ArticleNucleic acids research2015
Accurate multiplexing and filtering for high-throughput amplicon-sequencing.
Article in Nucleic acids research, 2015. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 90 papers, 1 of them a synthesis that pooled it.
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Who cites it
90 citing papers in PubMed, 1 synthesis or guideline pooled it.
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- A ribodepletion and tagging protocol to multiplex samples for RNA-seq based virus detection: application to the cassava virome.Virology journal · 2025Article
- Subterranean morphology underpins the degree of mycoheterotrophy, mycorrhizal associations, and plant vigor in a green orchid Oreorchis patens.The Plant journal : for cell and molecular biology · 2025Article
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- Plants, pollinators and their interactions under global ecological change: The role of pollen DNA metabarcoding.Molecular ecology · 2023Article
- Amplicon Sequencing Minimal Information (ASqMI): Quality and Reporting Guidelines for Actionable Calls in Biodefense Applications.Journal of AOAC International · 2023Article
- Ecological drivers of fine-scale distribution of arbuscular mycorrhizal fungi in a semiarid Mediterranean scrubland.Annals of botany · 2023Article
- Nanopore sequencing of PCR products enables multicopy gene family reconstruction.Computational and structural biotechnology journal · 2023Article
30 more citing papers are in PubMed but not listed here.
Corrections and comments
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Authors and funding
3 authors.
Funding
No grant is acknowledged in the PubMed record.
Abstract
Tagging amplicons with tag sequences appended to PCR primers allow the multiplexing of numerous samples for high-throughput sequencing (HTS). This approach is routinely used in HTS-based diversity analyses, especially in microbial ecology and biomedical diagnostics. However, amplicon library preparation is subject to pervasive sample sequence cross-contaminations as a result of tag switching events referred to as mistagging. Here, we sequenced seven amplicon libraries prepared using various multiplexing designs in order to measure the magnitude of this phenomenon and its impact on diversity analyses. Up to 28.2% of the unique sequences correspond to undetectable (critical) mistags in single- or saturated double-tagging libraries. We show the advantage of multiplexing samples following Latin Square Designs in order to optimize the detection of mistags and maximize the information on their distribution across samples. We use this information in designs incorporating PCR replicates to filter the critical mistags and to recover the exact composition of mock community samples. Being parameter-free and data-driven, our approach can provide more accurate and reproducible HTS data sets, improving the reliability of their interpretations.
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