However, in contrast to spike-in studies, the identities of the genes expected to show differential expression are not definitely known ahead of time. d Includes 14 334 probes where no gene name is listed by the manufacturer. Affymetrix is … The Affymetrix and Illumina Microarray Analysis laboratory provides state of the art Affymetrix GeneChip® and Illumina BeadArray technology for analysis of gene expression, gene regulation, and genetic variation (SNP and CNV analysis). 500K: Content Optimized SNP Selection ~2,200,000 SNPs~2,200,000 SNPs From Public & PerlegenFrom Public & … Gentleman, R.C., Carey, V.J., Bates, D.M., Bolstad, B., Dettling, M., Dudoit, S., Ellis, B., Gautier, L., Ge, Y., Gentry, J., et al. Larkin, J.E., Frank, B.C., Gavras, H., Sultana, R., Quackenbush, J. Irizarry, R.A., Warren, D., Spencer, F., Kim, I.F., Biswal, S., Frank, B.C., Gabrielson, E., Garcia, J.G., Geoghegan, J., Germino, G., et al. Despite the overall difference from the ‘known genes’, many of these probe sets do yield strong correlations with the dilution profile, as evidenced by the smaller peaks near 1 and −1, suggesting that they have biologically meaningful targets ( Figure 3C and D ). Probes that could not be matched across platforms represent probes which did not map to a ‘known’ gene, or to genes represented on only one platform. There are 41 pairs of probes in this set which show cross-platform rank correlations of <−0.5. To compare profiles across platforms, the Pearson correlation was used on non-log transformed data (the RMA data were transformed back from log 2 ), though the Spearman rank correlation yielded very similar results (Supplementary Data). Black bars at the side indicate large clusters of genes that appear to show clear dilution effects in both platforms. We interpret this as indicating that the Affymetrix array contains more probe sets that are ‘truly redundant’, at least as reflected in our tissue samples. While our annotation system tries quite hard to identify which transcript or set of transcripts a probe is likely to hybridize to, thereby identifying cases where we believe the platforms are measuring the same RNA, the resolution of this approach is limited. This could lead to different populations of transcripts being assayed in some cases. When comparing gene expression studies, we not only have to consider the interesting biological factors but a plethora of technical factors including diverse sample handling, target preparation and data processing methods, as well as microarray platform choice. The Santa Clara, California-based Affymetrix… Create assays tailored directly to specific … At Illumina, our goal is to apply innovative technologies to the analysis of genetic variation and function, making studies possible that were not even imaginable just a few years ago. The reason for the discordance between such findings and ours, as well as that of a number of other groups ( 19 – 26 ) is not always clear, especially as in some cases the data are not publicly available. The key features of the design are the use of a single pair of RNA samples for all analyses, mixed together in varying proportions and analyzed in technical replicates on each platform. The results were visualized with matrix2png ( 12 ). None declared. Arrays produced by Affymetrix are fabricated by in situ synthesis of 25mer oligonucleotides (2) while the Illumina process involves using standard oligonucleotide synthesis … We then filtered out probes which were expressed at low levels on both platforms (medians below the 25th percentile). If there was more than one probe(set) for a given gene, when doing comparisons we considered all possible combinations (to avoid repetition, we will use the term ‘probe’ even when we mean Affymetrix ‘probe set’, unless stated explicitly). In addition, the Affymetrix arrays are constructed in a specific layout, with each probe synthesized at a predefined location ( 2 ), while individual Illumina arrays must undergo a ‘decoding’ step in which the locations of each probe on the array are determined using a molecular address ( 1 ). The ATGC performs SNP genotyping microarray services using Affymetrix and Illumina platforms.. Pricing for SNP profiling microarray services can be found on the service pricing schedule.. Affymetrix… The rows of this matrix were subjected to hierarchical clustering using XCluster ( ), with average linkage and Euclidean distance. A potential remaining source of ‘disagreement’ could be differential cross-hybridization. Two BeadChips were used, each one containing eight arrays, so that each dilution series of six samples was run on an individual BeadChip. The dilution profile was described as a simple factor in a linear model used to fit each gene. When these two factors are taken into account, the agreement of the results across platforms is very high, though still not perfect. Therefore