Hippocampus Consortium M430v2 (Oct05) PDNN

    Summary:

PRELIMINARY: The October 2005 Hippocampus Consortium data set provides estimates of mRNA expression in the adult hippocampus of approximately 99 genetically diverse strains of mice including 68 BXD recombinant inbred strains, 13 CXB recombinant inbred strains, a set of 16 diverse inbred strains, and 2 reciprocal F1 hybrids. The hippocampus is an important and intriguing part of the forebrain that is crucial for memory formation, and that is often affected in epilepsy, Alzheimer's disease, and schizophrenia. Unlike most other parts of the brain, the hippocampus contains a remarkable population of stems cells that continue to generate neurons and glial cells even in adult mammals (Kempermann, 2005). This genetic analysis of transcript expression in the hippocampus (dentate gyrus, CA1-CA3, and parts of the subiculum) is a joint effort of 14 investigators that is supported by numerous agencies described in the acknowledgments section. Samples were processed using a total of 206 Affymetrix Mouse Expression 430 2.0 short oligomer microarrays (MOE430 2.0 or M430v2), of which 179 passed stringent quality control and error checking. This particular data set was processed using the position-dependent nearest neighbor method (PDNN) of Zhang and colleagues. To simplify comparison among the transforms we have used, the quantile normalized PDNN values from each arrray have been adjusted to an average expression of 8 units and a standard deviation of 2 units.

    About the strains used to generate this set of data:

This analysis has used 68 of BXD strains, the complete set of 13 CXB recombinant inbred strain sets, and a mouse diversity panel consisting of 16 inbred strains and a pair of reciprocal F1 hybrids (B6D2F1 and D2B6F1).

The BXD genetic reference population of recombinant inbred strains consists of approximately 80 strains. Approximately 800 classical phenotypes from sets of 10 to 70 of these strains been integrated in the GeneNetwork. The BXD strains in this data set include 29 of the BXD strains made by Benjamin Taylor at the Jackson Laboratory in the 1970s and 1990s (BXD1 through BXD40). All of these strains are fully inbred, many well beyond the 100th filial (F) generation of inbreeding. We have also included 39 inbred (25 strains at F20+) and nearly inbred (14 strains between F14 and F20) BXD lines generated by Lu and Peirce. All of these strains, including those between F14 and F20, have been genotyped at 13,377 SNPs.

The CXB is the first and oldest set of recombinant inbred strains. Over 500 classical phenotypes from these strains been integrated in the GeneNetwork. It is noteworthy that the CXB strains segregate for the hippocampal lamination defect (Hld),characterized by Nowakowski and colleagues (1984). All of the CXBs have been recently genotyped at 13,377 SNPs.

Mouse Diversity Panel (MDP). We have profiled a MDP consisting 16 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 (129, A, C57BL/6J, CAST, NOD, NZO, PWK, and WSB) have been included in the MDP (noted below in the list). Twelve MDP strains have been sequenced, or are currently being resequenced by Perlegen for the NIEHS. This panel will be extremely helpful in systems genetic analysis of a wide variety of traits, and will be a powerful adjunct in fine mapping modulators using what is essentially an association analysis of sequence variants.

1. 129S1/SvImJ
       Collaborative Cross strain sequenced by NIEHS; background for many knockouts; Phenome Project A list
2. A/J
       Collaborative Cross strain sequenced by Perlegen/NIEHS; parent of the AXB/BXA panel
3. AKR/J
       Sequenced by NIEHS; Phenome Project B list
4. BALB/cByJ
       STILL IN PROGRESS (samples did not pass quality control); Sequenced by NIEHS; maternal parent of the CXB panel; Phenome Project A list
5. BALB/cJ
       Widely used strain with forebrain abnormalities (callosal defects); Phenome Project A list
6. C3H/HeJ
       Sequenced by Perlegen/NIEHS; paternal parent of the BXH panel; Phenome Project A list
7. C57BL/6J
       Sequenced by NHGRI; parental strain of AXB/BXA, BXD, and BXH; Phenome Project A list
8. C57BL/6ByJ
       Paternal substrain of B6 used to generate the CXB panel
9. CAST/Ei
       Collaborative Cross strain sequenced by NIEHS; Phenome Project A list
10. DBA/2J
        Sequenced by Perlegen/NIEHS and Celera; paternal parent of the BXD panel; Phenome Project A list
11. KK/HIJ
        Sequenced by Perlegen/NIEHS
12. LG/J
        Paternal parent of the LGXSM panel
13. NOD/LtJ
        Collaborative Cross strain sequenced by NIEHS; Phenome Project B list; diabetic
14. NZO/HILtJ
        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
18. B6D2F1 and D2B6F1
    F1 hybrids generated by crossing C57BL/6J with DBA/2J

We have not combined data from reciprocal F1s because they have different Y chromosome and mitochondial haplotypes. Parent-of-origin effects (imprinting, maternal environment) may also lead to interesting differences in hippocampal transcript levels.

These strains are available from The Jackson Laboratory. BXD43 through BXD100 are available from Lu Lu and colleagues at UTHSC.

