High-throughput single-cell DNA sequencing of acute myeloid leukemia tumors with droplet microfluidics

Abstract

To enable the characterization of genetic heterogeneity in tumor cell populations, we developed a novel microfluidic approach that barcodes amplified genomic DNA from thousands of individual cancer cells confined to droplets. The barcodes are then used to reassemble the genetic profiles of cells from next-generation sequencing data. By using this approach, we sequenced longitudinally collected acute myeloid leukemia (AML) tumor populations from two patients and genotyped up to 62 disease relevant loci across more than 16,000 individual cells. Targeted single-cell sequencing was able to sensitively identify cells harboring pathogenic mutations during complete remission and uncovered complex clonal evolution within AML tumors that was not observable with bulk sequencing. We anticipate that this approach will make feasible the routine analysis of AML heterogeneity, leading to improved stratification and therapy selection for the disease.

Genomic testing in myeloid malignancy

“…In particular, it is likely to become increasingly important to determine which molecular alterations are occurring within the same disease clone—methods that rely on using both cytogenetic and sequencing‐based assays will not be able to address this challenge as effectively as comprehensive sequencing‐based methods. Single‐cell methods for assaying either DNA or RNA are currently being used to show the heterogeneity within patient samples . At present, these methods are too time‐consuming and costly for routine clinical application.…”

Microfluidics for Production of Particles: Mechanism, Methodology, and Applications

“…Copyright 2016, The Authors published by Springer Nature Limited, under a Creative Common Attribution 4.0 International License. (b) Reproduced with permission . Copyright 2018, The Authors published by Cold Spring Harbor Laboratory Press, under a Creative Common Attribution 4.0 International License.…”

Microfluidic Single‐Cell Omics Analysis

“…Single‐cell genome analysis illustrates the heterogeneity in inheritance and aberrance information at single‐cell level. Initially, targeted genes are analyzed by means of fluorescence or sequencing technologies . Kumaresan et al encapsulated single cell, primer‐coated microbeads and uniform PCR mix within a single droplet.…”

“…Therein, cell barcode was proposed by labeling each cell so as to pool all of them for library construction, with the capability to identify individual cells from sequencing data based on the unique cell barcode. After PCR amplification, the barcode information was delivered from hydrogel beads to products, which could be read by sequencing technology to identify targeted genes from thousands of single cancer cells ( Figure A) . Since limited number of genes are not enough for comprehensively understanding cell trajectory, single‐cell whole‐genome sequencing is highly urgent.…”

“…A) Schematic illumination of protease‐based droplet workflow for single‐cell genomic DNA amplification and barcoding. Reproduced with permission . Copyright 2018, Cold Spring Harbor Laboratory Press.…”