SJUT Cerebellum mRNA M430 (Mar05) PDNN
Accession number: GN55
This March 2005 data freeze provides estimates of mRNA expression in adult cerebellum of 48 lines of mice including 45 BXD recombinant inbred strains, C57BL/6J, DBA/2J, and F1 hybrids. Data were generated by a consortium of investigators at St. Jude Children's Research Hospital (SJ) and the University of Tennessee Health Science Center (UT). Cerebellar samples were hybridized in small pools (n = 3) to Affymetrix M430A and B arrays. Data were processed using the Position-Dependent Nearest Neighbor (PDNN) method developed by Zhang and colleagues (2003). To simplify comparison between transforms, PDNN values of each array were adjusted to an average of 8 units and a standard deviation of 2 units.
About the cases used to generate this set of data:
We have exploited a set of BXD recombinant inbred strains. All BXD lines are derived crossed between C57BL/6J (B6 or B) and DBA/2J (D2 or D). Both B and D parental strains have been almost fully sequenced (8x coverage for B6 by a public consortium and approximately 1.5x coverage for D by Celera Discovery Systems) and data for 1.75 millioin B vs D SNPs are incorporated into WebQTLs genetic maps for the BXDs. BXD2 through BXD32 were produced by Benjamin A. Taylor starting in the late 1970s. BXD33 through 42 were also produced by Taylor, but they were generated in the 1990s. These strains are all available from The Jackson Laboratory, Bar Harbor, Maine. BXD43 through BXD99 were produced 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).
Most BXD animals were generated in-house at the University of Tennessee Health Science Center by Lu Lu and Robert Williams using stock obtained from The Jackson Laboratory between 1999 and 2004. All BXD strains with numbers above 42 are new advanced intecross type BXDs (Peirce et al. 2004) that are current available from UTHSC. Additional cases were provided by Glenn Rosen, John Mountz, and Hui-Chen Hsu. These cases were bred either at The Jackson Laboratory (GR) or at the University of Alabama (JM and HCH).
About the tissue used to generate this set of data:
The March 2005 data set consists of a total of 102 array pairs (Affymetrix 430A and 430B) from 49 different genotypes. Each sample consists of whole cerebellum taken from three adult animals of the same age and sex. Two sets of technical replicates (BXD14 n = 2; BXD29 n = 3) were combined before generating group means; giving a total of 99 biologically independent data sets. The two reciprocal F1s (D2B6F1 and B6D2F1) were combined to give a single F1 mean estimate of gene expression. 430A and 430B arrays were processed in three large batches. The first batch (May03 data) consists of 17 samples from 17 strains balanced by sex (8M and 9F). The second batch consists of 29 samples, and includes biological replicates, 2 technical replicates, and data for 10 new strains. The third batch consists of 56 samples, and also includes biological replicates, 2 technical replicates, and data for 15 additional strains.
Replication and Sample Balance: Our goal is to obtain data for independent biological sample pools from both sexes for each strain. Six of 48 genotypes are still represented by single samples: BXD5, BXD13, BXD20, BXD23, BXD27 are female-only strains, whereas BXD25, BXD77, BXD90 are male-only. Ten strains are represented by three independent samples with the following breakdown by sex: C57BL/6J (1F 2M), DBA/2J (2F 1M), B6D2F1 (1F 2M), BXD2 (2F 1M), BXD11 (2F 1M), BXD28 (2F 1M), BXD36 (1F 1M), BXD40 (2F 1M), BXD51 (1F 2M), BXD60 (1F 2M), BXD92 (2F 1M).
The age range of samples is relatively narrow. Only 18 samples were taken from animals older than 99 days and only two samples are older than 7 months of age. BXD11 includes an extra (third) 441-day-old female sample and the BXD28 includes an extra 427-day-old sample.
RNA was extracted at UTHSC by Lu Lu, Zhiping Jia, and Hongtao Zhai.
All samples were subsequently processed at the Hartwell Center Affymetrix laboratory at SJCRH by Jay Morris.
The table below summarizes informaton on strain, sex, age, sample name, and batch number.
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.
Probe set data: The expression data were processed by Yanhua Qu (UTHSC) using the Position-Dependent Nearest Neighbor (PDNN) method developed by Zhang and colleagues (2003). The normalized CEL files were read into the PerfectMatch. The same simple steps described above were also applied to the initial PDNN probe set expression estimates. A 1-unit difference represents roughly a two-fold difference in expression level. Expression levels below 5 are usually close to background noise levels.
- 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 for the log base 2 values for the total set of 104 arrays (all three batches) 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 6: We corrected for technical variance introduced by three large batches at the probe level. To do this we determined the ratio of the batch mean to the mean of all three batches and used this as a single multiplicative probe-specific batch correction factor. The consequence of this simple correction is that the mean probe signal value for each of the three batches is the same.
- Step 7a: The 430A and 430B arrays include a set of 100 shared probe sets (a total of 2200 probes) that have identical sequences. These probes and probe sets provide a way to calibrate expression of the 430A and 430B arrays to a common scale. To bring the two arrays into alignment, we regressed Z scores of the common set of probes to obtain a linear regression correction to rescale the 430B arrays to the 430A array. In our case this involved multiplying all 430B Z scores by the slope of the regression and adding or subtracting a small offset. The result of this step is that the mean of the 430A expression is fixed at a value of 8, whereas that of the 430B chip is typically reduced to 7. The average of the merged 430A and 430B array data set is approximately 7.5.
- Step 7b: We recentered the merged 430A and 430B data sets to a mean of 8 and a standard deviation of 2. This involved reapplying Steps 3 through 5.
- Step 8: Finally, we computed the arithmetic mean of the values for the set of microarrays for each strain. Technical replciates were averaged before computing the mean for independent biological samples. Note, that we have not (yet) corrected for variance introduced by differences in sex, age, source of animals, 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 these variables.
About the chromosome and megabase position values:
The chromosomal locations of probe sets and gene markers on the 430A and 430B microarrays were determined by BLAT analysis using the Mouse Genome Sequencing Consortium May 2004 Assembly (see http://genome.ucsc.edu/cgi-bin/hgBlat?command=start&org=mouse). We thank Dr. Yan Cui (UTHSC) for allowing us to use his Linux cluster to perform this analysis.
Data source acknowledgment:
Data were generated with funds contributed equally by The UTHSC-SJCRH Cerebellum Transcriptome Profiling Consortium. Our members include:
- Tom Curran
- Dan Goldowitz
- Kristin Hamre
- Lu Lu
- Peter McKinnon
- Jim Morgan
- Clayton Naeve
- Richard Smeyne
- Robert Williams
- The Center of Genomics and Bioinformatics at UTHSC
- The Hartwell Center at SJCRH
Information about this text file:
This text file originally generated by RWW and YHQ, March 21, 2005. Updated by RWW, March 23, 2005.