Hamilton Eye Institute Mouse Eye M430v2 (Sept06) RMA Data Set

Accession number: GN162

    Summary:

RECOMMENDED EYE DATA SET. The HEIMED September 2006 RMA fixed data set provides estimates of mRNA expression in whole eyes of 84 lines of young adult mice generated using approximately 175 Affymetrix M430 2.0 arrays. This data set corrects probable errors in strain assignment that affected strains BXD2, BXD31, BXD89, and A/J. Data were generated at UTHSC with support from a grant from Dr. Barrett Haik, Director of the Hamilton Eye Institute (HEI). We used pooled RNA samples, usually two independent pools; one male, one female, for each strain. This data set was processed using the RMA protocol. To simplify comparison among different transforms, RMA values of each array have been adjusted to an average expression of 8 units and a standard deviation of 2 units. Values range from a low of 4.78 (Tcf15, probe set 1420281_at) to a high of 15.47 (crystallin gamma C, Crygc, probe set 1422674_s_at). Even probe sets with comparatively low expression can provide reliable data. For example probe set 1440397_at (Cacna2d1) has expression of only 5.5, but is associated with a cis QTL with an LRS of 79. This indicates a high signal to noise ratio and the detection of significant strain variation of the correct transcript.

An additional set of strains will be added in early fall 2008 to complete the HEIMED data set. We will be adding data for the following strains:

1. Three additional BXD strains (67 total). The three new strains are BXD56, BXD71, and BXD99.
2. More arrays for the parental strains, C57BL/6J and DBA/2J, and their reciprocal F1s (n = 4)
3. Ten new common strains of mice. The Mouse diversity panel will include a total of 29 strains (n = 27 strains plus B6 and D2
4. Seven KO lines (Rpe65, Nyx (NOB), Gabbr1, Gnb1, Gabra1, Gpr19, and Clcn3)

Users of these mouse eye data may also find the NEIBank collection of ESTs and SAGE data of substantial utility.

    About the cases used to generate this set of data:

We used a set of 64 BXD recombinant inbred strains, 18 conventional inbred strains including C57BL/6J (B6) and DBA/2J (D2), and their reciprocal F1s. BXD strains were generated by crossing C57BL/6J with DBA/2J. The BXDs are particularly useful for systems genetics because both parental strains have been sequenced (8x coverage of B6 and 1.5x coverage of D2). Physical maps in WebQTL incorporate approximately 2 million B6 vs D2 SNPs from Celera Genomics and from the Perlegen-NIEHS sequencing effort. BXD1 through BXD32 were bred by Benjamin A. Taylor starting in the late 1970s. BXD33 through 42 were bred by Taylor in the 1990s. These strains are available from The Jackson Laboratory.

Please note that BXD24/TyJ (JAX stock number 000031) used in this study is also known as BXD24b/TyJ and has nearly complete retinal degeneration. BXD24a/TyJ, a 1988 F80 stock that has now been rederived, does not have retinal degeneration (stock number 005243) and is an ideal coisogenic control.

BXD43 through BXD99 were bred by Lu Lu, Jeremy Peirce, Lee M. Silver, and Robert W. Williams in the late 1990s and early 2000s using advanced intercross progeny (Peirce et al. 2004). Many of the 50 new BXD strains are available from Lu Lu and colleagues.

Mouse Diversity Panel (MDP). In addition to the BXD strains, we have profiled a MDP consisting 18 inbred strains and a pair of reciprocal F1 hybrids; B6D2F1 and D2B6F1. These strains were selected for several reasons:

• genetic and phenotypic diversity, including use by the Phenome Project
• their use in making genetic reference populations including recombinant inbred strains, cosomic strains, congenic and recombinant congenic strains
• their use by the Complex Trait Consortium to make the Collaborative Cross (Nairobi/Wellcome, Oak Ridge/DOE, and Perth/UWA)
• genome sequence data from three sources (NHGRI, Celera, and Perlegen-NIEHS)
• availability from The Jackson Laboratory

All eight parents of the Collaborative Cross (129S1/SvImJ, A/J, C57BL/6J, CAST/EiJ, NOD/LtJ, NZO/HlLtJ, PWK/PhJ, and WSB/EiJ) have been included in the MDP. Twelve MDP strains have been sequenced or resequenced by Perlegen for the NIEHS (see the GeneNetwork SNP Browser for data, details, and a link to Perlegen Inc excellent data resources and browser).

