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

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


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


Sequence type

ABI 3730 Solexa

Now: ABI 3730 Future: ABI 3730 + Solexa


AssemblyCustom primers

Finished BAC sequenceBasecalling

Vector mask

shear

Library (shot-gun)

BAC DNA

Trace files

Quality Control

Sequencing

Finishing

Data flow about finishing BACs


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


Determine the order and orientation of contigs by using physical map


(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


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


Examples of problematic clones encountered

a. Inverted repeats



b. High GC regions or simple sequence repeats



c. Repetitive sequences


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?


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

Documents

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