Athurva Gore/LabNotes/ExomeReseq/2009-5-13

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Revision as of 23:39, 13 May 2009 by >Ajgore (→‎Procedure)
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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