1. 129S1/SvImJ
       Collaborative Cross strain sequenced by NIEHS; background for many knockouts (R1 ES cell line); Phenome Project A list. This strain (JAX No 002448, aka 129S1/Sv-++Kitl/+) also carries hypopigmentation mutations (white bellied chinchilla) of the tyrosinase gene on Chr 7 and a mutant allele of the steel (Kitl) gene. (JAX Stock Number: 002448)
2. A/J
       Collaborative Cross strain sequenced by Perlegen/NIEHS; parent of the AXB/BXA panel. A tyrosinase-negative albino (c) mutant
3. BALB/cByJ
        Sequenced by NIEHS; maternal parent of the CXB panel; Phenome Project A list. A tyrosinase-negative albino (c) mutant. (JAX Stock Number: 001026)
4. BXSB/MpJ
        A strain with interesting autoimmune disease associated with glomerulonephritis. (JAX Stock Number: 000740)
5. C3H/HeJ
       Sequenced by Perlegen/NIEHS; paternal parent of the BXH panel; Phenome Project A list. Important to note for this Eye dataset, C3H/HeJ is a Pdeb6 mutant with near total photoreceptor loss at as early as postnatal day 30.
6. C57BL/6J
       Sequenced by NIH/NHGRI; parental strain of AXB/BXA, BXD, and BXH; Phenome Project A list
7. CAST/EiJ (please note in an early data release, we listed CAST/Ei and CAST/EiJ as two different strains)
       Collaborative Cross strain sequenced by NIEHS; Phenome Project A list (JAX Stock Number: 000928).
8. DBA/2J
       Sequenced by Perlegen/NIEHS and Celera; paternal parent of the BXD panel; Phenome Project A list
9. FVB/NJ
       Sequenced by Perlegen/NIEHS and Celera; most common strain used to make transgenic mice due to large and easily injected oocyte; Phenome Project A list (JAX Stock Number: 001800).
10. KK/HlJ
        Sequenced by Perlegen/NIEHS
11. LG/J
        Paternal parent of the LGXSM panel
12. MOLF/EiJ
        A wild strain derived from the M. musculus molossinus subspecies of mice that has retinal degeneration affecting photoreceptors. There appears to have been some genetic contamination or admixture of this strain with conventional inbred strains in the very recent past (F. Pardo, personal communication to RWW, August 2006) (JAX Stock Number: 000550).
13. NOD/LtJ
        Collaborative Cross strain sequenced by NIEHS; Phenome Project B list; diabetic
14. NZO/HlLtJ
        Collaborative Cross strain
15. PWD/PhJ
        Sequenced by Perlegen/NIEHS; parental strain for a consomic set by Forjet and colleagues
16. PWK/PhJ
        Collaborative Cross strain; Phenome Project D list
17. WSB/EiJ
        Collaborative Cross strain sequenced by NIEHS; Phenome Project C list (JAX Stock Number: 001145).
18. B6D2F1 and D2B6F1, aka F1 in some graphs and tables
    F1 hybrids generated by crossing C57BL/6J with DBA/2J. These reciprocal F1 can be used to detect some imprinted genes.

    About the tissue used to generate this set of data:

Tissue preparation protocol. Animal were killed by rapid cervical dislocation. Eyes were removed immediately and placed in RNAlater at room temperature. Usually six eyes from animals with a common sex, age, and strain were stored in a single tube.

Each array was hybridized with a pool of RNA from 4 to 8 eyes from 2 to 4 animals. RNA was extracted at UTHSC by Zhiping Jia. If tissue was saved for RNA extraction at a later time, eyes were placed directly in RNAlater (Ambion, Inc.) and treated per the manufacturer’s directions. If eyes were used for immediate RNA extraction then we proceeded immediately to the next steps.

Dissecting and preparing eyes for RNA extraction

1. Place eyes for RNA extraction in RNA STAT-60 (Tel-Test Inc.) and process per manufacturer’s instructions (in brief form below).
2. Store RNA in 75% ethanol at –80 deg. C until use.

Total RNA was extracted with RNA STAT-60 (Tel-Test Inc.) according to the manufacturer's instructions. Briefly we:

1. homogenize tissue samples in the RNA STAT-60 (1 ml/50 to 100 mg tissue)
2. allowed the homogenate to stand for 5 min at room temperature
3. added 0.2 ml of chloroform per 1 ml RNA STAT-60
4. shook the sample vigorously for 15 sec and let the sample sit at room temperature for 3 min
5. centrifuged at 12,000 G for 15 min
6. transfered the aqueous phase to a fresh tube
7. added 0.5 ml of isopropanol per 1 ml RNA STAT-60
8. vortexed and allowed sample to stand at room temperature for 5-10 min
9. centrifuged at 12,000 G for 10-15 min
10. removed the supernatant and washed the RNA pellet with 75% ethanol
11. stored the pellet in 75% ethanol at -80 deg C until use

Sample Processing. Samples were processed in the VA Medical Center, Memphis, Rheumatology Disease Research Core Center lead by Drs. John Stuart and Weikuan Gu. All processing steps were performed by Dr. Yan Jiao. In brief, samples were purified using a standard sodium acetate in alcohol method (recommended by Affymetrix). The RNA quality was checked using a 1% agarose gel. The 18S and 28S bands had to be clear and the 28S band had to be more prominent. RNA concentation was measured using a spectrophotometer. The 260/280 ratios had to be greater than 1.7, and the majority were 1.8 or higher. We used a total of 8 micrograms of RNA as starting amount for cDNA synthesis using a standard Eberwine T7 polymerase method (Superscript II RT, Invitrogen Inc., Affy Part No 900431, GeneChip Expression 3' Amplification One-Cyle cDNA Synthesis Kit). The Affymetrix IVT labeling kit (Affy 900449) was used to generate labeled cRNA. At this point the cRNA was evaluated again using both the 260/280 ratio (values of 2.0 or above were acceptable) and 1% agarose gel inspection of the product (a size range from 200 to 7000 bp is considered suitable for use). We used 45 micrograms of labeled cRNA for fragmentation. Those samples that passed both QC steps (<10% usually fail) were then sheared using a fragmentation buffer included in the Affymetrix GeneChip Sample Cleanup Module (Part No.900371). After fragmentation, samples were either stored at -80 deg. centrigrade until use (one third roughly) or were immediately used for hybridization.

