中国农业科学院蔬菜花卉研究所 Institute of Vegetables and Flowers Chinese Academy of Agricultural Sciences Zhonghua Zhang Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences Tomato chr11 finishing

中国农业科学院蔬菜花卉研究所 Institute of Vegetables and Flowers Chinese Academy of Agricultural Sciences  Sequence type  Data flow about finishing BACs  Sequence gap closure strategies  Information on read sequences used and amount of finished BAC sequences  Examples of problematic clones encountered  Aims for chromosome finishing from workshop Outline

中国农业科学院蔬菜花卉研究所 Institute of Vegetables and Flowers Chinese Academy of Agricultural Sciences Sequence type ABI 3730 Solexa Now: ABI 3730 Future: ABI Solexa

中国农业科学院蔬菜花卉研究所 Institute of Vegetables and Flowers Chinese Academy of Agricultural Sciences Assembly Custom primers Finished BAC sequence Basecalling Vector mask shear Library (shot-gun) BAC DNA Trace files Quality Control Sequencing Finishing Data flow about finishing BACs

中国农业科学院蔬菜花卉研究所 Institute of Vegetables and Flowers Chinese Academy of Agricultural Sciences 1.Determine the order and orientation of contigs: (a). Pair-ends (b). physical map (c). homologous sequences such as potato (b). PCR using Custom primers (betaine etc.) 2. Sequencing gaps: (a). Walking using custom primers and BigDye Terminator (b). Difficult regions: Dye primers, dGTP chemistry, Sequitherm EXCEL etc. Sequence gap closure strategies

中国农业科学院蔬菜花卉研究所 Institute of Vegetables and Flowers Chinese Academy of Agricultural Sciences Determine the order and orientation of contigs by using physical map

中国农业科学院蔬菜花卉研究所 Institute of Vegetables and Flowers Chinese Academy of Agricultural Sciences (5) (1) (4) (3)(2)(6) (2) (1) (4) (3) Potato: Tomato: Potato:(1) (2) (3) (4) (5) (6) Tomato: (1) (2) (3) (4) Determine the order and orientation of contigs using other sequnces

中国农业科学院蔬菜花卉研究所 Institute of Vegetables and Flowers Chinese Academy of Agricultural Sciences Information on read sequences used and amount of finished BAC sequences

中国农业科学院蔬菜花卉研究所 Institute of Vegetables and Flowers Chinese Academy of Agricultural Sciences Examples of problematic clones encountered a. Inverted repeats

中国农业科学院蔬菜花卉研究所 Institute of Vegetables and Flowers Chinese Academy of Agricultural Sciences Examples of problematic clones encountered b. High GC regions or simple sequence repeats

中国农业科学院蔬菜花卉研究所 Institute of Vegetables and Flowers Chinese Academy of Agricultural Sciences Examples of problematic clones encountered c. Repetitive sequences

中国农业科学院蔬菜花卉研究所 Institute of Vegetables and Flowers Chinese Academy of Agricultural Sciences Aims for chromosome finishing from workshop 1.Technica and reagents may help sequence the difficult regions with high success rate 2.How to employ Small Insert Libraries and Transposon Insertion Libraries efficiently?

中国农业科学院蔬菜花卉研究所 Institute of Vegetables and Flowers Chinese Academy of Agricultural Sciences Acknowledgements Chinese Academy of Agricultural Sciences: Sanwen Huang, Jun He, Yongchen Du, Dongyu Qu Beijing Genomics Institute: Dongyuan Liu, Longfei Liu China Agricultural University: Wencai Yang Huazhong Agricultural University: Zhibiao Ye, Junhong Zhang South China Agricultural University: Guoping Wang Cornell university: Zhangjun Fei, Jim Giovannoni, Lukas Mueller Colorado State University : Steven Stack, Suzanne M. Royer