Athurva Gore/LabNotes/ExomeReseq/2009-5-13
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iPS Cancer?
- Obtained Illumina Array data from Dr. Zhang
- Used SAM and DAVID to try and identify GO Terms in differentially expressed genes.
- Say we want a desired FDR of 15% or lower; since we are just doing preliminary.
- SAM's multiclass feature is not useful here; it will call everything that is fibroblast-only "significant," leading to numbers that are far too large.
Very Lenient
- Very lenient analysis results in a large amount of GO Terms and GO Clusters.
- Delta of 2.3 was used for IPS_HESC
- Delta of 0.1 (very low, since figured a lot of genes would need to be called as "not similar")
- Will upload DAVID results to Wiki.
- Lots of very interesting GO terms and GO clusters
- However, inspection of several reveals that these values may be too lenient. Lots of things are called as significant that are not very.
Very Stringent
- Limited ESC_IPS differences to 475, limited GO differences to 250.
- In this case, only 3 or 4 clusters were obtained at all (only 23 diff. expressed genes)
- Most of them have very low scores...probably too stringent here
New Method
- It seems SAM has issues finding differences between fibroblasts and IPS that are meaningful
- Because so many genes are differentially expressed already, very hard to choose a proper delta value.
- FDR is predicted to be very high.
- Instead, will find differentially expressed genes between IPS and HESC first.
- Only look at these in Fibroblasts, then run SAM!
- Should help find any genes that do not match normal Fibroblasts or hESC in iPS cells.
- Should help find some new behavior.
Procedure
- First, run SAM on Illumina sequencing data for just iPS and hESC.
- Extract differentially expressed genes such that the FDR is 0.05
- Turned out to be a delta of 1.7 with 4365 differentially expressed genes.
- Extracted data for just this gene subset from Illumina data, and only for FIBROBLASTS and IPS.
- Saved file in NewMethod folder as csv
- Imported into R, ran SAM again.
- Interesting results.
- Pulled out at Delta=4 in order to obtain FDR of 0.2...FDR never went below 0.04 even after just one gene was being called significant. Delta of 4 gave around 50 genes to work with.
- In one way, this is decent news; means that iPS cells do not stray too far from fibroblasts...
- However, several of the GO groups are interesting.
- Differentially expressed genes (from SAM):
ILLUMINA_ID Gene Name Related Genes Species ILMN_1731745 ninjurin 2 RG Homo sapiens ILMN_1759087 olfactory receptor, family 10, subfamily v, member 1 RG Homo sapiens ILMN_1662021 hypothetical protein flj20403 similar to zinc finger protein 326 RG Homo sapiens ILMN_1702568 chromosome 9 open reading frame 28 RG Homo sapiens ILMN_1722855 vascular endothelial growth factor b RG Homo sapiens ILMN_1714980 mas-related gpr, member d RG Homo