Dealing with Ocular Pigmentation: Variable ocular pigmentation is a potential confound in a study of the whole eye transcriptome. Even the most careful RNA preparations taken from brown and beige colored mice tend to have faint residual pigmentation that affects hybridization signal. The key determinant of this interesting effect is the Tyrp1 (brown) locus on Chr 4 at about 80 Mb. Loci on Chr 4 that map at this location should be considered with skepticism and reviewed carefully. To address this problem Yan Jiao purified total RNA a second time using the Qiagen RNeasy MinElute Cleanup Kit (Cat No. 74204). This was done for 8 colored samples (R2534E2, R2578E2, R1441E2, R2537E2, R2536E2, R2589E2, R2539E2) in the first batch of arrays (the November 05 data set) of which 5 were finally included in this data set (cases in which the strain ID is labeled with asterisks in the table). This same protocol was used for all samples in the second and third batches added in April 2006 and September 2006.

Replication and Sample Balance: Our goal is to obtain data for independent biological sample pools from at least one of sample from each sex for all BXD strains. The three batches of arrays included in this September data set, collectively represent a reasonably well balanced sample of males and females belonging to 84 strains, but without within-strain-by-sex replication. Three strains are represented only by male sample pools (A/J, BXD29, BXD48). One strain is represented only by a female pool sample (BXD89). Please use the probe sets 1427262_at (Xist, high in females) and probe set 1426438_at (Ddx3y, high in males) as quantitative surrogates for the sex balance in this data set.

Batch Structure: This data set consists of a three batches: the original batch that makes up the November 2005 data set (n = XXX), a second batch of 63 arrays (R0857E through R2649E, and R2682E through R2742E, non-consecutive identifiers) run in January 2006 by Dr. Yan Jiao; and a third batch of 41 arrays (XXXX through YYYYY) run in August 2006 by Dr. Yan Jiao. The arrays in the first batches are from different lots. All arrays in the first batch were from Lot YYYYY (expiration date XX.YY.ZZ). All arrays in the second batch were from Lot 4016879 (expiration date 12.28.06). All NN arrays in the third batch were from Lot XXXXX (expiration date XX.YY.ZZ). We started working with a total of approximately 190 (???) arrays that passed initial crude quality control based on RNA quality and initial Affymetrix report file information such as 3'/5' ratio, scale factor, and percent present calls. A total of 177 (???) arrays were finally approved for inclusion in this September 2006 data set. The complex normalization procedure is described below.

The table below summarizes information on strain, age, sex, original CEL filename, Affymetrix quality control values, and source of mice. Columns labeled "PDNN 2Z outlier" and "RMA 2Z outlier" list the fraction of probe sets with values that deviated more than 2 z units from the mean. Scale factor, background average, present, absent, marginal and 3'/5' ratios for actin and Gapdh were collated from the Affymetrix Report (RPT) files.

IN PROGRESS: PLEASE NOTE THAT THIS TABLE IS NOW BEING UPDATED TO INCLUDE BATCHES 2 and 3 OF EARLY and MID 2006.

