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