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中国农业科学院蔬菜花卉研究所Institute of Vegetables and FlowersChinese Academy of Agricultural Sciences
Zhonghua Zhang
Institute of Vegetables and Flowers,
Chinese Academy of Agricultural Sciences
2008.04.24
Tomato chr11 finishing
中国农业科学院蔬菜花卉研究所Institute of Vegetables and FlowersChinese Academy of Agricultural Sciences
Sequence type Data flow about finishing BACs Sequence gap closure strategies Information on read sequences used and amount of fini
shed 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 FlowersChinese Academy of Agricultural Sciences
Sequence type
ABI 3730 Solexa
Now: ABI 3730 Future: ABI 3730 + Solexa
中国农业科学院蔬菜花卉研究所Institute of Vegetables and FlowersChinese Academy of Agricultural Sciences
AssemblyCustom primers
Finished BAC sequenceBasecalling
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 FlowersChinese 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 FlowersChinese 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 FlowersChinese 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 FlowersChinese Academy of Agricultural Sciences
Information on read sequences used and amount of finished BAC sequences
BAC Length(bp) Coverage No.reads aver.len (bp) Quality
C11HBa0139J14 118446 5.6 1398 508.0 q20
C11HBa0029C01 126094 6.0 1722 512.7 q20
C11HBa0249E07 125082 6.4 2188 468.7 q20
C11HBa0245M17 137274 6.7 1847 513.8 q20
C11HBa0323C14 152519 7.0 2531 456.8 q20
C11HBa0161D01 108762 7.0 1659 509.7 q20
C11HBa0062I24 157776 7.3 2325 510.0 q20
C11HBa0089M02 104753 7.5 1617 509.3 q20
C11HBa0168B23 131677 7.7 2315 438.0 q20
C11HBa0072I13 137657 8.0 2231 516.5 q20
C11HBa0190J03 124868 8.1 2184 478.0 q20
C11HBa0143O06 132613 8.1 2406 446.3 q20
C11HBa0303G16 144100 8.2 2511 468.2 q20
C11HBa0054I23 164552 8.5 3112 450.3 q20
C11SLm0052K14 100466 8.5 2001 428.5 q20
C05HBa0107A04 124025 9.7 2767 433.9 q20
C11HBa0107K14 104816 10.5 2311 489.0 q20
C11HBa0096D22 23535 12.9 1504 487.2 q20
123279 8.0 2146 479
中国农业科学院蔬菜花卉研究所Institute of Vegetables and FlowersChinese 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 FlowersChinese 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 FlowersChinese 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 FlowersChinese 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 FlowersChinese 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