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Summary
SUPERCEDED EYE DATA SET. The HEIMED April 2006 data set provides estimates of mRNA expression in whole eyes of 71 lines of young adult mice generated using 132 Affymetrix M430 2.0 arrays. 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 straion. 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.
About cases
We used a set of 55 BXD recombinant inbred strains, 14 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 14 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 are currently being resequenced by Perlegen for the NIEHS.
- 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. - A/J
Collaborative Cross strain sequenced by Perlegen/NIEHS; parent of the AXB/BXA panel. A tyrosinase-negative albino (c) mutant - BALB/cByJ
Sequenced by NIEHS; maternal parent of the CXB panel; Phenome Project A list. A tyrosinase-negative albino (c) mutant - 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 maturity. - C57BL/6J
Sequenced by NHGRI; parental strain of AXB/BXA, BXD, and BXH; Phenome Project A list - CAST/Ei
Collaborative Cross strain sequenced by NIEHS; Phenome Project A list - DBA/2J
Sequenced by Perlegen/NIEHS and Celera; paternal parent of the BXD panel; Phenome Project A list - KK/HlJ
Sequenced by Perlegen/NIEHS - LG/J
Paternal parent of the LGXSM panel - NOD/LtJ
Collaborative Cross strain sequenced by NIEHS; Phenome Project B list; diabetic - NZO/HlLtJ
Collaborative Cross strain - PWD/PhJ
Sequenced by Perlegen/NIEHS; parental strain for a consomic set by Forjet and colleagues - PWK/PhJ
Collaborative Cross strain; Phenome Project D list - WSB/EiJ
Collaborative Cross strain sequenced by NIEHS; Phenome Project C list - 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 tissue
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
- Place eyes for RNA extraction in RNA STAT-60 (Tel-Test Inc.) and process per manufacturer’s instructions (in brief form below).
- 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:
- homogenize tissue samples in the RNA STAT-60 (1 ml/50 to 100 mg tissue)
- allowed the homogenate to stand for 5 min at room temperature
- added 0.2 ml of chloroform per 1 ml RNA STAT-60
- shook the sample vigorously for 15 sec and let the sample sit at room temperature for 3 min
- centrifuged at 12,000 G for 15 min
- transfered the aqueous phase to a fresh tube
- added 0.5 ml of isopropanol per 1 ml RNA STAT-60
- vortexed and allowed sample to stand at room temperature for 5-10 min
- centrifuged at 12,000 G for 10-15 min
- removed the supernatant and washed the RNA pellet with 75% ethanol
- 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 batch added in April 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 first and second batches of array data, collectively represents a reasonably well balanced sample of males and females belonging to 62 strains, but without within-strain-by-sex replication. Six strains are represented only by male sample pools (BXD15, 28, 29, 55, 98, and DBA/2J. Four strains are represented only by a female pool sample (BXD1, 27, 73 and 86). 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 two batches: the original batch that makes up the November 2005 data set and a new batch of 63 arrays (R0857E through R2649E, and R2682E through R2742E, non-consecutive identifiers) run in January 2006 by Dr. Yan Jiao. The arrays in the two batches are from two different lots. All arrays in the second batch were from Lot 4016879 (expiration date 12.28.06). We started working with a total of 140 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 130 arrays were finally approved for inclusion in this April 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 BATCH 2 OF EARLY 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 |
2.60 |
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 |
0.80 |
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 |
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 |
|
10 |
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 |
|
11 |
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 |
|
12 |
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 |
|
13 |
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 |
|
14 |
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 |
|
15 |
R2560E1 |
BXD |
BXD19 |
60 |
F |
R2560E.CEL |
0.009 |
0.012 |
1.79 |
98 |
60.90% |
37.50% |
1.60% |
1.35 |
0.80 |
Glenn |
|
16 |
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 |
|
17 |
R2584E1 |
BXD |
BXD20 |
59 |
F |
R2584E.CEL |
0.011 |
0.017 |
2.07 |
84 |
59.30% |
39.10% |
1.60% |
1.40 |
0.76 |
Glenn |
|
18 |
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 |
|
19 |
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 |
|
20 |
R2558E1 |
BXD |
BXD23 |
60 |
F |
R2558E-2.CEL |
0.018 |
0.027 |
1.91 |
115 |
59.90% |
38.80% |
1.40% |
1.20 |
0.82 |
Glenn |
|
21 |
R2589E2 |
BXD |
BXD24 |
59 |
M |
R2589E2.CEL |
0.132 |
0.176 |
2.61 |
112 |
57.50% |
40.90% |
1.60% |
1.24 |
0.80 |
Glenn |
|
22 |
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 |
|
23 |
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 |
