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 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.

[1]  S. Pääbo,et al.  DNA damage promotes jumping between templates during enzymatic amplification. , 1990, The Journal of biological chemistry.

[2]  A. Meyerhans,et al.  DNA recombination during PCR. , 1990, Nucleic acids research.

[3]  S. Odelberg,et al.  Template-switching during DNA synthesis by Thermus aquaticus DNA polymerase I. , 1995, Nucleic acids research.

[4]  J. Shendure,et al.  Materials and Methods Som Text Figs. S1 and S2 Tables S1 to S4 References Accurate Multiplex Polony Sequencing of an Evolved Bacterial Genome , 2022 .

[5]  James R. Knight,et al.  Genome sequencing in microfabricated high-density picolitre reactors , 2005, Nature.

[6]  N. Rohland,et al.  Comparison and optimization of ancient DNA extraction. , 2007, BioTechniques.

[7]  E. Birney,et al.  Patterns of somatic mutation in human cancer genomes , 2007, Nature.

[8]  U. Stenzel,et al.  Targeted high-throughput sequencing of tagged nucleic acid samples , 2007, Nucleic acids research.

[9]  P. Mitra,et al.  Alta-Cyclic: a self-optimizing base caller for next-generation sequencing , 2008, Nature Methods.

[10]  U. Stenzel,et al.  Parallel tagged sequencing on the 454 platform , 2008, Nature Protocols.

[11]  Antony V. Cox,et al.  Identification of somatically acquired rearrangements in cancer using genome-wide massively parallel paired-end sequencing , 2008, Nature Genetics.

[12]  S. Quake,et al.  Single-Molecule DNA Sequencing of a Viral Genome , 2008, Science.

[13]  Nancy F. Hansen,et al.  Accurate Whole Human Genome Sequencing using Reversible Terminator Chemistry , 2008, Nature.

[14]  E. Birney,et al.  Patterns of somatic mutation in human cancer genomes , 2007, Nature.

[15]  Matthew J. Huentelman,et al.  IDENTIFICATION OF GENETIC VARIANTS USING BARCODED MULTIPLEXED SEQUENCING , 2008, Nature Methods.

[16]  Jonas Korlach,et al.  Selective aluminum passivation for targeted immobilization of single DNA polymerase molecules in zero-mode waveguide nanostructures , 2008, Proceedings of the National Academy of Sciences.

[17]  A. Børresen-Dale,et al.  COMPLEX LANDSCAPES OF SOMATIC REARRANGEMENT IN HUMAN BREAST CANCER GENOMES , 2009, Nature.

[18]  U. Stenzel,et al.  Direct multiplex sequencing (DMPS)--a novel method for targeted high-throughput sequencing of ancient and highly degraded DNA. , 2009, Genome research.

[19]  Daniel J. G. Lahr,et al.  Reducing the impact of PCR-mediated recombination in molecular evolution and environmental studies using a new-generation high-fidelity DNA polymerase. , 2009, BioTechniques.

[20]  Martin Kircher,et al.  Improved base calling for the Illumina Genome Analyzer using machine learning strategies , 2009, Genome Biology.

[21]  Richard Durbin,et al.  Sequence analysis Fast and accurate short read alignment with Burrows – Wheeler transform , 2009 .

[22]  Adrian W. Briggs,et al.  The Neandertal genome and ancient DNA authenticity , 2009, The EMBO journal.

[23]  Anne E Carpenter,et al.  Visualization of image data from cells to organisms , 2010, Nature Methods.

[24]  Philip L. F. Johnson,et al.  Genetic history of an archaic hominin group from Denisova Cave in Siberia , 2010, Nature.

[25]  Mark Stoneking,et al.  Detecting heteroplasmy from high-throughput sequencing of complete human mitochondrial DNA genomes. , 2010, American journal of human genetics.

[26]  Matthias Meyer,et al.  Illumina sequencing library preparation for highly multiplexed target capture and sequencing. , 2010, Cold Spring Harbor protocols.

[27]  D. Dressman,et al.  Heteroplasmic mitochondrial DNA mutations in normal and tumor cells , 2010, Nature.

[28]  Philip L. F. Johnson,et al.  A Draft Sequence of the Neandertal Genome , 2010, Science.

[29]  Andrew Menzies,et al.  The patterns and dynamics of genomic instability in metastatic pancreatic cancer , 2010, Nature.

[30]  S. Pääbo,et al.  Multiplexed DNA Sequence Capture of Mitochondrial Genomes Using PCR Products , 2010, PloS one.

[31]  Martin Kircher,et al.  High‐throughput DNA sequencing – concepts and limitations , 2010, BioEssays : news and reviews in molecular, cellular and developmental biology.

[32]  Adrian W. Briggs,et al.  Removal of deaminated cytosines and detection of in vivo methylation in ancient DNA , 2009, Nucleic acids research.