    About the animals and tissue used to generate this set of data:

BXD animals were obtained from UTHSC, UAB, or directly from The Jackson Laboratory (see Table 1 below). Animals were housed at UTHSC, Beth Israel Deaconess, or the Jackson Laboratory before sacrifice. Virtually all CXB animals were obtained directly at the Jackson Laboratory by Lu Lu. We thanks Muriel Davission for making it possible to collect these cases on site. Standard inbred strain stock was from The Jackson Laboratory, but most animals were housed or reared at UTHSC. Mice were killed by cervical dislocation and brains were removed and placed in RNAlater prior to dissection. Cerebella and olfactory bulbs were removed; brains were hemisected, and both hippocampi were dissected whole by Hong Tao Zhang in the Lu lab. Hippocampal samples are very close to complete (see Lu et al., 2001) but probably include variable amounts of subiculum and fimbria.

A pool of dissected tissue typically from six hippocampi and three naive adults of the same strain, sex, and age was collected in one session and used to generate cRNA samples. Two-hundred and one RNA samples were extracted at UTHSC by Zhiping Jia, four samples by Shuhua Qi (R2331H1, R2332H1, P2350H1, R2349H1), and one by Siming Shou (R0129H2).

A great majority of animals used in this study were between 45 and 90 days of age (average of 66 days, maximum range from 41 to 196 days; see Table 1 below).

Sample Processing: Samples were processed in the INIA Bioanalytical Core at the W. Harry Feinstone Center for Genomic Research, The University of Memphis, led by Thomas R. Sutter. All processing steps were performed by Shirlean Goodwin. In brief, RNA purity was evaluated using the 260/280 nm absorbance ratio, and values had to be greater than 1.8. The majority of samples were 1.9 to 2.1. RNA integrity was assessed using the Agilent Bioanalyzer 2100. We required an RNA integrity number (RIN) of greater than 8. This RIN value is based on the intensity ratio and amplitude of 18S and 28S rRNA signals. The standard Eberwine T7 polymerase method was used to catalyze the synthesis of cDNA template from polyA-tailed RNA using Superscript II reverse transcriptase (Invitrogen Inc.). The Enzo Life Sciences, Inc., BioArray High Yield RNA Transcript Labeling Kit (T7, Part No. 42655) was used to synthesize labeled cRNA. The cRNA was evaluated using both the 260/280 ratio (values of 2.0 or 2.1 are acceptable) and the Bioanalyzer output (a dark cRNA smear on the 2100 output centered roughly between 600 and 2000 nucleotides is required). Those samples that passed both QC steps (10% usually failed and new RNA samples had to be acquired and processed) were then sheared using a fragmentation buffer included in the Affymetrix GeneChip Sample Cleanup Module (Part No. 900371). Fragmented cRNA samples were either stored at -80 deg. C until use or were immediately injected onto the array. The arrays were hybridized and washed following standard Affymetrix protocols.

Replication and Sample Balance: We obtained a male sample pool and female sample pool from each isogenic group. While all strains were orginally represented by matched male and female samples, not all data sets passed the final quality control steps. Seventy-seven of 99 strains are represented by pairs or (rarely) trios of arrays. The first and last samples are technical replicates of a B6D2F1 hippocampal pool (aliquotes R1291H3 and R1291H4).

Experimental Design and Batch Structure: This data set consists arrays processed in six groups over a three month period (May 2005 to August 2005). Each group consists of 32 to 34 arrays.Sex, strain, and strain type (BXD, CXB, and MDP) were interleaved among groups to ensure reasonable balance and to minimize group-by-strain statistical confounds in group normalization. The two independent samples from a single strain were always run in different groups. All arrays were processed using a single protocol by a single operator, Shirlean Goodwin.

All samples in a group were labeled on one day, except for a few cases that failed QC on their first pass. The hybridization station accommodates up to 20 samples, and for this reason each group was split into a large first set of 20 samples and a second set of 12 to 14 samples. Samples were washed in groups of four and then held in at 4 deg C until all 20 (or 12-14) arrays were ready to scan. The last four samples out of the wash stations were scanned directly. Samples were scanned in sets of four.

    Data Table 1:

This table lists all arrays by order of processing (Run), Sample ID, Strain, Sex, Age, number of animals in each sample pool (Pool), F generation number when less than 30 (GenN, and the Source of animals. SampleID is the ID number of the pooled RNA sample with a H1 through H3 suffix to indicate the actual hippocampal RNA aliquot used to prepare cRNA. Grp is the sequential group processing number (1 - 6).

