title: PacBio sequencing of gene families - a case study with wheat gluten genes.
abstract: Amino acids in wheat (Triticum aestivum) seeds mainly accumulate in storage proteins called gliadins and glutenins. Gliadins contain α/β-, γ- and ω-types whereas glutenins contain HMW- and LMW-types. Known gliadin and glutenin sequences were largely determined through cloning and sequencing by capillary electrophoresis. This time-consuming process prevents us to intensively study the variation of each orthologous gene copy among cultivars. The throughput and sequencing length of Pacific Bioscience RS (PacBio) single molecule sequencing platform make it feasible to construct contiguous and non-chimeric RNA sequences. We assembled 424 wheat storage protein transcripts from ten wheat cultivars by using just one single-molecule-real-time cell. The protein genes from wheat cultivar Chinese Spring are comparable to known sequences from NCBI. We demonstrated real-time sequencing of gene families with high-throughput and low-cost. This method can be applied to studies of gene amplification and copy number variation among species and cultivars.
authors.affiliation: Waksman Institute of Microbiology, Rutgers University, 190 Frelinghuysen Road, Piscataway, NJ 08854, USA.
authors.forename: Wei
authors.initials: W
authors.lastname: Zhang
authors.forename: Paul
authors.initials: P
authors.lastname: Ciclitira
authors.forename: Joachim
authors.initials: J
authors.lastname: Messing
mesh_terms: D005903:Gliadin / Q000235:genetics; D005983:Glutens / Q000235:genetics*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods; D005810:Multigene Family / Q000235:genetics*; D010802:Phylogeny; D016415:Sequence Alignment / Q000379:methods; D017422:Sequence Analysis, DNA / Q000379:methods*; D013045:Species Specificity; D014908:Triticum / Q000235:genetics*
chemical_list: D005983:Glutens; D005903:Gliadin; C037302:glutenin
keywords: BAC; CAP3; CCS; CS; Chinese Spring; Contig assembly program version 3; Copy number variation; GRIN; Gene family; Gluten diversity; HLA; HMW; LMW; PacBio; PacBio sequencing; Pacific Bioscience; Repetitive sequence; SMRT; ZMW; bacterial artificial chromosome; circular consensus sequence; germplasm resources information network; high molecular weight; human leukocyte antigen; low molecular weight; single molecule, real time; zero mode waveguides