dc.contributor.author
Seitz, Volkhard
dc.contributor.author
Schaper, Sigrid
dc.contributor.author
Dröge, Anja
dc.contributor.author
Lenze, Dido
dc.contributor.author
Hummel, Michael
dc.contributor.author
Hennig, Steffen
dc.date.accessioned
2018-06-08T04:15:03Z
dc.date.available
2016-01-29T09:18:06.432Z
dc.identifier.uri
https://refubium.fu-berlin.de/handle/fub188/16894
dc.identifier.uri
http://dx.doi.org/10.17169/refubium-21075
dc.description.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.
en
dc.rights.uri
http://creativecommons.org/licenses/by-nc/4.0/
dc.subject.ddc
600 Technik, Medizin, angewandte Wissenschaften::610 Medizin und Gesundheit
dc.title
A new method to prevent carry-over contaminations in two-step PCR NGS library
preparations
dc.type
Wissenschaftlicher Artikel
dcterms.bibliographicCitation
Nucleic Acids Research. - 43 (2015), 20, Artikel Nr. e135
dcterms.bibliographicCitation.doi
10.1093/nar/gkv694
dcterms.bibliographicCitation.url
http://nar.oxfordjournals.org/content/43/20/e135
refubium.affiliation
Charité - Universitätsmedizin Berlin
de
refubium.mycore.fudocsId
FUDOCS_document_000000023824
refubium.note.author
Der Artikel wurde in einer Open-Access-Zeitschrift publiziert.
refubium.resourceType.isindependentpub
no
refubium.mycore.derivateId
FUDOCS_derivate_000000005929
dcterms.accessRights.openaire
open access