the failure of one platform to confirm a result on a rare transcript should be interpreted cautiously. Woo, Y., Affourtit, J., Daigle, S., Viale, A., Johnson, K., Naggert, J., Churchill, G. Lee, J.K., Bussey, K.J., Gwadry, F.G., Reinhold, W., Riddick, G., Pelletier, S.L., Nishizuka, S., Szakacs, G., Annereau, J.P., Shankavaram, U., et al. Portions of each aliquot were split in half for Affymetrix analysis as technical replicates. Both show pronounced peaks near correlations of −1 and 1, apparently reflecting probes whose targets are differentially expressed between the two samples. A more refined analysis takes into account the fact that genes that are not differentially expressed between the two tissues would present noisy expression profiles that would not be predicted to be reproducible across platforms. Michael Barnes, Johannes Freudenberg, Susan Thompson, Bruce Aronow, Paul Pavlidis, Experimental comparison and cross-validation of the Affymetrix and Illumina gene expression analysis platforms, Nucleic Acids Research, Volume 33, Issue 18, 1 October 2005, Pages 5914–5923, For this reason, we have discarded identifier cross-references as a primary means of matching probes across platforms. a Mfg, Manufacturer's annotations; BLAT, our own annotations computed using BLAT alignments to the genomic sequence. Oxford University Press is a department of the University of Oxford. The complete list of probes on both platforms, with their agreement statistics across platforms, is included as Supplementary Data. All rights reserved
 The online version of this article has been published under an open access model. Hybridization to the Sentrix HumanRef-8 Expression BeadChip (Illumina, Inc., San Diego, CA), washing and scanning were performed according to the Illumina BeadStation 500× manual (revision C). To analyze the relationship of cross-platform agreement with probe location, the distance between two probes was measured as the distance between the centers of their alignments on the genome. Samples were processed immediately following blood draw. This paper details results from an experiment comparing Affymetrix HG-U133 Plus 2.0 microarrays with the Illumina HumanRef-8 BeadArrays. The Affymetrix 6.0 and Illumina OmniExpress chip have similar genotyping accuracy and provide similar accuracy of imputed SNPs. For complete data see the Supplementary Data. In extreme cases, the GenBank accession number referenced by the manufacturer includes multiple genes. Specifically, probes that are expected to hybridize to different parts of the same transcript might yield different signals. Read the full 47 word article. Cells were isolated by Ficoll gradient centrifugation and total RNA was isolated using Trizol (Invitrogen Life Technologies, Carlsbad, CA) according to the manufacturer's protocol, aliquoted and stored at −80°C until use. This article and the information contained in BioCentury's publications and services are solely for your own personal, non … Biotinylated cRNA was prepared using the Illumina RNA Amplification Kit (Ambion, Inc., Austin, TX) according to the manufacturer's directions starting with ∼100 ng total RNA. We supplemented these annotations with our own sequence analysis based on comparing sequences with the Human genome sequence (assembly hg17, July 2004). This is especially true once the factors of gene expression level and probe placement on the genome are considered. Thus, if both platforms had two probes for a single gene, there were four comparative values generated. Unlike the Affymetrix platform, each Illumina BeadArray slide contains multiple arrays, allowing us to analyze a complete dilution series on one slide. The Illumina SNP chips include LD-based tagSNPs derived from over 2 million common SNPs (minor allele frequency greater than 0.05) in the HapMap data. I will be grateful for any suggestions. Gunderson, K.L., Kruglyak, S., Graige, M.S., Garcia, F., Kermani, B.G., Zhao, C., Che, D., Dickinson, T., Wickham, E., Bierle, J., et al. 2.6 years ago by. 5 months ago by. We expected that large numbers of genes would show an effect of mixing of the RNA samples on their relative expression levels, while other genes expressed at equal levels (or not expressed) would not show such a pattern. A likely explanation for some of the effects we see have to do with differences in the technologies, such as differences in RNA labeling protocols, or the linker and ‘bar code’ sequences on the Illumina arrays compared with the direct attachment of the Affymetrix sequences to the substrate. Furthermore, there are large numbers of probes which clearly agree across platforms. As shown in Figure 6B , when Affymetrix and Illumina probes align to very close or overlapping locations in the genome, they have a tendency to agree more, whereas probes that hybridize to distinct locations, even along the same gene, tend to