolnikov and Santiago, 2014).



Reference

Shkolnikov, V. and Santiago, J. G., "Coupling Isotachophoresis with Affinity Chromatography for Rapid and Selective Purification with High Column Utilization Part I: Theory.", Analytical Chemistry, 2014.(click here for pdf)

Shkolnikov, V. and Santiago, J. G., "Coupling Isotachophoresis with Affinity Chromatography for Rapid and Selective Purification with High Column Utilization Part II: Experimental Study.",Analytical Chemistry, 2014.(click here for pdf)

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