Chasing Ghosts: Detecting Repeatmediated Deletions in the Human Genome - PowerPoint PPT Presentation

Title: Chasing Ghosts: Detecting Repeatmediated Deletions in the Human Genome


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Chasing Ghosts Detecting Repeat-mediated
Deletions in the Human Genome

• Benjamin Good
• Supervisor Dixie Mager

2
Big Picture
?

• Improve the understanding of the basic mechanisms
  of evolution.

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?
?
?
?
3
Hypothesis 1

• Non-allelic homologous recombination (NAHR) plays
  an important role in evolution by mediating
  genomic rearrangements.

4
Biological mechanisms
Position 10

• Homologous recombination
• a normal and ubiquitous process occurring during
  meiosis
• Chromosome length is conserved

Position 10
5
NAHR
Repeat
Position 876

• Non-Allelic homologous recombination
• Indels
• Exchanges
• Inversions

Position 10
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Duplications and Deletions
D
B
A
E
C
F
g
e
d
f
b
a
c
C
B
A
D
E
F
e
d
g
b
a
c
f
B
A
f
g
Deletion
b
a
e
d
C
c
D
E
F
Duplication
7
Hypothesis 2

• Transposable elements increase the likelihood of
  rearrangements by making NAHR much more likely.

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Ideal Approach

• Catalogue all examples of NAHR within the genome.
• Quantify the relationship between transposable
  elements and these putative evolutionary events.

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Can TSDs be used?
D
B
A
E
C
F
Before insertion
D
B
A
E
C
F
After insertion
10
No

• Original hope was that the absence of target site
  duplications in the flanking sequences of the
  repeats could be used to infer recombination.
• Problems are that
• RepeatMasker does not reliably label the edges
  correctly
• The TSDs may accumulate mutations
• Repeats often insert on top of one another
  forming complex mosaics that are extremely
  difficult to characterize computationally.
• In any case, genome comparisons offer more
  conclusive evidence

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So, focus on what is possible

• Deletions in the human genome are visible through
  comparisons with other genomes.
• Identify putative deletions based on gaps in
  pair-wise alignments
• Check the borders of the gaps for repetitive
  elements and make inferences about the causes of
  the gaps.

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Comparative Approach
Baboon
T
U
D
B
A
E
C
F
S
Repeat
T
U
B
A
C
S
Human

• If repeats overlap edges of a gap it is strong
  evidence for a NAHR-mediated deletion in that
  genome
• Different TSDs additional evidence for NAHR

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2003 Primate Comparisons

• Bailey 2003 mentions the discovery of 9 Alu-Alu,
  5 L1-L1, and 1 L2-L2 mediated deletions. These
  deletions were identified using an alignment
  between baboon and a 4.8 MB region of human
  chromosome 7.
• An investigation of the Alus, revealed that only
  one of them was a deletion within the human.
• This element was the inspiration for the rest of
  the project.

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Human Baboon Alignment

• Generated using BL2SEQ at NCBI
• Human chimeric Alu
• Baboon sequence from Eichler lab.
• E -47
• E -23

Alu repStart 1 repLeft -97
AluSp/q repStart 251 repLeft-13
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Chimeric Alus

• A search for Alu-chimeras corresponding to this
  structure yielded 91 examples in human chromosome
  7
• But how to prove that they are associated with
  deletions?
• Back to genome comparisons.

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Site of a deletion in the Human Genome
Intron
Mouse Alignment
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Mouse View
Gap in mouse human alignment
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Mouse Baboon
Significant alignment between the mouse and the
baboon that has been lost in the human sequence.
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So What Now

• The deletion discovered by Bailey could have been
  discovered using only the mouse.
• Build a system that can scan the entire human
  genome for deletions based on the human-mouse
  alignment from UCSC

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Protocol 1 Accumulate local data

• 1) First, download HumanNet table containing
  coordinates of gaps in the mouse-human alignment.
  This is the mouse-human alignment from the
  perspective of the mouse

2) Download RepeatMasker annotation for the human
genome.
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Protocol 2 Main Script

• Take all gaps that are at least 500bp larger than
  their corresponding region in human.
• Find all human repeats whose left and right edges
  are close to the edges of the human coordinates
  for the gap.
• Find chimeric elements that span the gaps
• Characterize the contents of the mouse sequence
  corresponding to the gap.

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Searching For
Protein Q8C5C0 Signal transduction
Mouse 3000 bp
Human AluJb 287 bp
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Results

• Scanned entire genome in 8.5 hours
• Checked 271,690 gaps
• Identified 456 potentially repeat-mediated
  deletions.
• Of these, 43 were chimeric and 297 were derived
  from human Alu repeats.

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Chimeras Only 1 good

• Only one of the chimeras obeys the repeat
  structure discussed previously (front repeat
  missing the end and back repeat missing the
  start). This is the Bailey deletion.
• The others seem to be mostly complete repeats in
  close proximity to each other.

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Repeat Content SINE enrichment
Classes of repeats associated with mouse gaps
Classes of repeats in the human genome
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Alu Families as expected
Types of Alus associated with mouse gaps
Types of Alus in the human genome
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Gap Content

• 18 gaps contained exons from known genes. 9 of
  these correspond to mouse L1s
• 78 gaps contained expressed ESTs

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The Human Net (Kent 2003)

• 400 level 1, 49 level 2, 4 level 3

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Gaps and higher net levels
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Summary

• In this project, we have designed and implemented
  a method for cataloguing potential examples of
  repeat-mediated deletions within the human genome
  through comparative genomics. Many of the
  examples identified are from transcribed
  sequences and hence could have significant
  phenotypic effects.

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Future Directions

• Other primate genomes.
• 3-way comparisons for validation.
• Deletions in regulatory regions?

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Thanks to the Mager Lab!
Somewhere in the Philippines