INTRODUCTION

The Purigen Ionic® Purification System uses isotachophoresis (ITP) to extract, concentrate and purify nucleic acid from biological samples. ITP is a gentle, microfluidic, process that separates nucleic acid from impurities using electrophoretic mobility.

To evaluate the performance of the Ionic Purification System for extraction of nucleic acid from limited or low input samples, we purified DNA from a set of FFPE tissue blocks and cultured cells at counts of 10,000 cells per sample or less. Replicates of each sample were purified and extracted using a commercially available column-based purification kit for comparison. We assessed the quantity and quality of the purified DNA with the Qubit dsDNA High Sensitivity Assay and Qiagen MRef multicopy reference assay.

ISOTACHOPHORESIS

Using a cutting-edge technology based on isotachophoresis, the Ionic system separates nucleic acids freely in solution, without binding to or stripping nucleic acid from physical surfaces. Taking as little as 5 minutes of hands-on time per sample to purify nucleic acid from a range of sample types including cells and FFPE tissue, the Ionic System is a highly efficient platform that produces more nucleic acid of higher purity, consistently and reliably.

RESULTS – Cultured Cell Samples

Improved yield from as few as 10 cells per sample

Cells from COLO320, GM24385, and H2228 cell lines were cultured and allocated to individual sample tubes in the amounts of 100,000, 10,000, 1000, 100, and 10 cells with a replicate tube at each cell amount. DNA was purified from the replicate samples using the Ionic system and a market-leading column-based kit. The DNA concentration and yield from each sample was determined using the Qiagen Multicopy Reference Assay and a standard control. Samples purified using the Ionic system had 1.5 – 2x higher yields than those from the column-based kit.

In the table below, libraries were generated for each sample described in the figure above following the protocol for the AmpliSeq for Illumina Comprehensive Cancer Panel and sequenced on the Illumina MiSeq sequencer. Standard sequencing metrics show comparable results, with the Ionic System samples showing a slightly improved Fold-80 score, indicating an improved coverage uniformity.

RESULTS – FFPE Tissue Samples

Improved yield from a single 10 μM thick FFPE tissue section

Summary of properties for replicate sections from 8 FFPE tissue blocks purified by the Ionic system and a column-based kit for hybrid-capture based library preparation. Purified and sheared amounts were quantified by Qubit 1X dsDNA High Sensitivity Assay. PCR cycles were performed prior to hybridization capture according to SureSelect XT HS protocol recommendations. High-quality human reference Coriell DNA was used as a “gold standard” control.

Comparison of the percentage of PCR duplicates reported for sequenced libraries from the 16 samples described in Table 2 to the amount of input DNA used for each library and the recommend PCR cycles for each amount. Libraries from extracts purified using the Ionic system are represented with blue dots. Libraries from extracts purified using a column-based kit are represented with orange dots. Shading indicates the recommended number of PCR cycles with red, blue and green representing 14, 12, and 11 respectively.

SUMMARY

• • Higher yields of nucleic acid from a single FFPE tissue section or as few as 10 cells
• • Higher yields and quality results in better coverage uniformity and fewer PCR duplicates in downstream NGS
• • Isotachophoresis enables a simple, automated workflow for nucleic acid purification