sequencing gap - Rice Genome Annotation Project

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Library Construction• Break the genomic DNA from theorganism into many small fragments• The fragments are cloned into plasmidvectors• This collection of fragments is called alibraryTIGRTHE INSTITUTE FOR GENOMIC RESEARCH
Library Requirements1. Free vector should be at low or undetectable level.2. No chimeric clones. Chimeras occur two or more randomfragments from separate parts of the genome recombineand end up next to each other.3. The majority of the inserts should be of relatively uniformsize.4. Libraries need to be random and cover the wholegenome.TIGRTHE INSTITUTE FOR GENOMIC RESEARCH
Page 6: DNA Shearing with HydroShearHydroSh
Page 9 and 10: Design Features of the BstXI Adapto
Page 11 and 12: Template Production• Transform cl
Page 13 and 14: Template Production LaboratoryCurre
Page 15 and 16: Sequence Production LaboratoryCurre
Page 17 and 18: Sample ElectropherogramTIGRTHE INST
Page 19 and 20: What are phred quality values?The q
Page 21 and 22: TIGRTHE INSTITUTE FOR GENOMIC RESEA
Page 23 and 24: Assembling the fragmentsTIGRTHE INS
Page 25 and 26: Merging two sequencesoverlap (19 ba
Page 27 and 28: Forward-reverse constraints• The
Page 29 and 30: Degenerate contigs• Too deep• T
Page 31 and 32: Scaffolding• Given a set of non-o
Page 33 and 34: Linking information• Overlaps•
Page 35 and 36: Assembly gapsphysical gapgroup Agro
Page 37 and 38: Problems with the data• Incorrect
Page 39 and 40: Unifying view of assemblyAssemblySc
Page 41 and 42: BAC-by-BAC Sequencing and Closure
Page 43 and 44: BAC Shotgun SequencingBAC DNASequen
Page 45 and 46: Why Completeness is Important• Im
Page 47 and 48: Levels of Genome ClosureTIGRTHE INS
Page 49 and 50: TIGRTHE INSTITUTE FOR GENOMIC RESEA
Page 51 and 52: Sequencing GapsContig AUnderlyingse
Page 53 and 54:
Repetitive Areas• Repetitive area
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Mis-assembled repeatClones link dif
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Sequence editor• In this example
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Resultingcontig afterrepeatresoluti
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Collapsed Repeat-5.5 CE DipReadCove
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Sequence Validation: Editing• Fin
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Sequence Validation:Sequence covera
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Sequence Coverage in CloeTIGRTHE IN
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Clone Coverage ExampleCATCCGAAGACGA
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Finished BAC Ready for Annotation!B
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TIGRTHE INSTITUTE FOR GENOMIC RESEA
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HT TemplateLibraryConstructionHT Se
Page 77 and 78:
Schematic View of 3 Typical Scaffol
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Short Tandem RepeatsTIGRTHE INSTITU
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Inverted RepeatsTIGRTHE INSTITUTE F
Page 83 and 84:
Use of Transposon-mediated librarie
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Closure Challenges: SequencingThrou
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Homopolymeric tractsTIGRTHE INSTITU
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• Apply different sequencing chem
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Automated Finishing (cont’d)• L
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• Dead EndsBAC Project Challenges
Page 95 and 96:
Filling small gaps in a chromosome
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Project Organization• Database to
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sequencing gap - Rice Genome Annotation Project

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