Sort Run SampleID Strain Sex Age GenN Source Pool Grp Notes Data_Links_to_Affy_Files 1 4 R1509H1 BXD01 F 59 >50 GDR 4 1 u EXP RPT TXT CEL DAT 2 74 R1507H1 BXD01 M 58 >50 GDR 4 3   EXP RPT TXT CEL DAT 3 102 R1520H1 BXD02 F 56 >50 GDR 4 4   EXP RPT TXT CEL DAT 4 6 R1516H1 BXD02 M 61 >50 GDR 4 1 r EXP RPT TXT CEL DAT 5 8 R1593H2 BXD05 F 60 >50 GDR 3 1 r EXP RPT TXT CEL DAT 6 80 R1692H1 BXD05 M 60 >50 GDR 2 3   EXP RPT TXT CEL DAT 7 10 R1539H2 BXD06 F 59 >50 GDR 4 1 s EXP RPT TXT CEL DAT 8 127 R1538H1 BXD06 M 59 >50 GDR 4 4   EXP RPT TXT CEL DAT 9 12 R1518H1 BXD08 F 56 >50 GDR 4 1 t EXP RPT TXT CEL DAT 10 189 R1548H1 BXD08 M 59 >50 GDR 3 6   EXP RPT TXT CEL DAT 11 14 R1350H2 BXD09 F 86 >50 UMem 3 1 u EXP RPT TXT CEL DAT 12 117 R1351H3 BXD09 M 86 >50 UMem 3 4   EXP RPT TXT CEL DAT 13 173 R1531H1 BXD11 F 56 >50 GDR 4 6   EXP RPT TXT CEL DAT 14 16 R1367H1 BXD11 M 56 >50 GDR 4 1 r EXP RPT TXT CEL DAT 15 18 R1530H1 BXD12 F 58 >50 GDR 4 1 s EXP RPT TXT CEL DAT 16 119 R1567H1 BXD12 M 58 >50 GDR 4 4   EXP RPT TXT CEL DAT 17 177 R1529H1 BXD13 F 58 >50 GDR 4 6   EXP RPT TXT CEL DAT 18 20 R1662H1 BXD13 M 60 >50 GDR 3 1 NA EXP RPT TXT CEL DAT 19 22 R1280H2 BXD14 F 56 >50 LuLu 3 1 s EXP RPT TXT CEL DAT 20 121 R1544H1 BXD14 M 59 >50 GDR 4 4   EXP RPT TXT CEL DAT 21 179 R1524H1 BXD15 F 60 >50 GDR 4 6   EXP RPT TXT CEL DAT 22 24 R1515H1 BXD15 M 61 >50 GDR 4 1 s EXP RPT TXT CEL DAT 23 26 R1661H1 BXD16 F 61 >50 GDR 3 1 s EXP RPT TXT CEL DAT 24 123 R1594H1 BXD16 M 61 >50 GDR 3 4   EXP RPT TXT CEL DAT 25 181 R1568H1 BXD19 F 60 >50 GDR 4 6   EXP RPT TXT CEL DAT 26 28 R1471H1 BXD19 M 157 >50 JBo 2 1 t EXP RPT TXT CEL DAT 27 30 R1573H1 BXD20 F 59 >50 GDR 4 1 s EXP RPT TXT CEL DAT 28 32 R1347H2 BXD21 F 64 >50 UMem 3 1 s EXP RPT TXT CEL DAT 29 125 R1349H3 BXD21 M 64 >50 UMem 3 4   EXP RPT TXT CEL DAT 30 183 R1848H1 BXD22 F 196 >50 UAB 3 6   EXP RPT TXT CEL DAT 31 34 R1525H1 BXD22 M 59 >50 GDR 4 2   EXP RPT TXT CEL DAT 32 156 R1254H1 BXD23 F 66 >50 LuLu 4 5   EXP RPT TXT CEL DAT 33 36 R1337H2 BXD23 M 102 >50 UAB 3 2   EXP RPT TXT CEL DAT 34 38 R1343H2 BXD24 F 71 >50 UMem 2 2   EXP RPT TXT CEL DAT 35 94 R1517H1 BXD24 M 57 >50 GDR 4 3   EXP RPT TXT CEL DAT 36 40 R1366H1 BXD27 F 60 >50 GDR 4 2   EXP RPT TXT CEL DAT 37 158 R1849H1 BXD27 M 70 >50 UAB 2 5   EXP RPT TXT CEL DAT 38 76 R1353H1 BXD28 F 79 >50 UMem 3 3   EXP RPT TXT CEL DAT 39 42 R2332H1 BXD28 M 60 >50 GDR 3 2   EXP RPT TXT CEL DAT 40 44 R1532H1 BXD29 F 57 >50 GDR 4 2   EXP RPT TXT CEL DAT 41 160 R1356H1 BXD29 M 76 >50 UMem 3 5   EXP RPT TXT CEL DAT 42 96 R1242H2 BXD31 F 61 >50 LuLu 4 3   EXP RPT TXT CEL DAT 43 46 R1240H2 BXD31 M 61 >50 LuLu 3 2   EXP RPT TXT CEL DAT 44 162 R1470H1 BXD32 F 76 >50 UMem 2 5   EXP RPT TXT CEL DAT 45 48 R1508H2 BXD32 M 58 >50 GDR 4 2   EXP RPT TXT CEL DAT 46 50 R1345H3 BXD33 F 65 >50 UMem 2 2   EXP RPT TXT CEL DAT 47 97 R1581H1 BXD33 M 59 >50 GDR 4 3   EXP RPT TXT CEL DAT 48 52 R1527H1 BXD34 F 59 >50 GDR 4 2   EXP RPT TXT CEL DAT 49 168 R1339H1 BXD34 M 74 >50 UMem 3 5   EXP RPT TXT CEL DAT 50 88 R1469H1 BXD36 F 83 >50 UMem 3 3   EXP RPT TXT CEL DAT 51 54 R1363H1 BXD36 M 77 >50 UMem 3 2   EXP