title: Capture and Amplification by Tailing and Switching (CATS). An ultrasensitive ligation-independent method for generation of DNA libraries for deep sequencing from picogram amounts of DNA and RNA.
abstract: Massive parallel sequencing (MPS) technologies have paved the way into new areas of research including individualized medicine. However, sequencing of trace amounts of DNA or RNA still remains a major challenge, especially for degraded nucleic acids like circulating DNA. This together with high cost and time requirements impedes many important applications of MPS in medicine and fundamental science. We have established a fast, cheap and highly efficient protocol called 'Capture and Amplification by Tailing and Switching' (CATS) to directly generate ready-to-sequence libraries for MPS from nanogram and picogram quantities of both DNA and RNA. Furthermore, those DNA libraries are strand-specific, can be prepared within 2-3 h and do not require preliminary sample amplification steps. To exemplify the capacity of the technique, we have generated and sequenced DNA libraries from hundred-picogram amounts of circulating nucleic acids isolated from human blood plasma, one nanogram of mRNA-enriched total RNA from cultured cells and few nanograms of bisulfite-converted DNA. The approach for DNA library preparation from minimal and fragmented input described here will find broad application in diverse research areas such as translational medicine including therapy monitoring, prediction, prognosis and early detection of various human disorders and will permit high-throughput DNA sequencing from previously inaccessible material such as minute forensic and archeological samples.
authors.affiliation: Molecular Epidemiology; German Cancer Research Center DKFZ; Heidelberg, Germany; Molecular Biology of Breast Cancer; University Women's Clinic; Heidelberg, Germany.
authors.forename: Andrey
authors.initials: A
authors.lastname: Turchinovich
authors.affiliation: Molecular Epidemiology; German Cancer Research Center DKFZ; Heidelberg, Germany; Molecular Biology of Breast Cancer; University Women's Clinic; Heidelberg, Germany.
authors.forename: Harald
authors.initials: H
authors.lastname: Surowy
authors.affiliation: Molecular Genetics; German Cancer Research Center DKFZ; Heidelberg, Germany.
authors.forename: Andrius
authors.initials: A
authors.lastname: Serva
authors.affiliation: Molecular Genetics; German Cancer Research Center DKFZ; Heidelberg, Germany.
authors.forename: Marc
authors.initials: M
authors.lastname: Zapatka
authors.affiliation: Molecular Genetics; German Cancer Research Center DKFZ; Heidelberg, Germany.
authors.forename: Peter
authors.initials: P
authors.lastname: Lichter
authors.affiliation: Molecular Epidemiology; German Cancer Research Center DKFZ; Heidelberg, Germany; Molecular Biology of Breast Cancer; University Women's Clinic; Heidelberg, Germany.
authors.forename: Barbara
authors.initials: B
authors.lastname: Burwinkel
mesh_terms: D000818:Animals; D017173:Caenorhabditis elegans / Q000235:genetics; D045744:Cell Line, Tumor; D004247:DNA / Q000097:blood / Q000235:genetics; D015723:Gene Library; D059014:High-Throughput Nucleotide Sequencing / Q000191:economics / Q000379:methods*; D006801:Humans; D012313:RNA / Q000097:blood / Q000235:genetics; D017422:Sequence Analysis, DNA / Q000191:economics / Q000379:methods*; D017423:Sequence Analysis, RNA / Q000191:economics / Q000379:methods*; D013997:Time Factors
chemical_list: D012313:RNA; D004247:DNA