sapiens ILMN_1772387 toll-like receptor 2 RG Homo sapiens ILMN_1767233 ectodysplasin a2 receptor RG Homo sapiens ILMN_1795336 phosphotriesterase related RG Homo sapiens ILMN_1772727 g protein-coupled receptor 75 RG Homo sapiens ILMN_1679558 hypothetical protein flj20393 RG Homo sapiens ILMN_1780465 c-type lectin domain family 5, member a RG Homo sapiens ILMN_1743329 retinal pigment epithelium-derived rhodopsin homolog RG Homo sapiens ILMN_1752813 udp glucuronosyltransferase 1 family, polypeptide a6 RG Homo sapiens ILMN_1754553 mediator of rna polymerase ii transcription, subunit 19 homolog (yeast) RG Homo sapiens ILMN_1700766 flj45850 protein RG Homo sapiens ILMN_1736951 leucine-rich repeats and iq motif containing 2 RG Homo sapiens ILMN_1747683 aquaporin 4 RG Homo sapiens ILMN_1717579 chromosome 17 open reading frame 64 RG Homo sapiens ILMN_1708779 granulysin RG Homo sapiens ILMN_1764201 microtubule-associated protein 2 RG Homo sapiens ILMN_1724424 proline-rich transmembrane protein 2 RG Homo sapiens ILMN_1750234 protease, serine, 2 (trypsin 2) RG Homo sapiens ILMN_1726928 transcription elongation factor a (sii), 3 RG Homo sapiens ILMN_1752046 lymphocyte adaptor protein RG Homo sapiens ILMN_1717252 f-box and leucine-rich repeat protein 21 RG Homo sapiens ILMN_1680932 hypothetical protein flj12492 RG Homo sapiens ILMN_1709953 downstream neighbor of son RG Homo sapiens ILMN_1748338 g protein-coupled receptor 85 RG Homo sapiens ILMN_1700888 ectonucleotide pyrophosphatase/phosphodiesterase 1 RG Homo sapiens ILMN_1729433 lipase, member h RG Homo sapiens ILMN_1750497 g protein-coupled receptor 109a RG Homo sapiens ILMN_1757521 creatine kinase, muscle RG Homo sapiens ILMN_1706266 ring finger protein 157 RG Homo sapiens ILMN_1729203 protein disulfide isomerase-like protein of the testis RG Homo sapiens ILMN_1738849 solute carrier family 9 (sodium/hydrogen exchanger), member 2 RG Homo sapiens ILMN_1706590 myeloid/lymphoid or mixed-lineage leukemia (trithorax homolog, drosophila); translocated to, 1 RG Homo sapiens ILMN_1741371 transmembrane protein 8 (five membrane-spanning domains) RG Homo sapiens ILMN_1769538 flj36268 protein RG Homo sapiens ILMN_1718525 inter-alpha (globulin) inhibitor h4 (plasma kallikrein-sensitive glycoprotein) RG Homo sapiens ILMN_1732049 dolichyl-phosphate mannosyltransferase polypeptide 2, regulatory subunit RG Homo sapiens ILMN_1798957 chromosome 12 open reading frame 47 RG Homo sapiens ILMN_1699735 fibroblast growth factor 1 (acidic) RG Homo sapiens ILMN_1777658 scavenger receptor class f, member 1 RG Homo sapiens ILMN_1780172 chromosome 20 open reading frame 195 RG Homo sapiens ILMN_1722502 chaperonin containing tcp1, subunit 6a (zeta 1) RG Homo sapiens ILMN_1748884 transducer of erbb2, 2 RG Homo sapiens ILMN_1754489 f-box and leucine-rich repeat protein 20 RG Homo sapiens ILMN_1753648 tripartite motif-containing 51 RG Homo sapiens
