Double indexing overcomes inaccuracies in multiplex sequencing on the Illumina platform

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 t...

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Bibliographic Details
Published in:Nucleic acids research Vol. 40; no. 1; p. e3
Main Authors: Kircher, Martin, Sawyer, Susanna, Meyer, Matthias
Format: Journal Article
Language:English
Published: England Oxford University Press 01.01.2012
Subjects:
Gene Library
High-Throughput Nucleotide Sequencing - methods
Methods Online
Reproducibility of Results
Sequence Analysis, DNA - methods
ISSN:0305-1048, 1362-4962, 1362-4962
Online Access:Get full text
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Summary: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 ~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.
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ISSN:0305-1048
1362-4962
1362-4962
DOI:10.1093/nar/gkr771