sequencing gap - Rice Genome Annotation Project

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pHOS Vector Features• The sequencing primer sites immediately flank the cloningsite to avoid excessive re-sequencing of vector DNA.• PCR primer sites are located immediately outside of theSequencing primer sites to allow PCR amplification fortemplate preparation.• The vector continues to have the regular, good vectorfeatures such as a gene for replication (origin of replication),antibiotic resistance and multiple cloning sites where wecan cut the circular plasmid and insert desired features.TIGRTHE INSTITUTE FOR GENOMIC RESEARCH
Template Production• Transform clones intoE. coli• Plate and pick coloniesto isolate clones• Replicate and isolateplasmid for sequencing• 384 well HT plateprocessingTIGRTHE INSTITUTE FOR GENOMIC RESEARCH
Page 4 and 5: Library Construction• Break the g
Page 6: DNA Shearing with HydroShearHydroSh
Page 9: Design Features of the BstXI Adapto
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
Page 55 and 56: Mis-assembled repeatClones link dif
Page 57 and 58: Sequence editor• In this example
Page 59 and 60: Resultingcontig afterrepeatresoluti
Page 61 and 62:
Collapsed Repeat-5.5 CE DipReadCove
Page 63 and 64:
Sequence Validation: Editing• Fin
Page 65 and 66:
Sequence Validation:Sequence covera
Page 67 and 68:
Sequence Coverage in CloeTIGRTHE IN
Page 69 and 70:
Clone Coverage ExampleCATCCGAAGACGA
Page 71 and 72:
Finished BAC Ready for Annotation!B
Page 73 and 74:
TIGRTHE INSTITUTE FOR GENOMIC RESEA
Page 75 and 76:
HT TemplateLibraryConstructionHT Se
Page 77 and 78:
Schematic View of 3 Typical Scaffol
Page 79 and 80:
Short Tandem RepeatsTIGRTHE INSTITU
Page 81 and 82:
Inverted RepeatsTIGRTHE INSTITUTE F
Page 83 and 84:
Use of Transposon-mediated librarie
Page 85 and 86:
Closure Challenges: SequencingThrou
Page 87 and 88:
Homopolymeric tractsTIGRTHE INSTITU
Page 89 and 90:
• Apply different sequencing chem
Page 91 and 92:
Automated Finishing (cont’d)• L
Page 93 and 94:
• Dead EndsBAC Project Challenges
Page 95 and 96:
Filling small gaps in a chromosome
Page 97:
Project Organization• Database to
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sequencing gap - Rice Genome Annotation Project

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