title: Peregrine: A rapid and unbiased method to produce strand-specific RNA-Seq libraries from small quantities of starting material.
abstract: Use of second generation sequencing (SGS) technologies for transcriptional profiling (RNA-Seq) has revolutionized transcriptomics, enabling measurement of RNA abundances with unprecedented specificity and sensitivity and the discovery of novel RNA species. Preparation of RNA-Seq libraries requires conversion of the RNA starting material into cDNA flanked by platform-specific adaptor sequences. Each of the published methods and commercial kits currently available for RNA-Seq library preparation suffers from at least one major drawback, including long processing times, large starting material requirements, uneven coverage, loss of strand information and high cost. We report the development of a new RNA-Seq library preparation technique that produces representative, strand-specific RNA-Seq libraries from small amounts of starting material in a fast, simple and cost-effective manner. Additionally, we have developed a new quantitative PCR-based assay for precisely determining the number of PCR cycles to perform for optimal enrichment of the final library, a key step in all SGS library preparation workflows.
authors.affiliation: Biotechnology and Bioengineering, Sandia National Laboratories, Livermore, CA, USA.
authors.forename: Stanley A
authors.initials: SA
authors.lastname: Langevin
authors.forename: Zachary W
authors.initials: ZW
authors.lastname: Bent
authors.forename: Owen D
authors.initials: OD
authors.lastname: Solberg
authors.forename: Deanna J
authors.initials: DJ
authors.lastname: Curtis
authors.forename: Pamela D
authors.initials: PD
authors.lastname: Lane
authors.forename: Kelly P
authors.initials: KP
authors.lastname: Williams
authors.forename: Joseph S
authors.initials: JS
authors.lastname: Schoeniger
authors.forename: Anupama
authors.initials: A
authors.lastname: Sinha
authors.forename: Todd W
authors.initials: TW
authors.lastname: Lane
authors.forename: Steven S
authors.initials: SS
authors.lastname: Branda
mesh_terms: D001483:Base Sequence; D045744:Cell Line, Tumor; D019295:Computational Biology; D004926:Escherichia coli / Q000235:genetics*; D020869:Gene Expression Profiling / Q000379:methods*; D015723:Gene Library; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D048348:Reverse Transcription; D017423:Sequence Analysis, RNA / Q000379:methods*
keywords: Next generation sequencing (NGS); RNA-Seq; library preparation; quantitative PCR (qPCR); second generation sequencing (SGS)
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