title: A new workflow for whole-genome sequencing of single human cells.
abstract: Unbiased amplification of the whole-genome amplification (WGA) of single cells is crucial to study cancer evolution and genetic heterogeneity, but is challenging due to the high complexity of the human genome. Here, we present a new workflow combining an efficient adapter-linker PCR-based WGA method with second-generation sequencing. This approach allows comparison of single cells at base pair resolution. Amplification recovered up to 74% of the human genome. Copy-number variants and loss of heterozygosity detected in single cell genomes showed concordance of up to 99% to pooled genomic DNA. Allele frequencies of mutations could be determined accurately due to an allele dropout rate of only 2%, clearly demonstrating the low bias of our PCR-based WGA approach. Sequencing with paired-end reads allowed genome-wide analysis of structural variants. By direct comparison to other WGA methods, we further endorse its suitability to analyze genetic heterogeneity.
authors: Department of Pediatric Oncology, Hematology and Clinical Immunology, Medical Faculty, University of Duesseldorf, Duesseldorf, Germany.
authors: Vera
authors: V
authors: Binder
authors: Christoph
authors: C
authors: Bartenhagen
authors: Vera
authors: V
authors: Okpanyi
authors: Michael
authors: M
authors: Gombert
authors: Birte
authors: B
authors: Moehlendick
authors: Bianca
authors: B
authors: Behrens
authors: Hans-Ulrich
authors: HU
authors: Klein
authors: Harald
authors: H
authors: Rieder
authors: Pina Fanny
authors: PF
authors: Ida Krell
authors: Martin
authors: M
authors: Dugas
authors: Nikolas Hendrik
authors: NH
authors: Stoecklein
authors: Arndt
authors: A
authors: Borkhardt
mesh_terms: D002869:Chromosome Aberrations; D056915:DNA Copy Number Variations; D005787:Gene Frequency; D018740:Genetic Heterogeneity; D015894:Genome, Human; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D019656:Loss of Heterozygosity; D016133:Polymerase Chain Reaction / Q000379:methods; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA / Q000379:methods*; D059010:Single-Cell Analysis / Q000379:methods*; D057188:Workflow
keywords: CNV; LOH; allele dropout rate; single cell; whole-genome amplification