Parallel Single Cancer Cell Whole Genome Amplification Using Button-Valve Assisted Mixing in Nanoliter Chambers

Yoonsun Yang,Leon W M M Terstappen,Séverine Le Gac,Hoon Suk Rho,Joost F Swennenhuis,Xin-Yuan Guan

doi:10.1371/journal.pone.0107958

Abstract

The heterogeneity of tumor cells and their alteration during the course of the disease urges the need for real time characterization of individual tumor cells to improve the assessment of treatment options. New generations of therapies are frequently associated with specific genetic alterations driving the need to determine the genetic makeup of tumor cells. Here, we present a microfluidic device for parallel single cell whole genome amplification (pscWGA) to obtain enough copies of a single cell genome to probe for the presence of treatment targets and the frequency of its occurrence among the tumor cells. Individual cells were first captured and loaded into eight parallel amplification units. Next, cells were lysed on a chip and their DNA amplified through successive introduction of dedicated reagents while mixing actively with the help of integrated button-valves. The reaction chamber volume for scWGA 23.85 nl, and starting from 6–7 pg DNA contained in a single cell, around 8 ng of DNA was obtained after WGA, representing over 1000-fold amplification. The amplified products from individual breast cancer cells were collected from the device to either directly investigate the amplification of specific genes by qPCR or for re-amplification of the DNA to obtain sufficient material for whole genome sequencing. Our pscWGA device provides sufficient DNA from individual cells for their genetic characterization, and will undoubtedly allow for automated sample preparation for single cancer cell genomic characterization.

Highlights

For the characterization of tumors, the expression of specific proteins or genes is usually provided as present or absent, for instance, ER+ or ER2, HER2+ or HER, and EGFR mutation present or absent
Patients whose cancer cells have an amplification of the ERBB2 (Her2) gene are most likely to benefit from Her2 targeting drugs such as Trastuzumab
We presented a microfluidic device for the parallel processing of single cells to obtain sufficient amounts of DNA by whole genome amplification for downstream analysis

Summary

Introduction

For the characterization of tumors, the expression of specific proteins or genes is usually provided as present or absent, for instance, ER+ or ER2, HER2+ or HER-, and EGFR mutation present or absent. To investigate the genome of a single cell in an extensive and reliable way, the whole genome must be amplified while maintaining the original representation of the genes to perform downstream analysis such as whole genome sequencing [6,7,8], array comparative genome hybridization (aCGH) [9,10], or realtime quantitative PCR (RT-qPCR) [11,12]. At this time all these techniques require tens of nano-grams to a few micro-grams of material of the whole genome. This advantage has been highlighted for WGA of single bacterial cells using reaction volumes of 60 nl resulting in a lower background and higher coverage with less amplification bias [27]

Methods

Results

Conclusion