title: A new method to prevent carry-over contaminations in two-step PCR NGS library preparations.
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.
authors.affiliation: Institute of Pathology, Charité-University Medicine Berlin, Campus Benjamin Franklin, Berlin, Germany HS Diagnomics GmbH, Berlin, Germany volkhard.seitz@charite.de.
authors.forename: Volkhard
authors.initials: V
authors.lastname: Seitz
authors.affiliation: HS Diagnomics GmbH, Berlin, Germany.
authors.forename: Sigrid
authors.initials: S
authors.lastname: Schaper
authors.affiliation: HS Diagnomics GmbH, Berlin, Germany.
authors.forename: Anja
authors.initials: A
authors.lastname: Dröge
authors.affiliation: Institute of Pathology, Charité-University Medicine Berlin, Campus Benjamin Franklin, Berlin, Germany.
authors.forename: Dido
authors.initials: D
authors.lastname: Lenze
authors.affiliation: Institute of Pathology, Charité-University Medicine Berlin, Campus Benjamin Franklin, Berlin, Germany michael.hummel@charite.de.
authors.forename: Michael
authors.initials: M
authors.lastname: Hummel
authors.affiliation: HS Diagnomics GmbH, Berlin, Germany hennig@hsdiagnomics.de.
authors.forename: Steffen
authors.initials: S
authors.lastname: Hennig
mesh_terms: D002460:Cell Line; D019295:Computational Biology; D059018:DNA Contamination; D015723:Gene Library; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA