A new method to prevent carry-over contaminations in two-step PCR NGS library preparations

Nucleic Acids Res. 2015 Nov 16;43(20):e135. doi: 10.1093/nar/gkv694. Epub 2015 Jul 7.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Line
  • Computational Biology
  • DNA Contamination*
  • Gene Library
  • High-Throughput Nucleotide Sequencing*
  • Humans
  • Polymerase Chain Reaction / methods*
  • Sequence Analysis, DNA*