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• W1667734330 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." @default.
• W1667734330 created "2016-06-24" @default.
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• W1667734330 date "2015-07-07" @default.
• W1667734330 modified "2023-10-14" @default.
• W1667734330 title "A new method to prevent carry-over contaminations in two-step PCR NGS library preparations" @default.
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• W1667734330 doi "https://doi.org/10.1093/nar/gkv694" @default.
• W1667734330 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/4787772" @default.
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