Analysis of DNA Replication

1
Analysis of DNA Replication

• Dutta Lab
• University of Virginia
• July 13th, 2005

2
Previous Work

• Proc Natl Acad Sci U S A. 2005 May
  3102(18)6419-24. Epub 2005 Apr 21, Temporal
  profile of replication of human chromosomes. Jeon
  Y, Bekiranov S, Karnani N, Kapranov P, Ghosh S,
  MacAlpine D, Lee C, Hwang DS, Gingeras TR, Dutta
  A.
• Chromosome 21/22 replication profile was studied
  in the above work.
• The following analysis extends this work to the
  ENCODE regions and develops new analysis
  techniques

3
Overview

• Temporal Profiling of DNA Replication
• Replication Tracks (Available on UCSC GB)
• TR50 calculation
• Segregation
• Correlations with
• Gencode
• AT content
• DnaseI

4
Replication Tracks

• e.g. ENm005
• Generated with Affy GTrans utility
• regions exceeding 10-4 p-value.
• Difficult for direct correlations due to overlap
  across different time points.

5
TR50 Calculation
TR50 - Time at which 50 of the locus is
replicated In the example below, probe A has
a TR50 of 1.25hr (80 at 2hr, 0 at 0hr) probe
B has a TR50 of 6.33hr (100 at 8hr, 40 at 6hr)
Example
Probe Probe
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Specificity of Regions

• Some regions show a panS (not time point
  specific) phase pattern of replication
• To identify such regions we classify probes as
  specific or non-specific
• A probe is specific iff at least 50 of the total
  signal appears in a single time point.
• Utilizing this classification, we can identify
  broad regions as specific or non-specific

7
Segregation

• Various patterns of replication are seen in the
  DNA replication tracks
• We segregate regions into 4 classes
• Early Specific replication early in S-phase
• Mid Specific replication mid in S-phase
• Late Specific replication late in S-phase
• Pan-S Non-specific replication

8
Region Classifier

• We pass a sliding window (10,000 bp) across each
  region and accumulate within the window the ratio
  of non-specific vs. specific probes and the
  average TR50
• Once the ratio exceeds a threshold (e.g. 60) a
  Pan-S region is started
• In order to prevent thrashing, a tolerance (e.g.
  10) is used and the Pan-S region is not ended
  until we drop below thr-tol (e.g. 50)

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Specific Classification

• Within a specific region, we classify sub-regions
  as early, mid, or late based on the average TR50
• For ENCODE regions
• 23 Pan-S
• 77 Specific
• 35 Early
• 38 Mid
• 27 Late

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Advantages of Segregation

• Much more suited to correlations with other data
  than current replication tracks
• Automated identification of non-specific regions
• Provides an independent method to compare with
  the current replication tracks

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Examples
ENm005
ENm012
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Correlations Overview

• Segregation utility was just completed
• Not part of data freeze
• Currently working on correlations
• We will show previously completed correlations
  with TR50 data
• all Encode regions pooled together

13
Gencode VS TR50
For each region we calculate Gene Density for
each base pair based on GENCODE annotations. Gene
Density was calculated using a sliding window of
50KB. Lowess smoothing was done with Gene Density
on the x-axis and tr50 on the y axis and a
spearman rank correlation was calculated
(-0.283). We also plot a density profile, which
calculates number of probes with a given Gene
Density.
TR50
Gene Density
Frequency ( of probes)
Gene Density
14
AT content vs TR50
For each region we calculate AT content for each
base pair based on hg16. AT content was
calculated using a sliding window of 50bp. Lowess
smoothing was done with AT Content on the x-axis
and tr50 on the y axis and a spearman rank
correlation was calculated (1.0). We also plot a
density profile, which calculates number of
probes with a given AT content.
TR50
AT Content
Frequency ( of probes)
AT Content
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CorrelationDnaseI HS vs TR50

• For each DnaseI Hypersensitive site identified a
  window is chosen around it and average tr50 value
  is calculated
• This data is pooled from all the regions and a
  histogram of TR50s is plotted
• Also as a background model a histogram of TR50
  from all the regions is plotted
• A Mann-Whitney Test is performed on these two
  distributions

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CorrelationDnaseI HS with TR50

• DnaseI hypersensitive sites are associated with
  lower tr50 values as compared to the total
  distribution of the tr50s
• The medians of the two distributions are
  significantly different with a p-value of 0.0075

17
Acknowledgements

• Anindya Dutta PI
• Neerja Karnani PostDoc
• Patrick Boyle PostDoc
• Chris Taylor Doctoral Student
• Ankit Malhotra Doctoral Student