RPT TXT CEL DAT 52 92 R1855H1 BXD38 F 55 >50 GDR 3 3   EXP RPT TXT CEL DAT 53 56 R1510H1 BXD38 M 65 >50 UMem 3 2   EXP RPT TXT CEL DAT 54 58 R1528H2 BXD39 F 59 >50 GDR 4 2   EXP RPT TXT CEL DAT 55 99 R1514H1 BXD39 M 59 >50 GDR 4 3   EXP RPT TXT CEL DAT 56 100 R1522H1 BXD40 F 59 >50 GDR 4 4   EXP RPT TXT CEL DAT 57 60 R1359H1 BXD40 M 73 >50 UMem 3 2   EXP RPT TXT CEL DAT 58 62 R1519H1 BXD42 F 58 >50 GDR 4 2   EXP RPT TXT CEL DAT 59 101 R1512H1 BXD42 M 59 >50 GDR 4 4   EXP RPT TXT CEL DAT 60 5 R1334H2 BXD43 F 59 22 LuLu 4 1 r EXP RPT TXT CEL DAT 61 84 R1303H1 BXD43 M 63 24 LuLu 3 3   EXP RPT TXT CEL DAT 62 67 R1326H1 BXD44 F 65 20 LuLu 4 3   EXP RPT TXT CEL DAT 63 7 R1577H2 BXD44 M 56 20 LuLu 4 1 r EXP RPT TXT CEL DAT 64 103 R1399H2 BXD45 F 58 20 LuLu 3 4   EXP RPT TXT CEL DAT 65 191 R1465H1 BXD45 M 62 20 LuLu 4 6   EXP RPT TXT CEL DAT 66 105 R1316H1 BXD48 F 58 21 LuLu 3 4   EXP RPT TXT CEL DAT 67 78 R1575H3 BXD48 M 65 22 LuLu 4 3   EXP RPT TXT CEL DAT 68 175 R1879H1 BXD50 F 69 18 LuLu 3 6   EXP RPT TXT CEL DAT 69 13 R1944H2 BXD50 M 81 18 LuLu 2 1 r EXP RPT TXT CEL DAT 70 72 R2331H1 BXD51 F 66 25 LuLu 3 3   EXP RPT TXT CEL DAT 71 193 R1330H1 BXD51 M 65 21 LuLu 4 6   EXP RPT TXT CEL DAT 72 107 R2095H2 BXD55 F 61 18 LuLu 3 4   EXP RPT TXT CEL DAT 73 17 R1474H1 BXD55 M 57 15 LuLu 2 1 r EXP RPT TXT CEL DAT 74 109 R1331H1 BXD60 F 60 21 LuLu 4 4   EXP RPT TXT CEL DAT 75 19 R1281H2 BXD60 M 59 22 LuLu 4 1 s EXP RPT TXT CEL DAT 76 111 R1914H2 BXD61 F 63 20 LuLu 2 4   EXP RPT TXT CEL DAT 77 21 R1856H2 BXD61 M 94 19 LuLu 2 1 s EXP RPT TXT CEL DAT 78 23 R1246H1 BXD62 F 54 22 LuLu 4 1 s EXP RPT TXT CEL DAT 79 195 R1585H1 BXD62 M 64 20 LuLu 4 6   EXP RPT TXT CEL DAT 80 25 R1945H1 BXD63 F 107 21 LuLu 4 1 t EXP RPT TXT CEL DAT 81 197 R2093H1 BXD63 M 70 21 LuLu 2 6   EXP RPT TXT CEL DAT 82 27 R2062H2 BXD64 F 65 19 LuLu 2 1 u EXP RPT TXT CEL DAT 83 95 R2061H1 BXD64 M 87 17 LuLu 4 3   EXP RPT TXT CEL DAT 84 29 R2054H2 BXD65 F 55 20 LuLu 2 1 r EXP RPT TXT CEL DAT 85 199 R2056H1 BXD65 M 89 17 LuLu 2 6   EXP RPT TXT CEL DAT 86 31 R1941H2 BXD66 F 78 20 LuLu 4 1 r EXP RPT TXT CEL DAT 87 115 R1949H2 BXD66 M 96 21 LuLu 2 4   EXP RPT TXT CEL DAT 88 185 R2060H1 BXD67 F 54 20 LuLu 3 6   EXP RPT TXT CEL DAT 89 33 R2052H1 BXD67 M 61 20 LuLu 3 1 t EXP RPT TXT CEL DAT 90 142 R2074H1 BXD68 F 60 19 LuLu 3 5   EXP RPT TXT CEL DAT 91 35 R1928H1 BXD68 M 72 16 LuLu 2 2   EXP RPT TXT CEL DAT 92 37 R1439H3 BXD69 F 60 21 LuLu 3 2   EXP RPT TXT CEL DAT 93 86 R1559H1 BXD69 M 64 20 LuLu 3 3   EXP RPT TXT CEL DAT 94 144 R2134H1 BXD70 F 64 21 LuLu 2 5   EXP RPT TXT CEL DAT 95 39 R2063H1 BXD70 M 55 20 LuLu 3 2   EXP RPT TXT CEL DAT 96 113 R1277H1 BXD73 F 60 20 LuLu 2 4   EXP RPT TXT CEL DAT 97 41 R1443H2 BXD73 M 76 21 LuLu 3 2   EXP RPT TXT CEL DAT 98 43 R2055H2 BXD74 M 79 18 LuLu 4 2   EXP RPT TXT CEL DAT 99 146 R2316H1 BXD74 M 193 18 LuLu 2 5   EXP RPT TXT CEL DAT 100 45 R1871H1 BXD75 F 61 21 LuLu 4 2   EXP RPT TXT CEL DAT 101 90 R1844H2 BXD75 M 90 20 LuLu 4 3   EXP RPT TXT CEL DAT 102 47 R1948H2 BXD76 F 81 16 LuLu 3 2   EXP RPT TXT CEL DAT 103 166 R2094H1 BXD76 M 61 17 LuLu 3 5   EXP RPT TXT