TY - JOUR
T1 - Linear amplification for deep sequencing
AU - Hoeijmakers, Wieteke A.M.
AU - Bártfai, Richárd
AU - Françoijs, Kees Jan
AU - Stunnenberg, Hendrik G.
N1 - Funding Information:
acknowleDGMents The development of this method has been financially supported by the Netherlands Organisation for Scientific Research (ZonMw/NGI Horizon 93511023; NWO-Toptalent 021.001.011) and by the European Commission (EVIMalaR; ATLAS EU-FP7_221952). We thank E. Janssen-Megens for technical assistance, advice and operation of the GAII machine and D. van Soolingen and A. Schurch for providing Mycobacterium tuberculosis genomic DNA. Furthermore, we thank A. Salcedo-Amaya and A. Brinkman for useful discussions during the development of LADS and for critical reading of the manuscript, and we are grateful for the advice and help of all our colleagues in the molecular biology department.
PY - 2011/6
Y1 - 2011/6
N2 - Linear amplification for deep sequencing (LADS) is an amplification method that produces representative libraries for Illumina next-generation sequencing within 2 d. The method relies on attaching two different sequencing adapters to blunt-end repaired and A-tailed DNA fragments, wherein one of the adapters is extended with the sequence for the T7 RNA polymerase promoter. Ligated and size-selected DNA fragments are transcribed in vitro with high RNA yields. Subsequent cDNA synthesis is initiated from a primer complementary to the first adapter, ensuring that the library will only contain full-length fragments with two distinct adapters. Contrary to the severely biased representation of AT- or GC-rich fragments in standard PCR-amplified libraries, the sequence coverage in T7-amplified libraries is indistinguishable from that of nonamplified libraries. Moreover, in contrast to amplification-free methods, LADS can generate sequencing libraries from a few nanograms of DNA, which is essential for all applications in which the starting material is limited.
AB - Linear amplification for deep sequencing (LADS) is an amplification method that produces representative libraries for Illumina next-generation sequencing within 2 d. The method relies on attaching two different sequencing adapters to blunt-end repaired and A-tailed DNA fragments, wherein one of the adapters is extended with the sequence for the T7 RNA polymerase promoter. Ligated and size-selected DNA fragments are transcribed in vitro with high RNA yields. Subsequent cDNA synthesis is initiated from a primer complementary to the first adapter, ensuring that the library will only contain full-length fragments with two distinct adapters. Contrary to the severely biased representation of AT- or GC-rich fragments in standard PCR-amplified libraries, the sequence coverage in T7-amplified libraries is indistinguishable from that of nonamplified libraries. Moreover, in contrast to amplification-free methods, LADS can generate sequencing libraries from a few nanograms of DNA, which is essential for all applications in which the starting material is limited.
UR - https://www.scopus.com/pages/publications/79959882452
U2 - 10.1038/nprot.2011.345
DO - 10.1038/nprot.2011.345
M3 - Article
C2 - 21720315
AN - SCOPUS:79959882452
SN - 1754-2189
VL - 6
SP - 1026
EP - 1036
JO - Nature protocols
JF - Nature protocols
IS - 7
ER -