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A New Workflow for Whole‐Genome Sequencing of Single Human Cells
Author(s) -
Binder Vera,
Bartenhagen Christoph,
Okpanyi Vera,
Gombert Michael,
Moehlendick Birte,
Behrens Bianca,
Klein HansUlrich,
Rieder Harald,
Ida Krell Pina Fanny,
Dugas Martin,
Stoecklein Nikolas Hendrik,
Borkhardt Arndt
Publication year - 2014
Publication title -
human mutation
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 162
eISSN - 1098-1004
pISSN - 1059-7794
DOI - 10.1002/humu.22625
Subject(s) - biology , genome , multiple displacement amplification , human genome , genetics , computational biology , personal genomics , single cell sequencing , loss of heterozygosity , whole genome sequencing , dna sequencing , genomics , exome sequencing , allele , gene , mutation , polymerase chain reaction , dna extraction
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.