Double indexing overcomes inaccuracies in multiplex sequencing on the Illumina platform

By Martin Kircher, S. Sawyer, M. Meyer. Nucleic Acids Research, 2011

1,091 citations recorded by the source corpus.

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.

Subfields: Cancer Research, Genetics, Molecular Biology

View Paper: https://www.semanticscholar.org/paper/d1281ad32607fb0ade628ba1b32c8ae7f06ad24e

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Citations in the corpus

Not All Sequence Tags Are Created Equal: Designing and Validating Sequence Identification Tags Robust to Indels (2012, background): “These tag sets may also be used in a variety of configurations to improve the accuracy of tracking and assigning reads to samples [12] and enable concurrent sequencing of hundreds of thousands of samples.”
Metagenomics: Tools and Insights for Analyzing Next-Generation Sequencing Data Derived from Biodiversity Studies (2015, methodology): “34 In contrast, when analyzing 16S metagenomics data, this technology obviates the need for time-consuming noise removal algorithms required for pyrosequencing and makes analysis less error-prone.”
From the field to the laboratory: Controlling DNA contamination in human ancient DNA research in the high-throughput sequencing era (2017, neutral): “…libraries allows the detection of cross-contamination during library ampli ﬁ cation and HTS sequencing (Green et al. 2009; Knapp and Hofreiter 2010; Kircher, Sawyer, and Meyer 2012; Knapp, Stiller, and Meyer 2012), all steps prior to the ligation of barcoded adapters to the DNA templates are…”
Adapterama I: universal stubs and primers for 384 unique dual-indexed or 147,456 combinatorially-indexed Illumina libraries (iTru & iNext) (2019, methodology): “Dual indexing on the Illumina platform means that indexes can be used combinatorially ( Kircher, Sawyer & Meyer, 2012 ; Faircloth & Glenn, 2012 ).”
MiFish, a set of universal PCR primers for metabarcoding environmental DNA from fishes: detection of more than 230 subtropical marine species (2015, neutral): “[48] demonstrated such misassignment on the Illumina sequencing platform and the Illumina document (pub.”
Complete mitochondrial genome sequence of a Middle Pleistocene cave bear reconstructed from ultrashort DNA fragments (2013, methodology): “Libraries were amplified by PCR using AccuPrime Pfx DNA polymerase (Life Technologies) (43) following a double-indexing scheme described elsewhere (44).”
Analysis of the Human Tissue-specific Expression by Genome-wide Integration of Transcriptomics and Antibody-based Proteomics* (2013, background): “Barcode “Leakage”—As has been previously observed (15) multiplexing of samples on a single lane on the Illumina platform can sometimes lead to misidentification of barcodes, leading to what looks like a cross-contamination between samples.”