Genome finishing

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	==PCR==		==PCR==

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Revision as of 15:02, 4 September 2010

Contents 1 Annotation 1.1 사용 프로그램 1.2 통계 2 PCR 2.1 4 Gaps 2.2 pcr 2.3 Arrange Scaffold 2.4 Sequencing 3 Reference

Annotation

사용 프로그램

• rRNA : rnammer
• tRNA : tRNAscan-SE
• CDS : glimmer3, GeneMarkHMM, Prodigal, blastp
• oriC : ori-finder

• viewer : artemis, DNAPlotter

통계

• rRNA : 5 operon(16s,23s,5s) + 5s rRNA
• tRNA : 58
• CDS : 5042
• oriC : 1

PCR

4 Gaps

• 6gap1

• 6gap2
  • N : 2994.. 3061
  • gb|EEG30450.1| hypothetical protein CLOSTMETH_01926 [Clostridium methylpentosum DSM5476] Length = 1188 q: 4..2811, s: 158..1072
  • gb|ACV54125.1| Gene info linked to ACV54125.1 hypothetical protein Elen_0131 [Eggerthella lenta DSM 2243] Length=705 q:4828..6171, s:40..518

• 1gap
  • N : 3025..3049
  • gb|EDP12637.1| hypothetical protein CLOBOL_07199 [Clostridium bolteae ATCC BAA-613] Length = 1481 q: 924..3017, s: 7..697
  • gb|EEG74299.1| hypothetical protein CLOHYLEM_05557 [Clostridium hylemonae DSM 15053] Length = 199 q: 3680..4279, s: 1..199

• 3_7gap
  • Get PCR product!

pcr

8-1

8-6

8-3

3-7

7-8

8-2

2-8

8-5

5-8

8-4

4-8

6-7

1-3

1-6

L

L

L

L

L

L

L

L

L

L

L

L

L

P

P

A

P

A

A

P

A

P

P

A

?

• L: 454 PE link
• P: PCR
• A: Assemble

Arrange Scaffold

• Pieces
  • 8-1
  • 8-6
  • 8-3(RC)-7-8
  • 8-2-8
  • 8-5-8
  • 8-4-8

Sequencing

• Primer: Product

1. 5tail-8head: ok
2. 8tail-8head: 4tail-8head
3. 1tail-3head: 7head-3head - 1tail primer의 중간부터 끝 부분이 7 head에 붙은 것으로 생각됨.
4. 7head-3head: ok
5. 5kb control- c1_4-c1_5: ok *gap -> CGTCTAAAAAATTATAATTTTTTAGACG

Reference

• Finishing Overview - PDF
• Consed--A Finishing Package (Editor, Autofinish, Autoreport, Autoedit, and Align Reads To Reference Sequence)
• Genomics Education - see some tutorials for finishing with Consed
• Automated Finishing with Autofinish
• Finishing genomes with limited resources: lessons from an ensemble of microbial genomes
• Google Search Result: genome+sequencing+finishing+pcr+protocol