Bioinformatics Final Product claire

325 views

Published on

0 Comments
0 Likes
Statistics
Notes
  • Be the first to comment

  • Be the first to like this

No Downloads
Views
Total views
325
On SlideShare
0
From Embeds
0
Number of Embeds
2
Actions
Shares
0
Downloads
4
Comments
0
Likes
0
Embeds 0
No embeds

No notes for slide
  • The objective of this study is to use C. gigasas a model organism to characterize the distribution and identify potential functions of DNA methylation
  • Input=% methylation value
  • % methylation per CytosineGonad- most bases were 100% methylatedGill- variation in % methylation, most were 100% methylated however a small peak at low % methylation**Differences due to library prep (gill enriched for methylation first so much higher % meth)
  • Read coverage distribution- Histogram of read coverage per cytosine
  • Pairwise correlation score between the % methylation profiles.Scatterplots of % methylation scores.
  • PCA of two oyster tissue profiles, shows principal component 1 and 2 for each sample. Samples closer to each other in principal component space are similar in their methylation profiles.
  • Hypo=under the level or parHyper= over the limit or above normal level
  • Bioinformatics Final Product claire

    1. 1. DNA methylation coverage in two tissues of the Pacific Oyster Claire Ellis Bioinformatics Terminal Product 3/14/13
    2. 2. DNA methylation patterns in Crassostrea gigasEpigenetics describes DNAmodifications that change geneexpression without alteringnucleotide sequence.DNA methylation in organismsis extremely diverse, variableamong species, and can changegenome function under CH3external influences. DNA methylation Source: http://www.nist.gov/pml/div689/dna
    3. 3. Sequencing ApproachesBisulfite sequencing was used Bisulfite sequencingto examine DNA methylation Cm= methylated cytosinein gonad tissue C= unmethylated cytosineMBD-Seq was used to 5’ ACmGTTCGCTTGAG 3’examine DNA methylation in 3’ TGCmAAGCGAACTC 5’gill tissue (Mackenzie) Bisulfite Treatment 5’ ACmGTTUGUTTGAG 3’ 3’ TGCmAAGUGAAUTU 5’
    4. 4. ApproachBisulfite converted reads aligned to genome and %methylation value per base calculated by processingalignmentsmethylKit is an R package for DNA methylationanalysis and annotation from high-throughputbisulfite sequencing
    5. 5. methylKitGoal: obtain methylation coverage andexamine differential methylation betweengonad and gill tissues Read annotation files and perform basic statistical analyses for differentially methylated regions or bases
    6. 6. Methylation Statistics Histogram of % CpG methylation Histogram of % CpG methylation test test Gonad 94.7 Gill 16.8 40000 200000 12.5 30000 150000 9.8Frequency Frequency 8.1 20000 100000 7.1 6.4 5.4 4.5 4 4.2 10000 3.8 50000 2.4 2.6 2.7 1.8 1.7 1.6 2 1.4 4.4 1 0 0 0 0 0 0.3 0 0 0 0 0 0.3 0 0 0 0 0 0 0 20 40 60 80 100 0 20 40 60 80 100 % methylation per base % methylation per base
    7. 7. Coverage Statistics Histogram of CpG coverage Histogram of CpG coverage test test 91.6 Gonad 16.9 Gill 40000 200000 13.6 12.7 12.5 30000 150000 9.4Frequency Frequency 8.5 100000 20000 7.6 5.5 50000 10000 3.9 2.9 2 7.2 1.5 1 0.70.5 0 0 0.7 0 0.2 0 0 0 0 0 0 0.1 0 0 0 0 0 0 0 0.30.20.10.1 0 0 0 0 0 0 0 0.0 0.5 1.0 1.5 2.0 1.0 1.5 2.0 2.5 3.0 log10 of read coverage per base log10 of read coverage per base
    8. 8. CpG base correlation CpG base pearson cor. 0.0 0.2 0.4 0.6 0.8 1.0 1.0 ~/Desktop/TJGR_GonadPE_BS_v9_90_CG_methylkit_modified.txt 0.8 0.6 Gonad 0.068 0.068 0.4 0.2 1.0 ~/Desktop/TJGR_gillMBD_BS_v9_10x_methylkit_modified.tabular.txt 0.8 Gill 0.6y 0.4 0.2 0.0 0.2 0.4 0.6 0.8 1.0
    9. 9. Methylation Clustering CpG methylation clustering 0.8 0.6Height Blue= Gonad 0.4 Red= Gill 0.2 0.0 ethylkit_modified.txt t_modified.tabular.txt Samples Distance method: "correlation"; Clustering method: "ward"
    10. 10. PCA- Principal Component Analysis CpG methylation PCA Analysis 2e-12 ~/Desktop/TJGR_GonadPE_BS_v9_90_CG_methylkit_modified.txt 1e-12 Blue= Gonad 0e+00PC2 Red= Gill -1e-12 -2e-12 -60 -40 -20 0 20 40 60 PC1
    11. 11. ConclusionsAdditional analyses included examining type of differentialmethylation (hypo and hyper) Extracted bases with a q-value <0.01 and % methylation difference >25%The methylKit package was successfully used tocharacterize DNA methylationDifferences between gonad and gill methylation profilesmay be due to library prepWill use R script for future analyses comparing differentsamples’ methylation profiles
    12. 12. Methylation Statistics> getMethylationStats(gonad,plot=F,both.strands=F)methylation statistics per basesummary (gonad): Min. 1st Qu. Median Mean 3rd Qu. Max. 9.091 100.000 100.000 97.360 100.000 100.000Percentiles (gonad):0% 10% 20% 30% 40% 50% 60% 70% 80% 95% 99.5% 99.9% 100%9.09 100 100 100 100 100 100 100 100 100 100 100 100summary (gill): Min. 1st Qu. Median Mean 3rd Qu. Max. 0.00 54.55 78.70 69.56 91.89 100.000Percentiles (gill):0% 10% 20% 30% 40% 50% 60% 70% 80% 95% 99.5% 99.9% 100%0 21.4 46.6 61.1 70.9 78.7 84.6 90 93.7 100 100 100 100

    ×