title: Polymerase chain reaction preparation of template for massively parallel pyrosequencing.
abstract: Massively parallel pyrosequencing of DNA fragments immobilized on beads has been applied to genome survey sequencing and transcriptome analysis of a variety of eukaryotic organisms, including laboratory model species, agricultural crops and livestock, and species of interest to population biologists and ecologists. Preparation of sufficient high-quality template for sequencing has been an obstacle to sequence analysis of nucleic acids from tissues or cell types available in limited quantities. We report that the use of a biotinylated primer for polymerase chain reaction amplification allows removal of excess primer and poly(A) tract fragments from the sequencing templates, providing much higher yields of useful sequence information from pyrosequencing of amplified templates. This advance allows deep sequencing analysis of nucleic acids isolated from very small tissue samples. Massively parallel pyrosequencing is particularly useful for preliminary investigations of species that have not yet been the subject of significant genomic research, as genomic survey sequences and catalogs of expressed genes provide a means of linking the biology of less intensively studied species to that of more intensively studied model organisms. We obtained over 220 Mb of transcript DNA sequences from Abies fraseri (Pursh) Poir., a conifer species native to the southern Appalachian Mountains of eastern North America. Comparison of the resulting assembled putative transcripts with similar data obtained by other sequencing methods from other conifers demonstrates the utility of the improved sequencing template preparation.
authors.affiliation: Department of Forestry & Environmental Resources, North Carolina State University, Raleigh, NC 27695, USA. whetten@ncsu.edu
authors.forename: Ross W
authors.initials: RW
authors.lastname: Whetten
authors.forename: Valenzuela A
authors.initials: VA
authors.lastname: Sofía
authors.forename: John
authors.initials: J
authors.lastname: Frampton
mesh_terms: D028202:Abies / Q000235:genetics; D001483:Base Sequence; D019921:Biotinylation; D017931:DNA Primers / Q000737:chemistry; D018076:DNA, Complementary / Q000737:chemistry; D005052:Eucalyptus / Q000737:chemistry; D020869:Gene Expression Profiling; D008969:Molecular Sequence Data; D028223:Pinus / Q000737:chemistry; D041603:Pinus taeda / Q000737:chemistry; D018515:Plant Leaves / Q000737:chemistry; D010940:Plant Proteins / Q000737:chemistry; D018547:Plant Stems / Q000737:chemistry; D016133:Polymerase Chain Reaction / Q000379:methods*; D012313:RNA / Q000302:isolation & purification; D012015:Reference Standards; D017422:Sequence Analysis, DNA / Q000379:methods*
chemical_list: D017931:DNA Primers; D018076:DNA, Complementary; D010940:Plant Proteins; D012313:RNA