- GO Terms associated with this set:
Category Term Count % PValue Genes List Total Pop Hits Pop Total Fold Enrichment Bonferroni Benjamini FDR UP_SEQ_FEATURE repeat:LRR 3 3 6.12% 0.050375089032457555 ILMN_1754489, ILMN_1717252, ILMN_1772387, 34 132 12056 8.058823529411764 1.0 1.0 67.40654846247544 UP_SEQ_FEATURE disulfide bond 13 26.53% 0.01293759405941519 ILMN_1741371, ILMN_1759087, ILMN_1718525, ILMN_1743329, ILMN_1748338, ILMN_1700888, ILMN_1722855, ILMN_1708779, ILMN_1767233, ILMN_1750497, ILMN_1777658, ILMN_1750234, ILMN_1780465, 34 2257 12056 2.0423779613750686 1.0 1.0 24.60526798193786 SP_PIR_KEYWORDS transducer 6 12.24% 0.057294646924235346 ILMN_1759087, ILMN_1743329, ILMN_1772727, ILMN_1748338, ILMN_1714980, ILMN_1750497, 43 881 17599 2.787371644273157 1.0 0.9998747358559845 60.92937311117008 GOTERM_BP_ALL GO:0009620~response to fungus 2 4.08% 0.044754229278689765 ILMN_1708779, ILMN_1772387, 40 18 15360 42.66666666666667 1.0 1.0 58.350929107631956 GOTERM_CC_ALL GO:0005887~integral to plasma membrane 7 14.29% 0.059501566519986435 ILMN_1731745, ILMN_1741371, ILMN_1743329, ILMN_1772727, ILMN_1747683, ILMN_1780465, ILMN_1772387, 37 1246 15857 2.4076829638627393 1.0 1.0 61.39321623792138 UP_SEQ_FEATURE topological domain:Extracellular 14 28.57% 0.0022672796720831292 ILMN_1731745, ILMN_1759087, ILMN_1743329, ILMN_1747683, ILMN_1748338, ILMN_1700888, ILMN_1714980, ILMN_1772387, ILMN_1767233, ILMN_1750497, ILMN_1777658, ILMN_1772727, ILMN_1780465, ILMN_1738849, 34 2092 12056 2.3729614216623554 1.0 0.9999999994964114 4.80383877784748 SP_PIR_KEYWORDS g-protein coupled receptor 6 12.24% 0.04681987018050463 ILMN_1759087, ILMN_1743329, ILMN_1772727, ILMN_1748338, ILMN_1714980, ILMN_1750497, 43 832 17599 2.95153175313059 1.0 0.9999636833469407 53.41019833781681 UP_SEQ_FEATURE topological domain:Cytoplasmic 14 28.57% 0.013195267574960728 ILMN_1731745, ILMN_1759087, ILMN_1743329, ILMN_1747683, ILMN_1748338, ILMN_1700888, ILMN_1714980, ILMN_1772387, ILMN_1767233, ILMN_1750497, ILMN_1777658, ILMN_1772727, ILMN_1780465, ILMN_1738849, 34 2554 12056 1.9437099820351005 1.0 1.0 25.030994571356445 GOTERM_MF_ALL GO:0001871~pattern binding 3 6.12% 0.025350396961618844 ILMN_1722855, ILMN_1699735, ILMN_1772387, 36 120 16968 11.783333333333333 1.0 0.999999999979955 36.88587537377367 GOTERM_MF_ALL GO:0060089~molecular transducer activity 11 22.45% 0.029894078111325575 ILMN_1777658, ILMN_1759087, ILMN_1743329, ILMN_1772727, ILMN_1752046, ILMN_1780465, ILMN_1748338, ILMN_1714980, ILMN_1750497, ILMN_1767233, ILMN_1772387, 36 2557 16968 2.02763655325251 1.0 0.9999995240203935 41.95651509259159 UP_SEQ_FEATURE repeat:LRR 2 3 6.12% 0.055934508134846746 ILMN_1754489, ILMN_1717252, ILMN_1772387, 34 140 12056 7.598319327731093 1.0 1.0 71.3038083352595 GOTERM_MF_ALL GO:0004930~G-protein coupled receptor activity 6 12.24% 0.044329382679716686 