CEL DAT 104 98 R2262H1 BXD77 F 62 24 LuLu 3 3   EXP RPT TXT CEL DAT 105 49 R1423H1 BXD77 M 62 20 LuLu 4 2   EXP RPT TXT CEL DAT 106 51 R1947H1 BXD79 F 108 17 LuLu 2 2   EXP RPT TXT CEL DAT 107 169 R2092H1 BXD79 M 86 15 LuLu 3 5   EXP RPT TXT CEL DAT 108 164 R1880H1 BXD80 F 68 19 LuLu 3 5   EXP RPT TXT CEL DAT 109 53 R1881H2 BXD80 M 68 19 LuLu 3 2   EXP RPT TXT CEL DAT 110 55 R2075H1 BXD83 F 60 15 LuLu 3 2   EXP RPT TXT CEL DAT 111 187 R2076H1 BXD83 M 60 15 LuLu 3 6   EXP RPT TXT CEL DAT 112 171 R2077H1 BXD84 F 62 17 LuLu 2 6   EXP RPT TXT CEL DAT 113 57 R2135H3 BXD84 M 75 17 LuLu 2 2   EXP RPT TXT CEL DAT 114 59 R1473H1 BXD85 F 79 20 LuLu 4 2   EXP RPT TXT CEL DAT 115 129 R1597H1 BXD85 M 86 21 LuLu 3 4   EXP RPT TXT CEL DAT 116 130 R1415H1 BXD86 F 77 20 LuLu 3 4   EXP RPT TXT CEL DAT 117 61 R1419H1 BXD86 M 58 21 LuLu 3 2   EXP RPT TXT CEL DAT 118 131 R1946H2 BXD87 F 101 20 LuLu 2 4   EXP RPT TXT CEL DAT 119 63 R1710H1 BXD87 M 96 20 LuLu 3 2   EXP RPT TXT CEL DAT 120 64 R1872H2 BXD89 F 90 20 LuLu 2 2   EXP RPT TXT CEL DAT 121 132 R1850H2 BXD89 M 82 19 LuLu 4 4   EXP RPT TXT CEL DAT 122 65 R2058H1 BXD90 F 61 23 LuLu 3 2   EXP RPT TXT CEL DAT 123 133 R1453H1 BXD90 M 61 20 LuLu 2 4   EXP RPT TXT CEL DAT 124 66 R1301H2 BXD92 F 58 21 LuLu 3 2   EXP RPT TXT CEL DAT 125 134 R1309H1 BXD92 M 59 21 LuLu 3 4   EXP RPT TXT CEL DAT 126 148 R2057H1 BXD93 F 92 19 LuLu 2 5   EXP RPT TXT CEL DAT 127 9 R2059H1 BXD93 M 58 19 LuLu 4 1 s EXP RPT TXT CEL DAT 128 82 R2313H1 BXD94 F 59 14 LuLu 3 3   EXP RPT TXT CEL DAT 129 136 R2314H1 BXD94 M 59 14 LuLu 3 5   EXP RPT TXT CEL DAT 130 150 R1847H1 BXD96 F 70 20 LuLu 3 5   EXP RPT TXT CEL DAT 131 11 R1846H2 BXD96 M 63 20 LuLu 4 1 s EXP RPT TXT CEL DAT 132 138 R2053H1 BXD97 F 55 21 LuLu 3 5   EXP RPT TXT CEL DAT 133 15 R1927H2 BXD97 M 67 20 LuLu 3 1 r EXP RPT TXT CEL DAT 134 154 R1942H1 BXD98 F 62 19 LuLu 3 5   EXP RPT TXT CEL DAT 135 68 R1943H2 BXD98 M 62 19 LuLu 3 3   EXP RPT TXT CEL DAT 136 70 R2197H1 BXD99 F 70 14 LuLu 4 3   EXP RPT TXT CEL DAT 137 140 R2315H1 BXD99 M 84 14 LuLu 2 5   EXP RPT TXT CEL DAT 138 69 R2116H1 CXB1 F 55 >50 JAX 3 3   EXP RPT TXT CEL DAT 139 104 R2096H1 CXB1 M 55 >50 JAX 2 4   EXP RPT TXT CEL DAT 140 124 R2124H1 CXB10 F 53 >50 JAX 2 4   EXP RPT TXT CEL DAT 141 87 R2108H1 CXB10 M 53 >50 JAX 3 3   EXP RPT TXT CEL DAT 142 89 R2125H1 CXB11 F 58 >50 JAX 3 3   EXP RPT TXT CEL DAT 143 114 R2128H1 CXB11 M 58 >50 JAX 2 4   EXP RPT TXT CEL DAT 144 126 R2126H1 CXB12 F 47 >50 JAX 3 4   EXP RPT TXT CEL DAT 145 91 R2109H1 CXB12 M 47 >50 JAX 3 3   EXP RPT TXT CEL DAT 146 93 R2127H2 CXB13 F 56 >50 JAX 3 3   EXP RPT TXT CEL DAT 147 128 R2110H1 CXB13 M 56 >50 JAX 3 4   EXP RPT TXT CEL DAT 148 116 R2117H1 CXB2 F 62 >50 JAX 2 4   EXP RPT TXT CEL DAT 149 71 R2098H1 CXB2 M 68 >50 JAX 3 3   EXP RPT TXT CEL DAT 150 73 R2118H1 CXB3 F 47 >50 JAX 3 3   EXP RPT TXT CEL DAT 151 106 R2100H1 CXB3 M 47 >50 JAX 3 4   EXP RPT TXT CEL DAT 152 118 R2119H1 CXB4 F 58 >50 JAX 3 4   EXP RPT TXT CEL DAT 153 75 R2101H1 CXB4 M 58 >50 JAX 3 3   EXP RPT TXT CEL DAT 154 77 R0129H2 CXB5 M 70 >50 LuLu 3 3   EXP RPT TXT CEL DAT 155 108 R2131H1 CXB5 