Developing a New Nucleic Acid Extraction Technology to Enable High Quality DNA Sequencing for Formalin Fixed Paraffin Embedded Tissue Samples

Patient tissue has been collected and preserved as formalin fixed paraffin embedded tissue
(FFPET) samples for over 100 years and provides a way to reliably store samples for decades.
To the present day, the collection of FFPET remains a mainstay technique to support both
diagnostic and research applications. As a result, millions of these tissue samples have been
stored and remain available to the research community. At the same time, next generation DNA
sequencing (NGS) has quickly become an essential technique for the analysis of FFPET but
challenges remain in obtaining quality data from precious samples that are likely to be irreplaceable.
One problem with the reliable use of FFPET samples is the variability of yield, quality, and length
of purified DNA and RNA resulting from the multi-step process of purification from the tissue
sample, which includes 1) cell lysis and removal of paraffin; 2) treatment with protease; 3) reverse
cross linking of the nucleic acids; and finally, 4) purifying the nucleic acids from sample buffer.
Purification systems that rely on silicon or magnetic beads can often produce variable sample
quality: the paraffin or cell debris from FFPET can clog a column or prevent the nucleic acids from
binding the solid phase purification material.
To help improve the yield and quality of purified nucleic acids (NAs) from challenging samples,
Vellum Biotechnology is developing epitachophoresis (ETP) technology to purify NAs employing
a circular well and a liquid/liquid interface. Preliminary development and studies of early prototype
ETP systems conducted at Roche Molecular Systems in Pleasanton, California and Vellum,
supervised by our PI on this grant, Yann Astier, PhD, demonstrated favorable properties vs.
commercially available systems from ProMega and Qiagen including: 1) superior yields of nucleic
acids; 2) improved range of lengths of NAs; 3) shorter purification times; 4) label-free monitoring
of the purification process; 5) comparable quality of nucleic acids as evaluated by a qPCR LINE
assay score; and, importantly 6) improved sequencing quality. We propose to build on these
studies to create a more robust, scalable ETP system with the following Specific Aims:
Aim 1: Screen and select sample well components consisting of at least five outer well, 5
inner well barrier, three bottom membrane, and three electrode compositions to improve
system performance and manufacturability relative to a Promega and Qiagen commercial
product.
Aim 2: Benchmark the quality of extracted NA from FFPET samples for at least five
candidate well device prototypes integrated into a 12 well prototype consumable.

Grant Number: 1R44HG014445-01
NIH Institute/Center: NIH
Principal Investigator: Yann Astier