ID	tube ID	group_type	Strain	age	Sex	original CEL filename	PDNN 2Z outlier	RMA 2Z outlier	scale factor	background average	present	absent	marginal	AFFX-b- ActinMur(3'/5')	AFFX- GapdhMur(3'/5')	Source
1	R2533E1	GDP	129S1/SvImJ	60	M	R2533E.CEL	0.025	0.028	2.11	94	57.90%	40.50%	1.60%	1.37	0.78	UTM RW
2	R2595E1	GDP	129S1/SvImJ	59	F	R2595E.CEL	0.033	0.036	1.79	115	61.00%	37.50%	1.50%	1.46	0.77	UTM RW
3	R0754E2	GDP	A/J	60	M	R0754E.CEL	0.027	0.03	2.72	86	59.80%	38.70%	1.50%	1.36	0.76	JAX
4	R2546E1	GDP	A/J	66	F	R2545E.CEL	0.024	0.029	1.99	96	58.60%	39.70%	1.70%	1.47	0.78	UTM RW
5	R2601E1	GDP BXD	B6D2F1	73	F	R2601E.CEL	0.007	0.008	2.55	92	58.90%	39.60%	1.50%	1.44	0.78	UTM RW
6	R2602E1	GDP BXD	B6D2F1	73	M	R2602E.CEL	0.003	0.008	2.6	84	59.70%	38.80%	1.50%	1.37	0.78	UTM RW
7	R1672E1	GDP	BALB/cByJ	83	M	R1672E.CEL	0.043	0.039	2.22	111	59.90%	38.60%	1.50%	1.26	0.8	JAX
8	R1676E1	GDP	BALB/cByJ	83	F	R1676E.CEL	0.083	0.085	2.69	98	58.90%	39.60%	1.50%	1.46	0.74	JAX
9	R2704E	BXD	BXD1	59	F	R2704E.CEL	0.029	0.03	2.066	139.61	56.60%	41.90%	1.50%	1.31	0.81	GU
10	R2581E1	BXD	BXD11	65	F	R2581E.CEL	0.009	0.021	1.94	89	62.10%	36.40%	1.60%	1.55	0.81	UTM RW
11	R2612E	BXD	BXD11	70	M	R2612E.CEL	0.101	0.112	1.83	142.03	58.20%	40.50%	1.40%	1.78	0.81	GU
12	R2543E1	BXD	BXD12	63	M	R2543E.CEL	0.018	0.017	1.61	118	58.60%	39.90%	1.60%	1.43	0.77	UTM RW
13	R2742E	BXD	BXD12	71	F	R2742E.CEL	0.073	0.077	2.127	134.14	57.00%	41.60%	1.40%	1.64	0.78	GU
14	R2586E1	BXD	BXD13	60	F	R2586E.CEL	0.259	0.258	2.01	74	56.40%	42.00%	1.60%	2.85	3.81	Glenn
15	R877E	BXD	BXD13	76	M	R877E.CEL	0.026	0.067	1.558	125.63	61.20%	37.50%	1.20%	1.42	0.81	GU
16	R2557E1	BXD	BXD14	60	F	R2557E.CEL	0.012	0.027	1.83	99	62.50%	36.10%	1.40%	1.31	0.78	Glenn
17	R1128E	BXD	BXD14	65	M	R1128E.CEL	0.037	0.038	2.366	118.39	57.30%	41.30%	1.40%	1.45	0.81	GU
18	R2716E	BXD	BXD15	60	M	R2716E.CEL	0.035	0.037	2.015	150.83	56.40%	42.10%	1.60%	1.42	0.81	GU
19	R2567E1	BXD	BXD16	60	M	R2567E.CEL	0.048	0.058	2.24	82	56.70%	41.60%	1.70%	1.37	0.75	Glenn
20	R2711E	BXD	BXD16	61	F	R2711E.CEL	0.032	0.021	1.953	118.53	59.00%	39.60%	1.50%	1.45	0.8	GU
21	R2559E1	BXD	BXD18	59	M	R2559E.CEL	0.01	0.012	1.65	104	60.80%	37.70%	1.50%	1.27	0.78	Glenn
22	R2720E	BXD	BXD18	59	F	R2720E.CEL	0.014	0.019	2.32	99.93	59.50%	39.00%	1.50%	1.33	0.77	GU
23	R2560E1	BXD	BXD19	60	F	R2560E.CEL	0.009	0.012	1.79	98	60.90%	37.50%	1.60%	1.35	0.8	Glenn
24	R2713E	BXD	BXD19	60	M	R2713E.CEL	0.055	0.021	1.67	120.82	60.20%	38.30%	1.50%	1.45	0.8	GU
25	R2597E1	BXD	BXD2	61	M	R2597E.CEL	0.005	0.012	2.37	94	60.30%	38.30%	1.50%	1.34	0.77	Glenn
26	R1231E	BXD	BXD2	64	F	R1231E.CEL	0.044	0.037	2.197	138.73	57.30%	41.30%	1.40%	1.41	0.77	GU
27	R2584E1	BXD	BXD20	59	F	R2584E.CEL	0.011	0.017	2.07	84	59.30%	39.10%	1.60%	1.4	0.76	Glenn
28	R2731E	BXD	BXD20	60	M	R2731E.CEL	0.017	0.019	1.825	147	59.00%	39.50%	1.50%	1.4	0.8	GU
29	R2541E2	BXD	BXD21	61	M	R2541E2.CEL	0.049	0.084	2.63	125	56.00%	42.40%	1.50%	1.29	0.78	UTM RW
30	R2702E	BXD	BXD21	59	F	R2702E.CEL	0.009	0.008	1.811	128.65	59.40%	39.10%	1.40%	1.26	0.8	GU
31	R2553E1	BXD	BXD22	58	F	R2553E.CEL	0.004	0.01	1.95	111	59.90%	38.50%	1.50%	1.28	0.76	Glenn
32	R2700E	BXD	BXD22	59	M	R2700E.CEL	0.01	0.015	1.858	102.96	61.50%	37.10%	1.30%	1.48	0.79	GU
