Assessed Value Non-School Dist. Note that enrichment results are independent of the actual number of differentially expressed genes identified in each method and each platform. Show Details. Antisense remapping of U95Av2 and Uplus2 probes identified 6, and 21, probes with unique homology to the reverse strand of RefSeq transcripts. Step 2: probe clusters Neighboring probes that map to isoforms that are differentiated by alternative splicing, RNA editing, or non-representative hybridization can produce readouts of different molecular species leading to poor quantitative intensity evaluation for the probe cluster. Bangkok Bangkok is a license holder connected to this address. Jaradul Mortgage Inc was registered at this address. Neighboring probes that map to isoforms that are differentiated by alternative splicing, RNA editing, or non-representative hybridization can produce readouts of different molecular species leading to poor quantitative intensity evaluation for the probe cluster.
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To account for RNA isoforms, we constructed transcript-focused probe clusters by performing a non-supervised, single-linkage hierarchical clustering of the probes using Pearson correlation coefficient as a distance measure.
Measurement multiplicity, which is implicit to gene expression profiling technologies using multiple probes per target, can be used to assemble informative, transcript-specific probe-clusters that dramatically improve expression estimates in large cell-context-specific datasets.
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Gene expression data include B-cell samples profiled on U95A [ 10 ], B-cell samples profiled on U95Av2 [ 10 ], 75 lung carcinoma samples on U95Av2 and Uplus2 [ 3132 ], 51 and ovarian cancer samples on U95Av2 and Uplus2 [ 3334 ], 49 and 45 glioblastoma samples on U95Av2 GSE and Uplus2 [ 3536 ], 88 and prostate cancer samples on U95Av2 and Uplus2, 40 and breast carcinoma samples on U95Av2 and Uplus2 [ 2437 ], B-cell samples profiled on Uplus2 [ 21 ], 60 samples from 30 human tissues profiled on UA chips [ 38 ], and 55 human glial brain tumor samples hybridized on huex10stv1 Affymetrix exon arrays [ 29 ].
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|Number of probes, probe sets, probe clusters and genes represented on two popular Affymetrix GeneChip microarrays.
Results suggest that Cleaner is not effective for analyzing data derived from fewer than 20 microarray experiments. We estimated the probe-cluster FDR for each consistency score using permutation testing, where each probe cluster constructed after permuting sample labels individually for each probe was considered a false positive detection. Interestingly, the six MYB probe sets responding to MYB knock-down contributed to the Cleaner cluster, while all but two probes from the non-responding probe sets were discarded during the remapping process Figure 5c.
G spots have been shown to bias expression measurements [ 22 ], although not all probes containing G spots are flawed; we showed that Cleaner preferentially discards G-spot probes.
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A Bioconductor-based [ 39 ] implementation of MAS5 [ 40 ] was used to quantitatively estimate and normalize the intensity levels of probe clusters. To conclude, we note that according to differential expression analysis, expression consistency across platforms when using Cleaner annotation G spots have been shown to bias expression measurements [ 22 ], although not all probes containing G spots are flawed; we showed that Cleaner preferentially discards G-spot probes.
However, our methodology extends to other technologies that use multiple probes per target, including exon arrays and deep sequencing. Using relatively simple statistics, we were able to resolve ambiguity and correct for bias with no concern for its source.
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We showed that testing probe consistency across individual measurements helps identify biased or uninformative probes, leading to increased accuracy when estimating expression intensities on the transcript and gene levels. The color scale, from blue to red, depicts the correlation Pearson correlation coefficient between probe sets. Cleaner probe clusters conclusively indicated up-regulation for the two genes in germinal center Figure 4band down-regulation for the two genes after shRNA-mediated knock-down Figure 5c.
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