Dinh/Dinh 2012/NOTES/2012-1-4

From ZhangLabWiki
Jump to navigation Jump to search

iPSC Epigenetics[edit]

Sergio's iPSCs[edit]

  • Checked the localization of the CpGs found in shared aberrantly methylated genes:
File:Idiograph-sharedGenes-CGs.txt
File:Idiograph-sharedGenes-CGs.png
**Note: background are EST density by number.
  • Shared aberrantly methylated genes tend to be away from the centrimere region.

iPSCs from Yang Xu's lab[edit]

  • These iPSCs were reprogrammed using episomal vectors.
  • First, I checked the 'reprogramming completeness' of different iPSC lines (including ones from whole genome bisulfite sequencing). To do this, I limited the sites to only CGs with no known disrupting SNP AND were not found to be differentially methylated between Hues1, Hues3, and Hues8 (methylation data from BSPP sequencing only).
  • Below are the different pluripotent lines included for this analysis:
Source Pluripotent lines Cell type derived Vectors Method #CpGs
Xu lab (BSPP) 218HFF211 fetal liver fibroblasts OCT4,SOX2,NANOG,LIN28A Episomal 286691
Xu lab (BSPP) 218HFF411 fetal liver fibroblasts OCT4,SOX2,NANOG,LIN28A Episomal 266433
Xu lab (BSPP) 2HFF8d_10 fetal liver fibroblasts OCT4,SOX2,NANOG,LIN28A Episomal 308102
WGBS ADS-iPSC adipose derived stem cells OCT4, SOX2, KLF4, cMYC Retroviral 477356
Church lab (BSPP) PGP1-iPSC human adult fibroblasts OCT4, SOX2, KLF4, cMYC Retroviral 395985
WGBS FF-iPS-19-11 foreskin fibroblasts OCT4, SV40LT, NANOG, LIN28, KLF4 Episomal 291927
WGBS FF-iPS-19-7 foreskin fibroblasts OCT4, SV40LT, NANOG, LIN28, KLF4 Episomal 316709
WGBS FF-iPS-6-9 foreskin fibroblasts OCT4, SV40LT, NANOG, LIN28, cMYC, KLF4 Episomal 322250
WGBS IMR90-iPS fetal lung fibroblasts OCT4, SOX2, NANOG, LIN28A Lentiviral 284770
WGBS H9 human embryonic stem cells NA NA 266495
WGBS H9 Laurent human embryonic stem cells NA NA 408474
WGBS H1 human embryonic stem cells NA NA 305459
Xu lab (BSPP) H1 human embryonic stem cells NA NA 400577
Xu lab (BSPP) H3 human embryonic stem cells NA NA 305826
Xu lab (BSPP) H8 human embryonic stem cells NA NA 454397
File:Xu dendrogram.png
  • Next, I determined the sites which were aberrantly methylated using a Fisher Exact Test with Benjamini-Hochberg FDR of 0.01. Aberrant methylation were determined at sites where iPSCs were different from the combined Hues1, Hues3, and Hues8.
for f in mf_*.txt
do
       ./getCpgChsq+DiffMatrix_DD_*.pl $f 10 0.2 0.01 H1H3H8_filteredsites_noCG-SNP > muir.$f.dmTable_noSNP
       awk ' 
               function abs(value) { return (value<0?-value:value) };
               { if($2==1 && $3==1 && abs($4-$5) >= 0.2 && ( ($4<0 && $5>=0) || ($4>0 && $5<=0) )) print $0,"\tMEMORY_INCOMPLETE"; //$2 is result from test vs hESC and $3 is result from test vs somatic source
                 if($2==1 && $3==0 && abs($4-$5) >= 0.2 && abs($5) < 0.2 && abs($4) > abs($5) ) print $0, "\tMEMORY_COMPLETE";
                 if($2==1 && $3==1 && ( ($4<0 && $5<0) || ($4>0 && $5>0) )) print $0, "\tMUTATION";
                 if($2==0 && $3==1 && abs($4)<0.2) print $0,"\tPLURIPOTENCY";
               }
       ' muir.$f.dmTable_noSNP > muir.$f.memmuTable_noSNP
done;
  • The aberrantly methylated sites were further limited to those which have <0.1 standard deviation between all of H1 (WGBS), H9 (WGBS), H9_Laurent (WGBS), Hues1, Hues3, and Hues8
  • I uploaded the lists to [GREAT | http://www.great.stanford.edu] and obtained genes list for each of the 3 iPSC lines from Xu's lab.
  • I took the intersection of the three lists to obtain shared methylation memory and shared methylation mutation gene lists.
File:Xu shared mutation.txt 
  • 1/5/11 - I forgot to separate out the genes associated with chrXYM
  • BC018860 should be removed. (Shared mutated methylated on chrM in all three iPSCs.
File:Xu shared memory.txt
  • MAGEE1 and ZDHHC15 should be removed. (Shared memory methylated in all three iPSCs.)
  • Affy Expression Data, I got expression data from two samples, HuSCID and SCID.
  • First, I uploaded the shared memory/mutation gene lists to DAVID and found all associated Affymetric probe IDs for each gene.
  • Next, I annotated each probe as memory/mutation/normal.
  • I divided the probes list into two: upregulated in HuSCID or downregulated in HuSCID. Additionally, I required that upregulated probes in HuSCID must NOT have detection value of 'A' (absence) and downregulated probes in HuSCID must NOT have detection value of 'A' (absence) in SCID.
  • Next I calculated the fold change and rank them.
  • Here are the list of aberrantly methylated genes and their fold-change in expression value in HuSCID/SCID (tumor samples). File:AberrantlyMethylatedGenesProbesExpression.xls