A new workflow for whole-genome sequencing of single human cells.

Autor: Binder V; Department of Pediatric Oncology, Hematology and Clinical Immunology, Medical Faculty, University of Duesseldorf, Duesseldorf, Germany., Bartenhagen C, Okpanyi V, Gombert M, Moehlendick B, Behrens B, Klein HU, Rieder H, Ida Krell PF, Dugas M, Stoecklein NH, Borkhardt A
Jazyk: angličtina
Zdroj: Human mutation [Hum Mutat] 2014 Oct; Vol. 35 (10), pp. 1260-70. Date of Electronic Publication: 2014 Aug 18.
DOI: 10.1002/humu.22625
Abstrakt: 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.
(© 2014 WILEY PERIODICALS, INC.)
Databáze: MEDLINE
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