|
24 |
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 |
|
25 |
R2563E1 |
BXD |
BXD32 |
63 |
F |
R2563E.CEL |
0.023 |
0.025 |
1.55 |
102 |
61.90% |
36.70% |
1.40% |
1.50 |
0.80 |
UTM RW |
|
26 |
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 |
|
27 |
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 |
|
28 |
R2532E1 |
BXD |
BXD38 |
62 |
M |
R2532E.CEL |
0.002 |
0.006 |
2.04 |
94 |
59.80% |
38.70% |
1.50% |
1.37 |
0.80 |
UTM RW |
|
29 |
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 |
|
30 |
R2590E1 |
BXD |
BXD40 |
60 |
M |
R2590E.CEL |
0.007 |
0.012 |
2.71 |
77 |
59.10% |
39.30% |
1.50% |
1.40 |
0.77 |
Glenn |
|
31 |
R2596E1 |
BXD |
BXD42 |
59 |
M |
R2596E.CEL |
0.016 |
0.03 |
2.63 |
108 |
59.00% |
39.60% |
1.50% |
1.24 |
0.80 |
Glenn |
|
32 |
R2605E1 |
BXD |
BXD43 |
79 |
M |
R2607E.CEL |
0.006 |
0.01 |
1.82 |
131 |
60.50% |
38.20% |
1.30% |
1.32 |
0.80 |
UTM RW |
|
33 |
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 |
|
34 |
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 |
|
35 |
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 |
|
36 |
R2591E1 |
BXD |
BXD5 |
60 |
F |
R2591E.CEL |
0.052 |
0.014 |
1.70 |
136 |
58.50% |
40.00% |
1.50% |
1.33 |
0.78 |
Glenn |
|
37 |
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 |
|
38 |
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 |
|
39 |
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 |
|
40 |
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 |
|
41 |
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 |
|
42 |
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 |
|
43 |
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 |
|
44 |
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 |
|
45 |
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 |
|
46 |
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 |
|
47 |
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 |
|
48 |
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 |
|
49 |
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 |
|
50 |
R2545E1 |
BXD |
BXD89 |
67 |
M |
R2546E.CEL |
0.266 |
0.257 |
1.67 |
105 |
56.20% |
42.30% |
1.50% |
3.60 |
9.84 |
UTM RW |
|
51 |
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 |
|
52 |
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 |
|
53 |
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 |
|
54 |
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 |
|
55 |
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 |
|
56 |
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 |
|
57 |
R0872E2 |
GDP BXD |
C57BL/6J |
66 |
M |
R0872E.CEL |
0.014 |
0.023 |
3.13 |
89 |
58.90% |
39.60% |
1.50% |
1.30 |
0.79 |
UTM RW |
|
58 |
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 |
|
59 |
R2564E1 |
GDP |
CAST/Ei |
64 |
F |
R2564E.CEL |
0.124 |
0.105 |
1.94 |
89 |
58.50% |
39.90% |
1.60% |
1.60 |
0.77 |
JAX |
|
60 |
R2580E1 |
GDP |
CAST/Ei |
64 |
M |
R2580E.CEL |
0.123 |
0.109 |
2.09 |
95 |
58.20% |
40.10% |
1.70% |
1.40 |
0.76 |
JAX |
|
61 |
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 |
|
62 |
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 |
|
63 |
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 |
|
64 |
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 |
|
65 |
R2637E1 |
GDP |
KK/HIJ |
64 |
M |
R2637E.CEL |
0.056 |
0.036 |
2.19 |
103 |
59.40% |
39.00% |
1.50% |
1.30 |
0.79 |
UTM RW |
|
66 |
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 |
|
67 |
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 |
|
68 |
R1688E1 |
GDP |
NOD/LtJ |
66 |
F |
R1688E.CEL |
0.028 |
0.033 |
2.66 |
98 |
58.60% |
39.90% |
1.50% |
1.26 |
0.80 |
JAX |
|
69 |
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 |
|
70 |
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 |
|
71 |
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 |
|
72 |
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 |
|
73 |
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 |
|
74 |
R2544E1 |
GDP |
PWK/PhJ |
63 |
F |
R2544E.CEL |
0.174 |
0.175 |
2.20 |
108 |
54.90% |
43.50% |
1.70% |
1.36 |
0.82 |
JAX |
|
75 |
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 |
|
76 |
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 |
|
77 |
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 |
|
78 |
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 |
|
79 |
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 |
|
80 |
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 |
|
81 |
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 |
|
82 |
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 |
|
83 |
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 |
|
84 |
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 |
|
85 |
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 |
|
86 |
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 |
|
87 |
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 |
|
88 |
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 |
|
89 |
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 |
|
90 |
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 |