M 42 >50 JAX 3 4   EXP RPT TXT CEL DAT 156 79 R2120H1 CXB6 F 49 >50 JAX 3 3   EXP RPT TXT CEL DAT 157 120 R2102H1 CXB6 M 49 >50 JAX 3 4   EXP RPT TXT CEL DAT 158 110 R2121H1 CXB7 F 63 >50 JAX 2 4   EXP RPT TXT CEL DAT 159 81 R2104H2 CXB7 M 58 >50 JAX 2 3   EXP RPT TXT CEL DAT 160 83 R2122H1 CXB8 F 54 >50 JAX 3 3   EXP RPT TXT CEL DAT 161 122 R2105H1 CXB8 M 41 >50 JAX 3 4   EXP RPT TXT CEL DAT 162 85 R2123H1 CXB9 F 54 >50 JAX 3 3   EXP RPT TXT CEL DAT 163 112 R2106H1 CXB9 M 54 >50 JAX 3 4   EXP RPT TXT CEL DAT 164 135 R2028H2 129S1/SvImJ F 66 >50 JAX 3 5 EXP RPT TXT CEL DAT 165 170 R2029H1 129S1/SvImJ M 66 >50 LuLu 4 6 EXP RPT TXT CEL DAT 166 186 R2031H2 A/J F 57 >50 JAX 3 6   EXP RPT TXT CEL DAT 167 149 R2030H1 A/J M 57 >50 LuLu 4 5   EXP RPT TXT CEL DAT 168 151 R2032H2 AKR/J F 66 >50 LuLu 3 5   EXP RPT TXT CEL DAT 169 172 R2033H2 AKR/J M 67 >50 LuLu 3 6   EXP RPT TXT CEL DAT 170 188 R2034H2 BALB/cByJ F 63 >50 LuLu 2 6   EXP RPT TXT CEL DAT 171 152 R2035H2 BALB/cByJ M 63 >50 JAX 3 5   EXP RPT TXT CEL DAT 172 137 R2036H2 BALB/cJ F 51 >50 JAX 3 5   EXP RPT TXT CEL DAT 173 174 R2037H2 BALB/cJ M 51 >50 LuLu 2 6   EXP RPT TXT CEL DAT 174 190 R2038H2 C3H/HeJ F 63 >50 JAX 3 6   EXP RPT TXT CEL DAT 175 153 R2039H1 C3H/HeJ M 63 >50 LuLu 3 5   EXP RPT TXT CEL DAT 176 139 R2137H1 C57BL/6ByJ F 55 >50 LuLu 4 5   EXP RPT TXT CEL DAT 177 176 R2136H1 C57BL/6ByJ M 55 >50 LuLu 2 6   EXP RPT TXT CEL DAT 178 192 R2040H2 C57BL/6J F 64 >50 LuLu 2 6   EXP RPT TXT CEL DAT 179 2 R2041H2 C57BL/6J M 65 >50 LuLu 3 1 s EXP RPT TXT CEL DAT 180 155 R1449H2 C57BL/6J M 71 >50 LuLu 3 5   EXP RPT TXT CEL DAT 181 141 R2042H2 CAST/EI F 64 >50 LuLu 2 5   EXP RPT TXT CEL DAT 182 178 R2043H2 CAST/EI M 64 >50 JAX 2 6   EXP RPT TXT CEL DAT 183 165 R1602H2 DBA/2J F 60 >50 LuLu 3 5 EXP RPT TXT CEL DAT 184 203 R2044H2 DBA/2J F 63 >50 LuLu 3 6 EXP RPT TXT CEL DAT 185 3 R2045H2 DBA/2J M 65 >50 LuLu 2 1 s EXP RPT TXT CEL DAT 186 194 R1683H1 KK/HIJ F 72 >50 JAX 3 6   EXP RPT TXT CEL DAT 187 157 R1687H2 KK/HIJ M 72 >50 JAX 2 5   EXP RPT TXT CEL DAT 188 143 R2046H1 LG/J F 63 >50 JAX 5 5   EXP RPT TXT CEL DAT 189 180 R2047H1 LG/J M 63 >50 LuLu 2 6   EXP RPT TXT CEL DAT 190 196 R2048H1 NOD/LtJ F 77 >50 LuLu 4 6   EXP RPT TXT CEL DAT 191 159> R2049H2 NOD/LtJ M 76 >50 LuLu 4 5   EXP RPT TXT CEL DAT 192 182 R2350H1 NZ0/HILtJ M 96 >50 JAX 2 6   EXP RPT TXT CEL DAT 193 145 R2200H1 NZO/H1LtJ F 62 >50 DanG 4 5   EXP RPT TXT CEL DAT 194 198 R2050H1 PWD/PhJ F 65 >50 JAX 3 6   EXP RPT TXT CEL DAT 195 161 R2051H2 PWD/PhJ M 64 >50 JAX 2 5   EXP RPT TXT CEL DAT 196 147 R2322H1 PWK/PHJ F 63 >50 JAX 3 5.   EXP RPT TXT CEL DAT 197 184 R2349H1 PWK/PHJ M 83 >50 JAX 1 6   EXP RPT TXT CEL DAT 198 200 R2198H1 WSB/EiJ F 58 >50 LuLu 4 6   EXP RPT TXT CEL DAT 199 163 R2199H1 WSB/EiJ M 58 >50 JAX 3 5   EXP RPT TXT CEL DAT 200 201 R1289H1 B6D2F1 F 64 NA LuLu 4 6   EXP RPT TXT CEL DAT 201 1 R1291H3 B6D2F1 M 66 NA LuLu 4 s EXP RPT TXT CEL DAT 202 204 R1291H4 B6D2F1 M 66 NA JAX 3 6   EXP RPT TXT CEL DAT 203 167 R1595H1 D2B6F1 F 63 NA LuLu 3 5   EXP RPT TXT CEL DAT 204 202 R1286H1 D2B6F1 F 57 NA LuLu 3 6   EXP RPT TXT CEL DAT

