ArticleNucleic acids research2015
A new method to prevent carry-over contaminations in two-step PCR NGS library preparations.
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 25 papers.
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Who cites it
25 citing papers in PubMed.
- Protocol for optimized methylation context-sensitive enzyme ddRAD library preparation from partially degraded genomic plant DNA.STAR protocols · 2026Article
- Heavy Metals, Gut Microbiota, and Biochemical Markers: Unraveling the Complexities of Obesity.MicrobiologyOpen · 2025Article
- Blood microbiome signatures in systemic diseases: current insights, methodological pitfalls, and future horizons.Frontiers in cellular and infection microbiology · 2025Review
- T-cell receptors identified by a personalized antigen-agnostic screening approach target shared neoantigen KRAS Q61H.Frontiers in immunology · 2025Article
- A real-world multi-center RNA-seq benchmarking study using the Quartet and MAQC reference materials.Nature communications · 2024Article
- Microbiome confounders and quantitative profiling challenge predicted microbial targets in colorectal cancer development.Nature medicine · 2024Article
- Article
- Carryover Contamination-Controlled Amplicon Sequencing Workflow for Accurate Qualitative and Quantitative Detection of Pathogens: a Case Study on SARS-CoV-2.Microbiology spectrum · 2023Article
- A modified protocol with less clean-up steps increased efficiency and product yield of sequencing library preparation.3 Biotech · 2022Article
- Adaptive Immune Receptor Repertoire (AIRR) Community Guide to Repertoire Analysis.Methods in molecular biology (Clifton, N.J.) · 2022Article
- Bacteria in the amniotic fluid without inflammation: early colonization vs. contamination.Journal of perinatal medicine · 2021Article
- Toward Accurate and Robust Environmental Surveillance Using Metagenomics.Frontiers in genetics · 2021Article
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- Evidence for a role of RUNX1 as recombinase cofactor for TCRβ rearrangements and pathological deletions in ETV6-RUNX1 ALL.Scientific reports · 2020Article
- Technical Validation of a Hepatitis C Virus Whole Genome Sequencing Assay for Detection of Genotype and Antiviral Resistance in the Clinical Pathway.Frontiers in microbiology · 2020Article
- A core phyllosphere microbiome exists across distant populations of a tree species indigenous to New Zealand.PloS one · 2020Article
- Proof of concept: used malaria rapid diagnostic tests applied for parallel sequencing for surveillance of molecular markers of anti-malarial resistance in Bissau, Guinea-Bissau during 2014-2017.Malaria journal · 2019Article
- PacBio amplicon sequencing for metabarcoding of mixed DNA samples from lichen herbarium specimens.MycoKeys · 2019Article
- Sample tracking in microbiome community profiling assays using synthetic 16S rRNA gene spike-in controls.Scientific reports · 2018Article
- Comparative analysis of murine T-cell receptor repertoires.Immunology · 2018Review
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6 authors.
Funding
No grant is acknowledged in the PubMed record.
Abstract
Two-step PCR procedures are an efficient and well established way to generate amplicon libraries for NGS sequencing. However, there is a high risk of cross-contamination by carry-over of amplicons from first to second amplification rounds, potentially leading to severe misinterpretation of results. Here we describe a new method able to prevent and/or to identify carry-over contaminations by introducing the K-box, a series of three synergistically acting short sequence elements. Our K-boxes are composed of (i) K1 sequences for suppression of contaminations, (ii) K2 sequences for detection of possible residual contaminations and (iii) S sequences acting as separators to avoid amplification bias. In order to demonstrate the effectiveness of our method we analyzed two-step PCR NGS libraries derived from a multiplex PCR system for detection of T-cell receptor beta gene rearrangements. We used this system since it is of high clinical relevance and may be affected by very low amounts of contaminations. Spike-in contaminations are effectively blocked by the K-box even at high rates as demonstrated by ultra-deep sequencing of the amplicons. Thus, we recommend implementation of the K-box in two-step PCR-based NGS systems for research and diagnostic applications demanding high sensitivity and accuracy.
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