33	R2558E1	BXD	BXD23	60	F	R2558E-2.CEL	0.018	0.027	1.91	115	59.90%	38.80%	1.40%	1.2	0.82	Glenn
34	R1086E	BXD	BXD23	55	M	R1086E.CEL	0.043	0.034	2.233	125.05	58.60%	39.90%	1.50%	1.43	0.77	GU
35	R2589E2	BXD	BXD24	59	M	R2589E2.CEL	0.132	0.176	2.61	112	57.50%	40.90%	1.60%	1.24	0.8	Glenn
36	R2719E	BXD	BXD24	123	F	R2719E.CEL	0.112	0.111	1.47	140.38	61.50%	37.20%	1.30%	1.38	0.79	GU
37	R2573E1	BXD	BXD25	67	F	R2573E-2.CEL	0.055	0.063	3.15	72	57.90%	40.70%	1.40%	1.77	0.97	UAB
38	R2683E	BXD	BXD25	58	M	R2683E.CEL	0.068	0.068	1.777	115.64	58.30%	40.30%	1.40%	2.01	0.79	GU
39	R2703E	BXD	BXD27	60	F	R2703E.CEL	0.008	0.012	1.263	134.78	62.60%	36.10%	1.40%	1.44	0.78	GU
40	R2721E	BXD	BXD28	60	M	R2721E.CEL	0.04	0.048	2.065	157.39	56.10%	42.40%	1.50%	1.31	0.81	GU
41	R2562E1	BXD	BXD29	60	M	R2562E.CEL	0.007	0.01	1.65	116	59.90%	38.40%	1.70%	1.37	0.79	Glenn
42	R2598E1	BXD	BXD31	61	M	R2598E.CEL	0.006	0.013	1.99	106	60.90%	37.60%	1.50%	1.27	0.78	UTM RW
43	R1258E	BXD	BXD31	57	F	R1258E.CEL	0.037	0.036	2.063	117.09	59.00%	39.50%	1.50%	1.54	0.78	GU
44	R2563E1	BXD	BXD32	63	F	R2563E.CEL	0.023	0.025	1.55	102	61.90%	36.70%	1.40%	1.5	0.8	UTM RW
45	R1216E	BXD	BXD32	76	M	R1216E.CEL	0.05	0.049	2.23	111.99	58.80%	39.80%	1.40%	1.35	0.79	GU
46	R2542E1	BXD	BXD33	67	F	R2542E.CEL	0.058	0.062	2.13	97	56.50%	41.80%	1.60%	1.91	0.93	UTM RW
47	R857E	BXD	BXD33	77	M	R857E.CEL	0.078	0.108	1.737	113.98	61.90%	36.70%	1.30%	1.6	0.77	GU
48	R2585E1	BXD	BXD34	60	M	R2585E.CEL	0.024	0.032	2.64	75	58.30%	40.00%	1.70%	1.25	0.77	Glenn
49	R1451E	BXD	BXD34	61	F	R1451E.CEL	0.01	0.009	1.843	140.05	59.00%	39.50%	1.50%	1.42	0.81	GU
50	R2532E1	BXD	BXD38	62	M	R2532E.CEL	0.002	0.006	2.04	94	59.80%	38.70%	1.50%	1.37	0.8	UTM RW
51	R2710E	BXD	BXD38	55	F	R2710E.CEL	0.033	0.031	2.112	122.1	58.80%	39.80%	1.40%	1.37	0.78	GU
52	R2574E1	BXD	BXD39	70	F	R2574E.CEL	0.003	0.008	1.98	91	61.20%	37.30%	1.50%	1.39	0.78	UTM RW
53	R2695E	BXD	BXD39	59	M	R2695E.CEL	0.018	0.016	1.638	122.7	60.80%	37.80%	1.50%	1.42	0.8	GU
54	R2590E1	BXD	BXD40	60	M	R2590E.CEL	0.007	0.012	2.71	77	59.10%	39.30%	1.50%	1.4	0.77	Glenn
55	R2699E	BXD	BXD40	59	F	R2699E.CEL	0.014	0.015	1.827	105.23	61.70%	36.90%	1.40%	1.42	0.81	GU
56	R2596E1	BXD	BXD42	59	M	R2596E.CEL	0.016	0.03	2.63	108	59.00%	39.60%	1.50%	1.24	0.8	Glenn
57	R2696E	BXD	BXD42	58	F	R2696E.CEL	0.01	0.017	1.622	118.95	62.00%	36.60%	1.50%	1.53	0.79	GU
58	R2605E1	BXD	BXD43	79	M	R2607E.CEL	0.006	0.01	1.82	131	60.50%	38.20%	1.30%	1.32	0.8	UTM RW
59	R994E	BXD	BXD43	60	F	R994E.CEL	0.013	0.014	1.966	113.12	60.80%	37.80%	1.40%	1.66	0.8	GU
60	R2594E1	BXD	BXD44	63	F	R2594E.CEL	0.014	0.024	1.77	117	59.80%	38.80%	1.40%	1.35	0.85	UTM RW
61	R2610E	BXD	BXD44	68	M	R2610E.CEL	0.013	0.009	1.814	142.91	59.00%	39.50%	1.50%	1.35	0.8	GU
62	R2592E1	BXD	BXD45	62	M	R2592E.CEL	0.005	0.011	1.85	106	60.10%	38.60%	1.30%	1.43	0.85	UTM RW
63	R2732E	BXD	BXD45	63	F	R2732E.CEL	0.039	0.036	2.154	122.45	56.50%	42.10%	1.40%	1.8	0.83	GU
64	R2606E1	BXD	BXD48	78	M	R2606E.CEL	0.007	0.015	2.56	106	58.90%	39.70%	1.40%	1.35	0.83	UTM RW
65	R967E	BXD	BXD48	64	F	R967E.CEL	0.101	0.052	1.948	130.95	57.30%	41.20%	1.50%	1.63	0.81	GU