disagree more. For the Illumina platform, application of the same filter that yielded 940 pairs of probes (2.6% of the total) for the cross-platform comparison yielded 10 pairs of probes (0.2%), while for Affymetrix it yielded 103 pairs (0.3%). For full access to this pdf, sign in to an existing account, or purchase an annual subscription. This could lead to incorrectly predicting the same hybridization pattern for two probes located at nearby locations in the genome. Quality was assessed for each sample using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA) at their respective core facilities according to internal quality control measures. Affymetrix is a brand of DNA microarray products sold by Thermo Fisher Scientific that originated with an American biotechnology research and development and manufacturing company of the same name. All sequences used are provided as Supplementary Data or are available from the manufacturers. Gray bars indicate examples of clusters that appear to show dilution effects in one platform but not consistently in the other. We designate all other potential transcripts ‘unassigned probes’. 0. ( 4 ) also do not document any consideration of the impact of expression level on agreement. Note that in this figure, if a gene occurs multiple times on one platform, it is shown in all possible valid comparisons with matching probes on the other platform. In addition to the difference in oligonucleotide physical attachment, the two platforms are also very different in probe selection and design procedure. These often represented alignments to sequences duplicated in the assembly (e.g. Second, careful bioinformatics analysis of each platform is necessary to maximize the precision of the comparison. Following informed consent (approved by Cincinnati Children's Hospital Medical Center Internal Review Board), ∼50 ml whole blood was collected from 30 adult, apparently healthy, volunteers using Acid Citrate Dextrose as an anti-coagulant. Tan, P.K., Downey, T.J., Spitznagel, E.L., Jr, Xu, P., Fu, D., Dimitrov, D.S., Lempicki, R.A., Raaka, B.M., Cam, M.C. Schematic representation of the experimental design. It is mission critical for us … Illumina microarray technology (also known as BeadArray technology) uses silica microbeads. The specific numbers are that: For variants with MAF >1%, the average r 2 correlation between imputed and true genotypes will be 0.8879 (Affymetrix) and 0.8590 (Illumina). Tan et al . For example, we note that Tan et al . Affymetrix expression level log 2 > 7), the effect of location on agreement is enhanced slightly (rank correlation 0.25), indicating that the effect cannot be completely attributed to associated differences in expression level. Affymetrix is dedicated to developing state-of-the-art technology for acquiring, analyzing, and managing complex genetic information for use in biomedical research. However, there was no significant enrichment in alignments shorter or longer than the median in the set of 940 pairs ( P > 0.05, Fisher's exact test), suggesting that there is no overall pattern of alignment statistics that can explain the relatively anomalous behavior of these 940 pairs. Each BLAT hit was further scored based on two criteria. - Affymetrix - Illumina . Our study reinforces the idea that a failure to consider annotations and expression levels sufficiently carefully can help explain some of the observed differences. Note that many probes are not specific for a single transcript of a gene. Each hit was then given an overall score equal to ( m − g )/ s , where m is the number of matches, g is the number of gaps in the alignment, and s is the size of the query sequences. The Illumina data were extracted using software provided by the manufacturer. For Affymetrix microarray analysis, samples were run in the CCHMC Affymetrix core facility. Agreement was strongly correlated with the level of expression of a gene. These hits were associated with genes as follows. Become a monthly donor and receive a shirt, Information for our supporters in response to COVID-19. Indeed, if we first filter the data to remove comparisons between probes, of which at least one do not show a significant dilution effect (FDR 0.05, removing 48% of the comparisons), rejection of 88% null hypotheses yields an FDR of <0.05 (i.e. We hypothesized that reproducibility within each platform (for those genes with multiple probes predicted to target them) would show the same trends as reproducibility across platforms. This is because many of the weakly expressed probes do show (apparent) dilution effects. Figure 5B suggests that a large fraction of the ‘failures’ of the platforms to agree can be accounted for by probes which show a weak or no dilution effect. Barczak, A., Rodriguez, M.W., Hanspers, K., Koth, L.L., Tai, Y.C., Bolstad, B.M., Speed, T.P., Erle, D.J. The set of probes we found to be