Genome finishing

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Revision as of 01:30, 17 August 2010 (view source) Csbl (Talk | contribs) (→PCR) ← Older edit		Latest revision as of 22:23, 3 April 2011 (view source) Gnusnah (Talk | contribs) (→Beautiful figures)
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		+	==Annotation==
		+
		+	===사용 프로그램===
		+	*rRNA : rnammer
		+	*tRNA : tRNAscan-SE
		+	*CDS : glimmer3, GeneMarkHMM, Prodigal, blastp
		+	*oriC : ori-finder
		+
		+
		+	*viewer : artemis, DNAPlotter
		+
		+
		+	*Total predicted CDS: 4576
		+	*Hit - hypothetical protein: 1682
		+	*No Hit: 991
		+
		+	===통계===
		+	*rRNA : 5 operon(16s,23s,5s) + 5s rRNA (green)
		+	*tRNA : 58 (blue)
		+	*CDS : 4576 (red)
		+	*oriC : 1 (cyan)
		+
		+	[[File:EL612.jpg]]
		+
	==PCR==		==PCR==
		+
		+	===4 Gaps===
		+	*6gap1
		+	** repeat region
		+
		+	*6gap2
		+	**N : 2994.. 3061
		+	**gb|[http://www.ncbi.nlm.nih.gov/protein/224949241 EEG30450.1]| hypothetical protein CLOSTMETH_01926 [Clostridium methylpentosum DSM5476]  Length = 1188  q: 4..2811, s: 158..1072
		+	**gb|[http://www.ncbi.nlm.nih.gov/protein/257473805 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|[http://www.ncbi.nlm.nih.gov/protein/158434870 EDP12637.1]| hypothetical protein CLOBOL_07199 [Clostridium bolteae ATCC BAA-613]    Length = 1481  q:  924..3017, s: 7..697
		+	** gb|[http://www.ncbi.nlm.nih.gov/protein/225161680 EEG74299.1]| hypothetical protein CLOHYLEM_05557 [Clostridium hylemonae DSM 15053]    Length = 199    q:  3680..4279, s: 1..199
		+
		+	** filled!!
		+
		+	*3_7gap
		+	**Get PCR product!
		+
		+	===pcr===
	{| class="wikitable" style="text-align:center" border="1"		{| class="wikitable" style="text-align:center" border="1"
	|+		|+
-	|8-1||8-6||8-3||3-7||7-8||8-2||2-8||8-5||5-8||8-4||4-8	+	|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||||L||L||L||L||L||L||L||L||L||L
	|-		|-
-	|P||P||A||P||A||A||P||A||P||P||A	+	|P||P||A||P||A||A||P||A||P||P||A|| || ||?
	|}		|}
	*L: 454 PE link		*L: 454 PE link
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	#7head-3head: ok		#7head-3head: ok
	#5kb control- c1_4-c1_5: ok *gap -> CGTCTAAAAAATTATAATTTTTTAGACG		#5kb control- c1_4-c1_5: ok *gap -> CGTCTAAAAAATTATAATTTTTTAGACG
		+
		+	==Submission==
		+	[http://www.ncbi.nlm.nih.gov/projects/genome/assembly/submission/faq.shtml FAQ]
		+	[http://www.ncbi.nlm.nih.gov/genomes/static/Annotation_pipeline_README.txt PGAAP]
		+
		+	==Beautiful figures==
		+	*Drawing Software: Artemis, CGView
		+	*CGView command
		+	**perl cgview_xml_builder.pl -sequence NC_015213.gbk -output NC_015213.xml -gc_skew T -gc_content T  -size x-large
		+	**java -jar -Xmx4000m ../cgview.jar -i NC_015213.xml -o NC_015213.svg -f svg
	==Reference==		==Reference==
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	*[http://www.biomedcentral.com/1471-2164/11/242 Finishing genomes with limited resources: lessons from an ensemble of microbial genomes]		*[http://www.biomedcentral.com/1471-2164/11/242 Finishing genomes with limited resources: lessons from an ensemble of microbial genomes]
	*[http://www.google.com/search?q=genome+sequencing+finishing+pcr+protocol Google Search Result]: genome+sequencing+finishing+pcr+protocol		*[http://www.google.com/search?q=genome+sequencing+finishing+pcr+protocol Google Search Result]: genome+sequencing+finishing+pcr+protocol
		+	*[http://insilicogen.com/blog/36 Good Blog]
		+	*Stothard P, Wishart DS. Circular genome visualization and exploration using CGView. Bioinformatics 21:537-539.[http://wishart.biology.ualberta.ca/cgview/]

---

Latest revision as of 22:23, 3 April 2011

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 Submission 4 Beautiful figures 5 Reference

Annotation

사용 프로그램

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

• viewer : artemis, DNAPlotter

• Total predicted CDS: 4576
• Hit - hypothetical protein: 1682
• No Hit: 991

통계

• rRNA : 5 operon(16s,23s,5s) + 5s rRNA (green)
• tRNA : 58 (blue)
• CDS : 4576 (red)
• oriC : 1 (cyan)

PCR

4 Gaps

• 6gap1
  • repeat region

• 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

• • filled!!

• 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

Submission

FAQ PGAAP

Beautiful figures

• Drawing Software: Artemis, CGView
• CGView command
  • perl cgview_xml_builder.pl -sequence NC_015213.gbk -output NC_015213.xml -gc_skew T -gc_content T -size x-large
  • java -jar -Xmx4000m ../cgview.jar -i NC_015213.xml -o NC_015213.svg -f svg

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
• Good Blog
• Stothard P, Wishart DS. Circular genome visualization and exploration using CGView. Bioinformatics 21:537-539.[1]