66	R2591E1	BXD	BXD5	60	F	R2591E.CEL	0.052	0.014	1.7	136	58.50%	40.00%	1.50%	1.33	0.78	Glenn
67	R2714E	BXD	BXD5	58	M	R2714E.CEL	0.047	0.014	1.404	144.35	60.60%	37.90%	1.50%	1.43	0.79	GU
68	R2603E1	BXD	BXD51	66	F	R2603E.CEL	0.007	0.02	2.49	115	57.70%	40.80%	1.50%	1.24	0.79	UTM RW
69	R1042E	BXD	BXD51	62	M	R1042E.CEL	0.028	0.027	2.352	104.12	58.70%	39.90%	1.40%	1.53	0.82	GU
70	R2690E	BXD	BXD55	65	M	R2690E.CEL	0.081	0.067	1.887	164.01	56.10%	42.30%	1.60%	1.43	0.8	GU
71	R2570E1	BXD	BXD6	65	F	R2570E.CEL	0.013	0.017	1.99	87	58.50%	40.00%	1.50%	1.46	0.76	UTM RW
72	R2694E	BXD	BXD6	58	M	R2694E.CEL	0.012	0.018	1.983	97.23	61.60%	37.10%	1.30%	1.39	0.82	GU
73	R2534E2	BXD	BXD61	70	F	R2534E2.CEL	0.03	0.058	2.47	118	57.90%	40.60%	1.50%	1.42	0.79	UTM RW
74	R2684E	BXD	BXD61	62	M	R2684E.CEL	0.031	0.032	2.01	131.03	57.00%	41.50%	1.50%	1.34	0.78	GU
75	R2611E1	BXD	BXD64	68	M	R2611E.CEL	0.067	0.068	2.29	92	58.00%	40.50%	1.50%	1.57	1.06	UTM RW
76	R943E-2	BXD	BXD64	56	F	R943E-2.CEL	0.024	0.021	1.591	141.34	60.10%	38.40%	1.50%	1.32	0.76	GU
77	R2583E1	BXD	BXD65	60	M	R2583E.CEL	0.027	0.03	2.49	70	56.90%	41.50%	1.60%	1.67	1.01	UTM RW
78	R2689E	BXD	BXD65	63	F	R2689E.CEL	0.008	0.008	1.721	142.44	59.90%	38.60%	1.50%	1.38	0.76	GU
79	R2536E2	BXD	BXD66	64	F	R2536E2.CEL	0.067	0.139	2.74	109	56.10%	42.30%	1.70%	1.28	0.79	UTM RW
80	R1207E	BXD	BXD66	83	M	R1207E.CEL	0.017	0.012	1.681	136.86	60.40%	38.10%	1.50%	1.45	0.77	GU
81	R2551E1	BXD	BXD68	67	F	R2551E.CEL	0.294	0.291	2.49	92	54.30%	44.10%	1.60%	2.91	1.55	UTM RW
82	R2726E	BXD	BXD68	64	M	R2726E.CEL	0.125	0.025	1.811	153.09	58.70%	39.80%	1.50%	1.39	0.78	GU
83	R2593E1	BXD	BXD69	59	F	R2593E.CEL	0.027	0.038	1.67	128	59.20%	39.50%	1.30%	1.47	0.92	UTM RW
84	R2727E	BXD	BXD69	65	M	R2727E.CEL	0.01	0.008	1.578	143.86	60.30%	38.30%	1.40%	1.34	0.77	GU
85	R2537E2	BXD	BXD70	59	M	R2537E2.CEL	0.049	0.092	2.93	99	58.00%	40.50%	1.60%	1.29	0.75	UTM RW
86	R975E	BXD	BXD70	64	F	R975E.CEL	0.028	0.024	1.841	137.97	58.00%	40.50%	1.40%	1.36	0.79	GU
87	R2779E	BXD	BXD73	64	F	R2779E.CEL	0.012	0.038	1.746	121.11	59.60%	39.00%	1.40%	1.5	0.8	GU
88	R2565E1	BXD	BXD75	61	F	R2565E.CEL	0.118	0.124	1.79	102	58.00%	40.50%	1.50%	2.31	3.47	UTM RW
89	R1397E-re	BXD	BXD75	58	M	R1397E-re.CEL	0.032	0.01	1.449	189.71	59.60%	39.00%	1.40%	1.39	0.82	GU
90	R2538E1	BXD	BXD8	77	F	R2538E.CEL	0.033	0.056	1.91	102	61.20%	37.30%	1.50%	1.52	0.79	UTM RW
91	R2709E	BXD	BXD8	61	M	R2709E.CEL	0.012	0.011	1.99	99.79	60.90%	37.60%	1.50%	1.42	0.76	GU
92	R2579E1	BXD	BXD80	65	F	R2579E.CEL	0.013	0.026	2.42	72	59.20%	39.40%	1.50%	1.73	0.82	UTM RW
93	R2686E	BXD	BXD80	61	M	R2686E.CEL	0.046	0.05	2.342	119.63	56.00%	42.60%	1.50%	1.38	0.79	GU
94	R2692E	BXD	BXD85	63	F	R2692E.CEL	0.006	0.007	1.423	160.87	60.20%	38.30%	1.40%	1.46	0.79	GU
95	R2715E	BXD	BXD85	91	M	R2715E.CEL	0.007	0.008	1.488	142.6	61.20%	37.30%	1.40%	1.5	0.78	GU
96	R1405E	BXD	BXD86	58	F	R1405E.CEL	0.053	0.052	2.351	119.34	56.40%	42.20%	1.40%	1.64	0.81	GU
97	R2540E1	BXD	BXD87	63	M	R2540E.CEL	0.014	0.034	2.33	93	61.10%	37.40%	1.40%	1.22	0.81	UTM RW
98	R2724E	BXD	BXD87	63	F	R2724E.CEL	0.013	0.019	1.906	113.71	60.70%	37.90%	1.40%	1.45	0.79	GU
99	R2545E1	BXD	BXD89	67	M	R2546E.CEL	0.266	0.257	1.67	105	56.20%	42.30%	1.50%	3.6	9.84	UTM RW