|
91 |
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 |
|
92 |
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 |
|
93 |
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 |
|
94 |
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 |
|
95 |
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 |
|
96 |
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 |
|
97 |
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 |
|
98 |
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 |
|
99 |
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 |
|
100 |
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 |
|
101 |
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 |
|
102 |
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 |
|
103 |
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 |
|
104 |
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 |
|
105 |
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 |
|
106 |
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 |
|
107 |
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 |
|
108 |
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 |
|
109 |
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 |
|
110 |
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 |
|
111 |
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 |
|
112 |
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 |
|
113 |
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 |
|
114 |
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 |
|
115 |
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 |
|
116 |
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 |
|
117 |
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 |
|
118 |
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 |
|
119 |
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 |
|
120 |
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 |
|
121 |
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 |
|
122 |
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 |
|
123 |
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 |
|
124 |
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 |
|
125 |
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 |
|
126 |
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 |
|
127 |
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 |
|
128 |
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 |
|
129 |
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 |
|
130 |
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 |
|
131 |
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 |
|
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 platform
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 two 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 (C3H/HeJ and BXD24) and samples from wild subspecies such as 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 problem and errors than to informative biological variation. Approximately 8 arrays total were discarded in batches 1 and 2 combined.
After this process, the acceptable set of arrays was renormalized using all step as above, starting with the original RMA procedure, etc.
We then categorized arrays into XXX major "technical groups" depending on expression patterns as noted in scatterplots. This process of defining technical groups was done in DataDesk by manually "typing" arrays. These technical groups are apparently due to subtle within-batch effect that we do not yet understand and that cannot be corrected by quantile normalization. These XXX major technical groups are not obviously related to strain, sex, age, or any other known biological effect or variable. They are also not obviously related to any of the Affymetrix QC data types (3'/5' ratios, gain, etc.). Once the technical groups were defined, we forced the means of each probe set in the XX technical groups to the same value. This simple process partially removes a technical error of unknown origin in large expression array data sets.
We reviewed the final data set using a new method developed by RW Williams, Jeremy Peirce, and Hongqiang Li. For the full set of 140 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. 1000) represented the QTL harvest for the full data set. We then dropped a single array from the data set (n = 139 arrays), 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., 950 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 (1000-950). Values ranged from -90 (good0 to +38 (bad). 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 final method to polish a data set. By applying this procedure we discovered that a set of XX (7?) arrays could be excluded while simultaneously improving the total number of QTLs with values above 50.
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.
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 Weikuan Gu.
Notes
This study includes the following datasets:
-
Eye M430v2 (Apr06) RMA
-
Eye M430v2 (Apr06) PDNN
-
Eye M430v2 (Apr06) MAS5
-
Eye M430v2 (Sep06) RMA
This text file originally generated by RWW, May 26, 2006. Updated by RWW, May 27, 2006.