Chasing Ghosts: Detecting Repeatmediated Deletions in the Human Genome

1
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.

?
?
?
?
?
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
6
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.

8
Ideal Approach

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

9
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

11
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.

12
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

13
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.

14
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
15
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.

16
Site of a deletion in the Human Genome
Intron
Mouse Alignment
17
Mouse View
Gap in mouse human alignment
18
Mouse Baboon
Significant alignment between the mouse and the
baboon that has been lost in the human sequence.
19
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

20
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.
21
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.

22
Searching For
Protein Q8C5C0 Signal transduction
Mouse 3000 bp
Human AluJb 287 bp
23
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.

24
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.

25
Repeat Content SINE enrichment
Classes of repeats associated with mouse gaps
Classes of repeats in the human genome
26
Alu Families as expected
Types of Alus associated with mouse gaps
Types of Alus in the human genome
27
Gap Content

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

28
The Human Net (Kent 2003)

• 400 level 1, 49 level 2, 4 level 3

29
Gaps and higher net levels
30
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.

31
Future Directions

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

32
Thanks to the Mager Lab!
Somewhere in the Philippines