100	R1433E	BXD	BXD89	63	F	R1433E.CEL	0.029	0.026	2.241	115.86	57.70%	40.80%	1.50%	1.41	0.78	GU
101	R2569E1	BXD	BXD9	67	M	R2569E.CEL	0.256	0.239	1.75	87	55.10%	43.40%	1.50%	2.82	3.14	UTM RW
102	R2708E	BXD	BXD9	60	F	R2708E.CEL	0.024	0.045	1.966	126.46	57.70%	40.70%	1.50%	1.4	0.84	GU
103	R2578E2	BXD	BXD90	61	F	R2578E2.CEL	0.041	0.062	2.79	92	58.60%	39.80%	1.60%	1.52	0.77	UTM RW
104	R859E	BXD	BXD90	72	M	R859E.CEL	0.028	0.02	1.847	152.22	57.90%	40.70%	1.40%	1.36	0.77	GU
105	R2554E1	BXD	BXD96	67	M	R2554E.CEL	0.005	0.008	2.18	93	60.20%	38.30%	1.50%	1.46	0.77	UTM RW
106	R2733E	BXD	BXD96	67	F	R2733E.CEL	0.024	0.054	1.7	113.99	62.10%	36.60%	1.30%	1.4	0.78	GU
107	R2577E1	BXD	BXD97	55	M	R2577E.CEL	0.065	0.069	2.07	77	59.50%	39.10%	1.40%	1.87	1.29	UTM RW
108	R2649E	BXD	BXD97	74	F	R2649E.CEL	0.029	0.032	2.343	119.04	57.50%	41.20%	1.40%	1.53	0.8	GU
109	R2688E	BXD	BXD98	67	M	R2688E.CEL	0.032	0.03	1.772	145.24	58.50%	40.00%	1.50%	1.48	0.81	GU
110	R1700E1	GDP	C3H/HeJ	83	F	R1700E.CEL	0.152	0.168	2.98	69	60.80%	37.90%	1.40%	1.48	0.78	UTM RW
111	R1704E1	GDP	C3H/HeJ	83	M	R1704E.CEL	0.154	0.165	2.58	88	60.10%	38.60%	1.30%	1.38	0.84	UTM RW
112	R0872E2	GDP BXD	C57BL/6J	66	M	R0872E.CEL	0.014	0.023	3.13	89	58.90%	39.60%	1.50%	1.3	0.79	UTM RW
113	R2607E1	GDP BXD	C57BL/6J	67	F	R2605E.CEL	0.008	0.018	2.43	115	58.60%	40.00%	1.40%	1.31	0.76	UTM RW
114	R2564E1	GDP	CAST/Ei	64	F	R2564E.CEL	0.124	0.105	1.94	89	58.50%	39.90%	1.60%	1.6	0.77	JAX
115	R2580E1	GDP	CAST/Ei	64	M	R2580E.CEL	0.123	0.109	2.09	95	58.20%	40.10%	1.70%	1.4	0.76	JAX
116	R2600E1	GDP BXD	D2B6F1	72	F	R2600E.CEL	0.008	0.02	2.47	95	58.10%	40.20%	1.70%	1.41	0.78	UTM RW
117	R2604E1	GDP BXD	D2B6F1	69	M	R2604E.CEL	0.005	0.014	2.66	90	59.40%	39.20%	1.50%	1.28	0.79	UTM RW
118	R2572E1	GDP BXD	DBA/2J	65	M	R2572E.CEL	0.091	0.106	2.41	79	55.50%	42.90%	1.60%	1.37	0.79	UTM RW
119	R2636E1	GDP	KK/HIJ	64	F	R2636E.CEL	0.044	0.043	2.61	93	58.90%	39.50%	1.50%	1.39	0.76	UTM RW
120	R2637E1	GDP	KK/HIJ	64	M	R2637E.CEL	0.056	0.036	2.19	103	59.40%	39.00%	1.50%	1.3	0.79	UTM RW
121	R0999E1	GDP	LG/J	57	F	R0999E.CEL	0.021	0.023	2.45	82	59.40%	39.10%	1.50%	1.38	0.79	UTM RW
122	R1004E1	GDP	LG/J	65	M	R1004E.CEL	0.025	0.028	2.44	92	58.70%	39.80%	1.50%	1.38	0.79	UTM RW
123	R1688E1	GDP	NOD/LtJ	66	F	R1688E.CEL	0.028	0.033	2.66	98	58.60%	39.90%	1.50%	1.26	0.8	JAX
124	R2566E1	GDP	NOD/LtJ	76	M	R2566E-2.CEL	0.036	0.04	3.03	69	59.80%	38.80%	1.50%	1.38	0.75	UTM RW
125	R2535E1	GDP	NZO/H1LtJ	62	F	R2535E.CEL	0.037	0.062	1.89	86	60.40%	38.20%	1.40%	1.41	0.85	JAX
126	R2550E1	GDP	NZO/HILtJ	96	M	R2550E.CEL	0.025	0.029	1.79	87	60.70%	37.80%	1.50%	1.52	0.82	JAX
127	R2634E1	GDP	PWD/PhJ	62	F	R2635E.CEL	0.126	0.114	3.29	90	55.90%	42.50%	1.60%	1.57	0.81	JAX
128	R2635E1	GDP	PWD/PhJ	62	M	R2634E.CEL	0.15	0.137	3.72	80	54.20%	44.10%	1.70%	1.53	0.85	JAX
129	R2544E1	GDP	PWK/PhJ	63	F	R2544E.CEL	0.174	0.175	2.2	108	54.90%	43.50%	1.70%	1.36	0.82	JAX
130	R2549E1	GDP	PWK/PhJ	83	M	R2549E.CEL	0.103	0.087	2.28	84	57.30%	41.20%	1.50%	1.57	0.83	JAX
131	R2368E1	GDP	WSB/EI	67	F	R2368E.CEL	0.041	0.047	2.57	86	59.50%	39.10%	1.40%	1.29	0.74	UTM RW
132	R2547E1	GDP	WSB/Ei	67	M	R2547E.CEL	0.041	0.039	2.14	90	58.20%	40.10%	1.60%	1.32	0.77	UTM RW

