Introduction to Genomic Sequencing Assembly Annotation Diego Martinez

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Introduction to Genomic SequencingAssemblyAnnot
ationDiego Martinez
                                                                        
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Why do we want to know the sequence of an entire
genome??

• To know all the genes then proteins, then
  pathways
• We can understand the biochemistry of the
  organism
• We can understand diseases
• Evolution
• Regulation
• know all the upstream/downstream regions for
  proteins to bind and control transcription

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Convinced???? Good. Lets Sequence!!!

• 2 ways
• Map (Public)
• Several long steps tiling
• Expensive because they generated a complete map
• Whole Genome Shotgun (Private)
• Direct/cuts out some steps, missed 103 genes
• Repeats!
• Synthesis approach

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Generate Data
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Core principles

• BAC-end sequences allow one to know physical
  association of sequence
• More coverage leads to better sequence
• Better algorithms make it easier
• Longer sequence reads are critical

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Figure 2.5 Relationships of chromosomes to
genome sequencing markers
Sixteen overlapping clones represent 1,408 BACs
needed to span the 163 Mb X chromosome. (Avg
insert 146 kb)
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Assemblyput it all back together

• Assemble
• BAC assemblies, Phrap (Phil Green, UW)
• Celera, WGS Celera Assembler
• both find overlaps of same sequence,
  build regions (contigs)
• put contigs together using paired end
  information Order and Orient into large
  Scaffolds (also called super contigs.)
• Whole Genome Shotgun automated,
  without tiling
• Finishing

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Problems even Map-based couldnt fix
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Which method worked best?

• WGS failed with highly repetitive regions
• WGS, however, reduced overall workload for
  sequencing
• Use hybrid approach
• WGS used for 6-fold coverage
• Reduced number of BACs needed to sequence by 93

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Annotation

• Need to make it useable and fun!!!
• What is annotation?
• Find sequence features in the genome
• find genes (focus here)
• The act or process of furnishing critical
  commentary or explanatory notes.
• pseudogenes
• repeats
• reg. elements(very difficult, still in its
  infancy)
• attempt to describe gene function

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Figure 2.6 alternative splicing
NADPH oxidase
H channel
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Table 2.1 How annotation can be used to
infer/understand biological niche
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Example of annotation - What is a gene?
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Functional Annotation What does the protein
do?

• Found Genes
• Basic approach By similarity to known protein.
• Old Style Best Blast Hit
• Can lead to funny incorrect annotations

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Funny examples
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Critical residues/multiple sequence
alignment(lysozyme)
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Gene Family Expansion
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Signaling Pathways
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Phanerochaete chrysosporium

• Degrades lignin
• 30 million base pair (30 MB)
• This was the 1st basidiomycete so gene finding
  was a
• big challenge
• Estimate 11,777 genes

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Genome Facts!
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http//genome.jgi-psf.org/Phchr1/Phchr1.info.html
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Phylogenies of Genes
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Genome Evolution and RIP