    Downloading all data:

All data links (right-most column above) will be made active as sooon as the global analysis of these data by the Consoritum has been accepted for publication. Please see text on Data Sharing Policies, and Conditions and Limitations, and Contacts. Following publication, download a summary text file or Excel file of the PDNN probe set data. Contact RW Williams regarding data access probelms.

    About the array platform:

Affymetrix Mouse Genome 430 2.0 array: The 430v2 array consists of 992936 useful 25-nucleotide probes that estimate the expression of approximately 39,000 transcripts and the majority of known genes and expressed sequence tags. The array sequences were selected late in 2002 using Unigene Build 107 by Affymetrix. The UTHSC group has recently reannotated all probe sets on this array, producing more accurate data on probe and probe set targets. All probes were aligned to the most recent assembly of the Mouse Genome (Build 34, mm6) using Jim Kent's BLAT program. Many of the probe sets have been manually curated by Jing Gu and Rob Williams.

    About data processing:

Harshlight was used to examine the image quality of the array (CEL files). Bad areas (bubbles, scratches, blemishes) of arrays were masked.

First pass data quality control: Affymetrix GCOS provides useful array quality control data including:

1. The scale factor used to normalize mean probe intensity. This averaged 3.3 for the 179 arrays that passed and 6.2 for arrays that were excluded. The scale factor is not a particular critical parameter.
2. The average background level. Values averaged 54.8 units for the data sets that passed and 55.8 for data sets that were excluded. This factor is not important for quality control.
3. The percentage of probe sets that are associated with good signal ("present" calls). This averaged 50% for the 179 data sets that passed and 42% for those that failed. Values for passing data sets extended from 43% to 55%. This is a particularly important criterion.
4. The 3':5' signal ratios of actin and Gapdh. Values for passing data sets averaged 1.5 for actin and 1.0 for Gapdh. Values for excluded data sets averaged 12.9 for actin and 9.6 for Gapdh. This is a highly discriminative QC criterion, although one must keep in mind that only two transcripts are being tested. Sequence variation among strains (particularly wild derivative strains such as CAST/Ei) may affect these ratios.

The second step in our post-processing QC involves a count of the number of probe sets in each array that are more than 2 standard deviations (z score units) from the mean across the entire 206 array data sets. This was the most important criterion used to eliminate "bad" data sets. All 206 arrays were processed togther using standard RMA and PDNN methods. The count and percentage of probe sets in each array that were beyond the 2 z theshold was computed. Using the RMA transform the average percentage of probe sets beyond the 2 z threshold for the 179 arrays that finally passed of QC procedure was 1.76% (median of 1.18%). In contrast the 2 z percentage was more than 10-fold higher (mean of 22.4% and median 20.2%) for those arrays that were excluded. This method is not very senstive to the transformation method that is used. Using the PDNN transform the average percent of probe sets exceeding was 1.31% for good arrays and was 22.6% for those that were excluded. In our opinion, this 2 z criterion is the most useful criterion for the final decision of whether or not to include arrays, although again, allowances need to be made for wild strains that one expects to be different from the majority of conventional inbred strains. For examploe, if a data set has excellent characteristics on all of the Affymetrix GCOS metrics listed above, but generates a high 2 z percentage, then one whould include the ssample if one can verify that there are no problems in sample and data set identification.

The entire procedure can be reapplied once the initial outlier data sets have been eliminated to detect any remaining outlier data sets.

DataDesk was used to examine the statistical quality of the of the probe level (CEL) data after step 5 below. DataDesk allows a rapid detection of subsets of probes that are particular sensitive to still unknown factors in array processing. Arrays can then be categorized at the probe level into "reaction classes." A reaction class is a group of arrays for which the expression of essentially all probes are colinear over the full range of log2 values. A single but large group of arrays (n = 32) processed in essentially the identical manner by a single operator can produce arrays belonging to as many as four different reaction classes. Reaction classes are NOT related to strain, age, sex, treatment, or any known biological parameter (technical replicates can belong to different reaction classes). We do not yet understand the technical origins of reaction classes. The number of probes that contribute to the definition of reaction classes is quite small (<10% of all probes). We have categorized all arrays in this data set into one of 5 reaction classes. These have then been treated as if they were separate batches. Probes in these data type "batches" have been aligned to a common mean as described below.

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. <0L>

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.