    About downloading this data set:

This data set is not yet available as a bulk download. Please contact Robert W. Williams to request special data access.

    About the array platfrom:

Affymetrix Mouse Genome 430 2.0 arrays: The 430 2.0 array consists of 992936 25-nucleotide probes that estimate the expression of approximately 39,000 transcripts (many are near duplicates). The array sequences were selected late in 2002 using Unigene Build 107. The array nominally contains the same probe sequences as the old M430A and 430B array pair. However, we have found that roughy 75000 probes differ between those on A and B arrays and those on the new 430 2.0.

    About data processing:

Probe (cell) level data from the CEL file: These CEL values produced by GCOS are 75% quantiles from a set of 91 pixel values per cell. The CEL files were processed using the RMA protocol. We processed the three batches together in RMA.

• Step 1: We added an offset of 1.0 unit to each cell signal to ensure that all values could be logged without generating negative values. We then computed the log base 2 of each cell.
• Step 2: We performed a quantile normalization of the log base 2 values for the total set of arrays using the same initial steps used by the RMA transform.
• Step 3: We computed the Z scores for each cell value.
• Step 4: We multiplied all Z scores by 2.
• Step 5: We added 8 to the value of all Z scores. The consequence of this simple set of transformations is to produce a set of Z scores that have a mean of 8, a variance of 4, and a standard deviation of 2. The advantage of this modified Z score is that a two-fold difference in expression level corresponds approximately to a 1 unit difference.
• Step 7: Finally, when appropriate, we computed the arithmetic mean of the values for the set of microarrays for each strain. Technical replicates were averaged before computing the mean for independent biological samples.

After RMA processing all arrays were rank-order normalized. This second round of quantile normalization removes much residual non-linearity across arrays and forces every array to have the same distribution of values as the mean of all arrays. Comparative array data quality was then evaluated in DataDesk. Outlier arrays were flagged by visual inspection in DataDesk, usually by means of an analysis of scatter plots and more quantitatively by generating a correlation matrix of all arrays. Those arrays with mean correlation <0.96 versus all other arrays indicates trouble or a biological outlier). In some cases, outliers were expected, such as samples from strains with retinal degeneration (FVB/NJ, NOD/LtJ, MOLF/EiJ, C3H/HeJ and BXD24) and samples from wild subspecies such as WSB/EiJ, CAST/Ei, PWD/Ph, and PWK/Ph. However, when arrays were anomolous both within strain and across strains, they were often simply discarded. We tended to keep arrays that "conformed" to the expectation. The assumption in these cases is that anomolous data are much more likely due to experimental problems and errors than to informative biological variation. Approximately 11 (CHECK) arrays total were discarded in batches 1, 2, and 3 combined.

After this process, the acceptable set of arrays was renormalized using all step as above, starting with the original RMA procedure, etc.

We reviewed the data set using a new method developed by RW Williams, Jeremy Peirce, and Hongqiang Li. For the full set of arrays that passed standard QC protocols described above, we computed the strain means for the BXD strains, B6, D2, and F1s. Using this set of strain means we then computed LRS scores for all 45101 probe sets and counted the number of transcripts that generated QTLs with LRS values greater than 50. This value (e.g. 1800) represented the QTL harvest for the full data set. We then dropped a single array from the data set, recomputed strain means, and recomputed the number of transcripts with LRS scores great than 50. This value is expected to typically reduce the number of QTLs that reach the criterion level (e.g., 1750 QTLs > 50). This process was repeated for every array to obtain an array-specific difference value--the effect of removing that array on the total QTL count. For example, the loss of a single array might cause a decrease in 50 QTLs. Values ranged from approximately -90 (good arrays) to +40 (bad arrays). This procedure is similar in some ways to a jackknife protocol, although we are not using this procedure to esimate an error term, but rather as a method to polish a data set.

During this final process we discovered that nearly XX arrays in the second batch had been mislabeled at some point in processing. We computed the correct strain membership of each array using a large number of Mendelian probe sets (more than 50) and comparing their match to standard SNP and microsatellite markers and the original array data set of November 2005. This allowed us to rescue a large number of arrays that were of very high quality.

A third batch of approximately 40 arrays were processed by Yan Jiao and Wiekuan Gu in August 2006. These complete data set assembled by Hongqiang Li. This process again included a correction for a batch effect.

For this June 2006 data set Hongqiang Li used a new batch correction method that stabilizes the range of expression in each batch. For each of the three large batches, we extracted the minumum and maximum raw probe expression (CEL file level) value. We then adjusted raw probe values in each batch to have the same range as the largest batch (Batch 1) using a simple linear interpolation. These procedure generated new correct CEL files which were then used with RMA. (note added by RWW and HQL, Oct 19, 2006)

    Data source acknowledgment:

Support for acquisition of microarray data sets was generously provided by Dr. Barrrett Haik, Chair of the Department of Ophthalmology, and director of the Hamilton Eye Institute. Support for the continued development of GeneNetwork was provided by a NIDA/NIMH/NIAAA Human Brain Project grant. All arrays were processed at the VA Medical Center, Memphis by Yan Jiao and Weikuan Gu.

    Information about this text file:

This text file originally generated by RWW, May 26, 2006. Updated by RWW, Oct 10, 2006.