Single cell on-chip whole genome amplification via micropillar arrays for reduced amplification bias.

Harvey C. Tian,Harold G. Craighead,Jaime J. Benitez,Victor M. Ugaz

doi:10.1371/journal.pone.0191520

Abstract

Single cell whole genome amplification is susceptible to amplification biases that impact the accuracy of single cell sequencing data. To address this, we have developed a microfluidic device for the isolation and purification of genomic DNA from individual cells. The device uses a micropillar array to physically capture single cells and its chromosomal DNA upon extraction. The extracted DNA is immobilized within the micropillar array in a way that allows isothermal amplification. In this system, whole genome amplification of the single cell is carried out under a continual fluid flow within the microfluidic channel. We have demonstrated the process for amplification of individual human cancer cell genomes from the HeLa cell line. By sampling select gene loci along the human genome and performing whole exome sequencing, we demonstrate improved genome coverage and reduced amplification bias compared to amplification of single cells deposited in wells by fluorescence activated cell sorting.

Highlights

Single cell analysis has become increasingly important for understanding and diagnosing disease.[1,2,3,4,5,6] For instance, cellular level aberrations have been shown to play critical roles in tumor heterogeneity, cancer metastasis, drug resistance, and cell fate.[7,8,9,10,11,12] Investigating these aberrations and differentiating between cell types within a population may give rise to improved treatments, single cell handling and analysis remains difficult
Amplification bias stemming from chimera formation and non-linear enrichment remain an issue in single cell whole genome amplification (WGA) through multiple displacement amplification (MDA).[16,17,18]
We have described a simple, valveless, and scalable micropillar-based microfluidic device capable of on-chip single cell processing and WGA

Summary

Introduction

Single cell analysis has become increasingly important for understanding and diagnosing disease.[1,2,3,4,5,6] For instance, cellular level aberrations have been shown to play critical roles in tumor heterogeneity, cancer metastasis, drug resistance, and cell fate.[7,8,9,10,11,12] Investigating these aberrations and differentiating between cell types within a population may give rise to improved treatments, single cell handling and analysis remains difficult. Because there are only picogram quantities of DNA within a single cell, existing due to sensitivity limits, existing workflows cannot sequence single cell genomes directly without amplification. Among the most widely used single cell WGA amplification techniques is multiple displacement amplification (MDA), which relies on a combination of random sequence primers and the strand-displacement properties of the Phi polymerase to isothermally amplify DNA.[14,15] amplification bias stemming from chimera formation and non-linear enrichment remain an issue in single cell WGA through MDA.[16,17,18] This bias can be averaged out when analyzing monodisperse multi-cell population samples because of the multiple copies of each gene from the many cells.

Methods

Results

Conclusion