title: pBACode: a random-barcode-based high-throughput approach for BAC paired-end sequencing and physical clone mapping.
abstract: Applications that use Bacterial Artificial Chromosome (BAC) libraries often require paired-end sequences and knowledge of the physical location of each clone in plates. To facilitate obtaining this information in high-throughput, we generated pBACode vectors: a pool of BAC cloning vectors, each with a pair of random barcodes flanking its cloning site. In a pBACode BAC library, the BAC ends and their linked barcodes can be sequenced in bulk. Barcode pairs are determined by sequencing the empty pBACode vectors, which allows BAC ends to be paired according to their barcodes. For physical clone mapping, the barcodes are used as unique markers for their linked genomic sequence. After multi-dimensional pooling of BAC clones, the barcodes are sequenced and deconvoluted to locate each clone. We generated a pBACode library of 94,464 clones for the flounder Paralichthys olivaceus and obtained paired-end sequence from 95.4% of the clones. Incorporating BAC paired-ends into the genome preassembly improved its continuity by over 10-fold. Furthermore, we were able to use the barcodes to map the physical locations of each clone in just 50 pools, with up to 11 808 clones per pool. Our physical clone mapping located 90.2% of BAC clones, enabling targeted characterization of chromosomal rearrangements.
authors.affiliation: MOE Key Laboratory of Bioinformatics, Center for Synthetic and Systems Biology, School of Life Sciences, Tsinghua University, Beijing 100084, China.|PTN (Peking University-Tsinghua University-National Institute of Biological Sciences) Joint Graduate Program, Beijing 100084, China.|School of Life Sciences, Peking University, Beijing 100084, China.
authors.forename: Xiaolin
authors.initials: X
authors.lastname: Wei
authors.affiliation: MOE Key Laboratory of Bioinformatics, Center for Synthetic and Systems Biology, School of Life Sciences, Tsinghua University, Beijing 100084, China.|PTN (Peking University-Tsinghua University-National Institute of Biological Sciences) Joint Graduate Program, Beijing 100084, China.
authors.forename: Zhichao
authors.initials: Z
authors.lastname: Xu
authors.affiliation: Beidaihe Central Experiment Station, Chinese Academy of Fishery Sciences, Qinhuangdao 066100, China.
authors.forename: Guixing
authors.initials: G
authors.lastname: Wang
authors.affiliation: Beidaihe Central Experiment Station, Chinese Academy of Fishery Sciences, Qinhuangdao 066100, China.
authors.forename: Jilun
authors.initials: J
authors.lastname: Hou
authors.affiliation: MOE Key Laboratory of Bioinformatics, Center for Synthetic and Systems Biology, School of Life Sciences, Tsinghua University, Beijing 100084, China.|PTN (Peking University-Tsinghua University-National Institute of Biological Sciences) Joint Graduate Program, Beijing 100084, China.
authors.forename: Xiaopeng
authors.initials: X
authors.lastname: Ma
authors.affiliation: Beidaihe Central Experiment Station, Chinese Academy of Fishery Sciences, Qinhuangdao 066100, China.
authors.forename: Haijin
authors.initials: H
authors.lastname: Liu
authors.affiliation: National Key Laboratory of Crop Genetic Improvement and College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China.
authors.forename: Jiadong
authors.initials: J
authors.lastname: Liu
authors.affiliation: National Key Laboratory of Crop Genetic Improvement and College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China.
authors.forename: Bo
authors.initials: B
authors.lastname: Chen
authors.affiliation: National Key Laboratory of Crop Genetic Improvement and College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China.
authors.forename: Meizhong
authors.initials: M
authors.lastname: Luo
authors.affiliation: Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 100081, China.
authors.forename: Bingyan
authors.initials: B
authors.lastname: Xie
authors.affiliation: Novogene Bioinformatics Institute, Beijing 100083, China.
authors.forename: Ruiqiang
authors.initials: R
authors.lastname: Li
authors.affiliation: Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences, Shenzhen, Guangdong 518120, China.
authors.forename: Jue
authors.initials: J
authors.lastname: Ruan
authors.affiliation: MOE Key Laboratory of Bioinformatics, Center for Synthetic and Systems Biology, School of Life Sciences, Tsinghua University, Beijing 100084, China.
authors.forename: Xiao
authors.initials: X
authors.lastname: Liu
mesh_terms: D000818:Animals; D022202:Chromosomes, Artificial, Bacterial; D003001:Cloning, Molecular; D005432:Flounder / Q000235:genetics; D015723:Gene Library; D016678:Genome; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D020161:Physical Chromosome Mapping / Q000379:methods*; D012441:Saccharomyces cerevisiae / Q000235:genetics; D017422:Sequence Analysis, DNA / Q000379:methods*