We performed a quantile normalization of the log base 2 values for all arrays using the same initial steps used by the RMA transform.

We computed the Z scores for each cell value.

We multiplied all Z scores by 2.

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 (probe brightness level) corresponds approximately to a 1 unit difference.

inally, 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. Note, that we have not (yet) corrected for variance introduced by differences in sex or any interaction terms. We have not corrected for background beyond the background correction implemented by Affymetrix in generating the CEL file. We eventually hope to add statistical controls and adjustments for some of these variables.

Probe set data from the CHP file: The expression values were generated using PDNN. The same simple steps described above were also applied to these values. Every microarray data set therefore has a mean expression of 8 with a standard deviation of 2. A 1 unit difference represents roughly a two-fold difference in expression level. Expression levels below 5 are usually close to background noise levels.

Probe level QC: Log2 probe data of all arrays were inspected in DataDesk before and after quantile normalization. Inspection involved examining scatterplots of pairs of arrays for signal homogeneity (i.e., high correlation and linearity of the bivariate plots) and looking at all pairs of correlation coefficients. XY plots of probe expression and signal variance were also examined. Probe level array data sets were organized into reaction groups. Arrays with probe data that were not homogeneous when compared to other arrays were flagged.

Probe set level QC: The final normalized individual array data were evaluated for outliers. This involved counting the number of times that the probe set value for a particular array was beyond two standard deviations of the mean. This outlier analysis was carried out using the PDNN, RMA and MAS5 transforms and outliers across different levels of expression. Arrays that were associated with an average of more than 8% outlier probe sets across all transforms and at all expression levels were eliminated. In contrast, most other arrays generated fewer than 5% outliers.

Validation of strains and sex of each array data set: A subset of probes and probe sets with a Mendelian pattern of inheritance were used to construct a expression correlation matrix for all arrays and the ideal Mendelian expectation for each strain constructed from the genotypes. There should naturally be a very high correlation in the expression patterns of transcripts with Mendelian phenotypes within each strain, as well as with the genotype strain distribution pattern of markers for the strain.

Sex of the samples was validated using sex-specific probe sets such as Xist and Dby.

    Data source acknowledgment:

Data were generated with funds provided by a variety of public and private source to members of the Consortium. All of us thank Muriel Davisson, Cathy Lutz, and colleagues at the Jackson Laboratory for making it possible for us to add all of the CXB strains, and one or more samples from KK/HIJ, WSB/Ei, NZO/HILtJ, LG/J, CAST/Ei, PWD/PhJ, and PWK/PhJ to this study. We thank Yan Cui at UTHSC for allowing us to use his Linux cluster to align all M430 2.0 probes and probe sets to the mouse genome. We thank Hui-Chen Hsu and John Mountz for providing us BXD tissue samples, as well as many strains of BXD stock. We thanks Douglas Matthews (UMem in Table 1) and John Boughter (JBo in Table 1) for sharing BXD stock with us. Members of the Hippocampus Consortium thank the following sources for financial support of this effort:

• David C. Airey, Ph.D.
  Grant Support: Vanderbilt Institute for Integratie Genomics
  Department of Pharmacology
  david.airey at vanderbilt.edu
• Lu Lu, M.D.
  Grant Support: NIH U01AA13499, U24AA13513
• Fred H. Gage, Ph.D.
  Grant Support: NIH XXXX
• Dan Goldowitz, Ph.D.
  Grant Support: NIAAA INIA AA013503
  University of Tennessee Health Science Center
  Dept. Anatomy and Neurobiology
  email: dgold@nb.utmem.edu
• Shirlean Goodwin, Ph.D.
  Grant Support: NIAAA INIA U01AA013515
• Gerd Kempermann, M.D.
  Grant Support: The Volkswagen Foundation Grant on Permissive and Persistent Factors in Neurogenesis in the Adult Central Nervous System
  Humboldt-Universitat Berlin
  Universitatsklinikum Charite
  email: gerd.kempermann at mdc-berlin.de
• Kenneth F. Manly, Ph.D.
  Grant Support: NIH P20MH062009 and U01CA105417
• Richard S. Nowakowski, Ph.D.
  Grant Support: R01 NS049445-01
• Yanhua Qu, Ph.D.
  Grant Support: NIH U01CA105417
• Glenn D. Rosen, Ph.D.
  Grant Support: NIH P20
• Leonard C. Schalkwyk, Ph.D.
  Grant Support: MRC Career Establishment Grant G0000170
  Social, Genetic and Developmental Psychiatry
  Institute of Psychiatry,Kings College London
  PO82, De Crespigny Park London SE5 8AF
  L.Schalkwyk@iop.kcl.ac.uk
• Guus Smit, Ph.D.
  Dutch NeuroBsik Mouse Phenomics Consortium
  Center for Neurogenomics & Cognitive Research
  Vrije Universiteit Amsterdam, The Netherlands
  e-mail: guus.smit at falw.vu.nl
  Grant Support: BSIK 03053
• Thomas Sutter, Ph.D.
  Grant Support: INIA U01 AA13515 and the W. Harry Feinstone Center for Genome Research
• Stephen Whatley, Ph.D.
  Grant Support: XXXX
• Robert W. Williams, Ph.D.
  Grant Support: NIH U01AA013499, P20MH062009, U01AA013499, U01AA013513

    About this text file:

This text file originally generated prospectively by RWW on July 30 2005. Updated by RWW July 31, 2005.