most highly reproducible can be used by others to help increase confidence in analyses of other data sets using these platforms. A summary of the annotations used in this study is given in Table 1 . If these values are different on the two platforms, then agreement may be lower if the predicted transcripts are indeed present in the tissues we studied. At an FDR of 0.05, 37% of the cross-platform comparisons result in rejection of the null hypothesis of no correlation (the threshold correlation to achieve significance is ∼0.56). Affymetrix GeneChip ® System for Exon-level Gene Expression Analysis. We considered this plausible because Affymetrix probe sets assay more sequence and often include probes spread fairly widely (a median of 481 genomic bases from the 5′ end of the 5′-most probe to the 3′ end of the 3′-most probe) compared with the Illumina platform, which use a single 50 bp probe that almost always maps to 50 contiguous bases in our analysis. These probes appear wholly unremarkable based on the parameters we have focused on (expression level and location), compared with the 899 other probe pairs (the complete list of the 940 probe pairs are provided as Supplementary Data). Finding cures. Genes that are expressed at low levels are not as likely to be reproducible across platforms. Next, we counted the number of different transcripts predicted to be hybridized by each probe (assuming for the moment that all RNAs are equally likely to be detected, regardless of 3′ location of the probe). Concordance was also improved when probes on the two platforms could be identified as being likely to target the same set of transcripts of a given gene. As an example of the latter situation, if one probe targets a previously unannotated transcript while another does not, our system might measure them as assaying the same transcripts while there is in fact a difference. Karolchik, D., Baertsch, R., Diekhans, M., Furey, T.S., Hinrichs, A., Lu, Y.T., Roskin, K.M., Schwartz, M., Sugnet, C.W., Thomas, D.J., et al. It is mission critical for us … SAN DIEGO & SANTA CLARA, Calif., Jan 10, 2008 - Affymetrix and Illumina, Inc. (NASDAQ:ILMN) announced today that they have entered into a settlement agreement to resolve … This is because noise will have a stronger influence on their measurement, making detecting a dilution effect difficult. These results shed light on the causes or failures of agreement across microarray platforms. Summary statistics for array designs studied, comparing two annotation methods. The full sets of annotations we derived are available as Supplementary Data, along with additional details of the results of the BLAT analysis. 1,2,3 Higher specificity and … both platforms indicate a strong dilution effect for the gene, but in the opposite direction. As for the effect of expression level, the effect remains after removing probes which failed the dilution effect filter. Search for other works by this author on: © The Author 2005. 6 – Bead T. Affymetrix GeneChip Mapping 500K Array Set . On both platforms, the probes not showing dilution effects tend to express at low levels, whereas highly expressed probes show strong dilution effects. Other ties often involved closely related genes, probably reflecting duplications (e.g. Saving children. The Affymetrix GeneChip Exon Array system provides, for the first time, exon-level expression profiling of … RNA was extracted using TRI reagent, purified using RNeasy columns (Qiagen, Valencia, CA), aliquoted and stored at −80°C until use. This set of cross-validated probes, though identified using conservative criteria on only one set of samples, could be considered as a starting point for identifying probes that perform well on these platforms. To our knowledge, this study is the first to examine the comparison between in situ synthesized oligonucleotide arrays with bead-based oligonucleotide arrays. To examine this in more detail, we sought to identify provisionally ‘unexplained’ cases of disagreement by filtering the full set of results, using partly arbitrary criteria. Cross-platform agreement measured by the rank correlation of expression levels as a function of expression level (log 2 ) ( A ) and distance between probes in base pairs ( B ). Very similar results overall were obtained when using annotations provided by the manufacturers (Supplementary Data). The laboratory offers both single sample analysis on cartridges or multi-sample analysis using PEG arrays on the Affymetrix GeneTitan system. The final ‘best’ match for a probe was the transcript closest to the probe's 3′ end and with the largest non-intronic overlap. Indeed, as shown in Figure 3C and D , probes targeted at known (annotated in RefSeq or in the ‘Known Gene’ table of the Golden Path database) genes have a stronger tendency to show a clear dilution effect.

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