ILMN_1759087, ILMN_1743329, ILMN_1772727, ILMN_1748338, ILMN_1714980, ILMN_1750497, 36 953 16968 2.967471143756558 1.0 0.9999999919856024 55.63350341762754 UP_SEQ_FEATURE transmembrane region 18 36.73% 0.002001285675137048 ILMN_1731745, ILMN_1741371, ILMN_1759087, ILMN_1743329, ILMN_1752813, ILMN_1747683, ILMN_1732049, ILMN_1748338, ILMN_1700888, ILMN_1714980, ILMN_1750497, ILMN_1772387, ILMN_1767233, ILMN_1777658, ILMN_1772727, ILMN_1679558, ILMN_1780465, ILMN_1738849, 34 3211 12056 1.9877260153516407 1.0 1.0 4.251872992840566 GOTERM_MF_ALL GO:0001584~rhodopsin-like receptor activity 6 12.24% 0.02634256599594032 ILMN_1759087, ILMN_1743329, ILMN_1772727, ILMN_1748338, ILMN_1714980, ILMN_1750497, 36 828 16968 3.4154589371980677 1.0 0.9999999954475052 38.027567684179154 GOTERM_BP_ALL GO:0009410~response to xenobiotic stimulus 2 4.08% 0.09680892049365422 ILMN_1752813, ILMN_1708779, 40 40 15360 19.200000000000003 1.0 1.0 85.74158716169707 SP_PIR_KEYWORDS glycoprotein 18 36.73% 0.004458676320668984 ILMN_1741371, ILMN_1718525, ILMN_1759087, ILMN_1743329, ILMN_1752813, ILMN_1747683, ILMN_1748338, ILMN_1700888, ILMN_1722855, ILMN_1729433, ILMN_1714980, ILMN_1772387, ILMN_1767233, ILMN_1777658, ILMN_1724424, ILMN_1772727, ILMN_1780465, ILMN_1738849, 43 3807 17599 1.9351256253779758 0.9914649707007087 0.9914649707007087 6.870422526756103 SP_PIR_KEYWORDS membrane 20 40.82% 0.025822530859489445 ILMN_1731745, ILMN_1741371, ILMN_1759087, ILMN_1743329, ILMN_1752813, ILMN_1747683, ILMN_1732049, ILMN_1700888, ILMN_1748338, ILMN_1729433, ILMN_1714980, ILMN_1750497, ILMN_1772387, ILMN_1767233, ILMN_1777658, ILMN_1724424, ILMN_1772727, ILMN_1679558, ILMN_1780465, ILMN_1738849, 43 5272 17599 1.552652009739916 0.999999999999227 0.9990623331254216 34.07913985791367 UP_SEQ_FEATURE repeat:LRR 1 3 6.12% 0.055934508134846746 ILMN_1754489, ILMN_1717252, ILMN_1772387, 34 140 12056 7.598319327731093 1.0 1.0 71.3038083352595 SP_PIR_KEYWORDS mitogen 2 4.08% 0.08906667883999794 ILMN_1722855, ILMN_1699735, 43 39 17599 20.988670244484197 1.0 0.999996004345615 77.36990492265086 SP_PIR_KEYWORDS receptor 9 18.37% 0.04950725285928249 ILMN_1777658, ILMN_1759087, ILMN_1743329, ILMN_1772727, ILMN_1748338, ILMN_1714980, ILMN_1750497, ILMN_1767233, ILMN_1772387, 43 1731 17599 2.1279674337995247 1.0 0.9998791523826503 55.4590073052319 GOTERM_BP_ALL GO:0006805~xenobiotic metabolic process 2 4.08% 0.08755462431164601 ILMN_1752813, ILMN_1708779, 40 36 15360 21.333333333333336 1.0 1.0 82.67139825680376 SP_PIR_KEYWORDS leucine-rich repeat 4 8.16% 0.023014335903258815 ILMN_1754489, ILMN_1736951, ILMN_1717252, ILMN_1772387, 43 256 17599 6.394985465116279 0.9999999999833731 0.9997447667578405 30.986331464416928 GOTERM_CC_ALL GO:0031226~intrinsic to plasma membrane 7 14.29% 0.06257872248122895 ILMN_1731745, ILMN_1741371, ILMN_1743329, ILMN_1772727, ILMN_1747683, ILMN_1780465, ILMN_1772387, 37 1262 15857 2.3771576647963335 1.0 0.9999999999993398 