title: Linear amplification for deep sequencing.
abstract: 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.
authors: Department of Molecular Biology, Faculty of Science, Nijmegen Center for Molecular Life Sciences, Radboud University, Nijmegen, The Netherlands.
authors: Wieteke A M
authors: WA
authors: Hoeijmakers
authors: Richárd
authors: R
authors: Bártfai
authors: Kees-Jan
authors: KJ
authors: Françoijs
authors: Hendrik G
authors: HG
authors: Stunnenberg
mesh_terms: D001482:Base Composition; D001483:Base Sequence; D004247:DNA / Q000737:chemistry; D017931:DNA Primers; D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D015723:Gene Library; D018503:Genome, Protozoan; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D009169:Mycobacterium tuberculosis / Q000235:genetics*; D010963:Plasmodium falciparum / Q000235:genetics*; D011401:Promoter Regions, Genetic; D017422:Sequence Analysis, DNA / Q000379:methods*; D014764:Viral Proteins / Q000235:genetics
chemical_list: D017931:DNA Primers; D014764:Viral Proteins; D004247:DNA; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases