Double indexing overcomes inaccuracies in multiplex sequencing on the Illumina platform

被引：793

作者：

Kircher, Martin ^{[1
]}

Sawyer, Susanna ^{[1
]}

Meyer, Matthias ^{[1
]}

机构：

[1] Max Planck Inst Evolutionary Anthropol, Dept Evolutionary Genet, D-04103 Leipzig, Germany

来源：

NUCLEIC ACIDS RESEARCH | 2012年 / 40卷 / 01期

关键词：

DNA-POLYMERASE; NEANDERTHAL GENOME; CANCER GENOMES; IDENTIFICATION; RECOMBINATION; PATTERNS; PCR;

D O I：

10.1093/nar/gkr771

中图分类号：

Q5 [生物化学]; Q7 [分子生物学];

学科分类号：

070307 [化学生物学]; 071010 [生物化学与分子生物学];

摘要：

Due to the increasing throughput of current DNA sequencing instruments, sample multiplexing is necessary for making economical use of available sequencing capacities. A widely used multiplexing strategy for the Illumina Genome Analyzer utilizes sample-specific indexes, which are embedded in one of the library adapters. However, this and similar multiplex approaches come with a risk of sample misidentification. By introducing indexes into both library adapters (double indexing), we have developed a method that reveals the rate of sample misidentification within current multiplex sequencing experiments. With similar to 0.3% these rates are orders of magnitude higher than expected and may severely confound applications in cancer genomics and other fields requiring accurate detection of rare variants. We identified the occurrence of mixed clusters on the flow as the predominant source of error. The accuracy of sample identification is further impaired if indexed oligonucleotides are cross-contaminated or if indexed libraries are amplified in bulk. Double-indexing eliminates these problems and increases both the scope and accuracy of multiplex sequencing on the Illumina platform.

引用

页数：8

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