63.307078417720106 GOTERM_MF_ALL GO:0004888~transmembrane receptor activity 9 18.37% 0.006275755892878886 ILMN_1777658, ILMN_1759087, ILMN_1743329, ILMN_1772727, ILMN_1748338, ILMN_1714980, ILMN_1750497, ILMN_1767233, ILMN_1772387, 36 1388 16968 3.056195965417867 0.9999999864724688 0.9999999864724688 10.670445055462363 GOTERM_CC_ALL GO:0031224~intrinsic to membrane 18 36.73% 0.06759206385198491 ILMN_1731745, ILMN_1741371, ILMN_1759087, ILMN_1743329, ILMN_1752813, ILMN_1747683, ILMN_1732049, ILMN_1748338, ILMN_1700888, ILMN_1714980, ILMN_1750497, ILMN_1772387, ILMN_1767233, ILMN_1777658, ILMN_1724424, ILMN_1772727, ILMN_1780465, ILMN_1738849, 37 5378 15857 1.4344024202707728 1.0 0.999999746198835 66.23619399038067 PIR_SUPERFAMILY PIRSF036848:conserved protein with F-box/LRR-repeat, Skp2 type 2 4.08% 0.02445880409797784 ILMN_1754489, ILMN_1717252, 20 9 6919 76.87777777777778 1.0 1.0 36.09156185764799 UP_SEQ_FEATURE glycosylation site:N-linked (GlcNAc...) 15 30.61% 0.02576746177407038 ILMN_1741371, ILMN_1718525, ILMN_1759087, ILMN_1743329, ILMN_1752813, ILMN_1747683, ILMN_1748338, ILMN_1700888, ILMN_1714980, ILMN_1772387, ILMN_1767233, ILMN_1777658, ILMN_1772727, ILMN_1780465, ILMN_1738849, 34 3085 12056 1.7240919058060824 1.0 1.0 43.23178380844887 SP_PIR_KEYWORDS transmembrane 19 38.78% 0.009679126085189019 ILMN_1731745, ILMN_1741371, ILMN_1759087, ILMN_1743329, ILMN_1752813, ILMN_1747683, ILMN_1732049, ILMN_1700888, ILMN_1748338, ILMN_1714980, ILMN_1750497, ILMN_1772387, ILMN_1767233, ILMN_1777658, ILMN_1724424, ILMN_1772727, ILMN_1679558, ILMN_1780465, ILMN_1738849, 43 4452 17599 1.7466986355753358 0.9999685844429379 0.9943950417430579 14.351966656994787 GOTERM_CC_ALL GO:0016021~integral to membrane 18 36.73% 0.06535743736236507 ILMN_1731745, ILMN_1741371, ILMN_1759087, ILMN_1743329, ILMN_1752813, ILMN_1747683, ILMN_1732049, ILMN_1748338, ILMN_1700888, ILMN_1714980, ILMN_1750497, ILMN_1772387, ILMN_1767233, ILMN_1777658, ILMN_1724424, ILMN_1772727, ILMN_1780465, ILMN_1738849, 37 5357 15857 1.4400254277050994 1.0 0.9999999967879639 64.95870291365749 GOTERM_MF_ALL GO:0004872~receptor activity 10 20.41% 0.024445864559518166 ILMN_1777658, ILMN_1759087, ILMN_1743329, ILMN_1772727, ILMN_1780465, ILMN_1748338, ILMN_1714980, ILMN_1750497, ILMN_1767233, ILMN_1772387, 36 2113 16968 2.2306357469632436 1.0 0.9999999999999997 35.82774717503101 INTERPRO IPR000276:Rhodopsin-like GPCR superfamily 6 12.24% 0.02736075111434335 ILMN_1759087, ILMN_1743329, ILMN_1772727, ILMN_1748338, ILMN_1714980, ILMN_1750497, 42 747 17845 3.4126984126984126 1.0 1.0 41.56899259261204 GOTERM_MF_ALL GO:0004871~signal transducer activity 11 22.45% 0.029894078111325575 ILMN_1777658, ILMN_1759087, ILMN_1743329, ILMN_1772727, ILMN_1752046, ILMN_1780465, ILMN_1748338, ILMN_1714980, ILMN_1750497, ILMN_1767233, ILMN_1772387, 36 2557 16968 2.02763655325251 1.0 0.9999995240203935 41.95651509259159