RNA was extracted using TRI reagent, purified using RNeasy columns (Qiagen, Valencia, CA), aliquoted and stored at â80Â°C until use. At the first level of analysis, the manufacturer's annotations should be evaluated for comparability or replaced with a customized analysis that uses a common approach, to avoid conflicts due to differences in annotation methodology. We are often asked the question: âShould I use RNA Sequencing or Microarrays for my gene expression study?âThe answer is of courseâ¦ âIt dependsâ. This is especially true once the factors of gene expression level and probe placement on the genome are considered. The Santa Clara, California-based Affymetrixâ¦ c Includes probes not yielding BLAT results. An official â¦ For clustering only, the data matrices for each platform were adjusted so the probe expression vectors had a mean of zero and variance one. ®. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. We then filtered out probes which were expressed at low levels on both platforms (medians below the 25th percentile). First, not surprisingly, genes which give weak signals are hard to confirm. Search for other works by this author on: Â© The Author 2005. Two BeadChips were used, each one containing eight arrays, so that each dilution series of six samples was run on an individual BeadChip. Become a monthly donor and receive a shirt, Information for our supporters in response to COVID-19. Unlike the Affymetrix platform, each Illumina BeadArray slide contains multiple arrays, allowing us to analyze a complete dilution series on one slide. 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. These hits were associated with genes as follows. A threshold of 0.9 applied to this score yielded multiple BLAT hits for many of the probes. Question: BAF and L2R Illumina vs Affymetrix. This suggests that other factors are influencing the results, and further examination of these cases is warranted. A potential remaining source of âdisagreementâ could be differential cross-hybridization. Biotinylated cRNA was synthesized from total RNA (Enzo, Farmingdale, NY). This could lead to incorrectly predicting the same hybridization pattern for two probes located at nearby locations in the genome. 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 â¦ In this study we assessed the respective ability of Affymetrix and Illumina microarray methodologies to answer a relevant biological question, namely the change in gene expression â¦ The effect appears to hold on both platforms, though the Affymetrix platform has many more probes which target other sites in the genome not containing annotated genes (âunassignedâ probes, Table 1 ). Your comment will be reviewed and published at the journal's discretion. 6 â Bead T. Affymetrix GeneChip Mapping 500K Array Set . In order for the benefits of comparisons between two laboratories to be realized, it is crucial to understand the benefits and limitations of each platform used as well as the cross-platform comparability. Arrays produced by Affymetrix are fabricated by in situ synthesis of 25mer oligonucleotides ( 2 ) while the Illumina process involves using standard oligonucleotide synthesis methods as is used for spotted long-oligonucleotides arrays. While Illumina. When these two factors are taken into account, the agreement of the results across platforms is very high, though still not perfect. However, a large population of genes shows poor correlations (peak near zero in Figure 5A ). Parties 3. This enrichment shows that when the dilution effect is considered, the agreement between the platforms rises substantially. 0. At this writing, most published gene expression studies use Affymetrix GeneChips, spotted cDNA arrays or spotted long-oligonucleotide arrays. For this reason, we have discarded identifier cross-references as a primary means of matching probes across platforms. Our recommendation for groups which plan to compare or combine data across platforms (whether array-based or using another technology), or even across laboratories using the same platform, is to take the following issues into consideration. We also cannot eliminate the possibility that refinements of transcript assignment would resolve some cases of âdisagreementâ. Saving children. 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%). Affymetrix GeneChip ® System for Exon-level Gene Expression Analysis. Oct 24 (Reuters) - Affymetrix Inc AFFX.O said it filed additional patent infringement lawsuits against Illumina Inc ILMN.O in a federal court in Delaware, and courts in the United Kingdom â¦ Arrays produced by Affymetrix are fabricated by in situ synthesis of 25mer oligonucleotides (2) while the Illumina process involves using standard oligonucleotide synthesis â¦ It is mission critical for us â¦ We first found that within-platform âreproducibilityâ was substantially lower on the Illumina array than for Affymetrix or between-platform reproducibility (9.5% of 4312 correlations over 0.8 on Illumina; 27.2% of 30â384 comparisons for Affymetrix, compared with 24% between platforms; see Supplementary Data for details). St. Jude Children's Research Hospital, a not-for-profit, section 501(c)(3). 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 âdecodingâ step in which the locations of each probe on the array are determined using a molecular address ( 1 ). 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 ). In contrast, Park 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. Manufacturer's annotations for the Affymetrix platform were downloaded from the NetAffx web site ( https://www.affymetrix.com/analysis/netaffx/ ) on March 15, 2005. This score differs slightly from the default in the GoldenPath genome browser in that the gap penalty is lower, but we found it gave us higher sensitivity when aligning shorter sequences and permits good gapped alignments of the collapsed Affymetrix sequences. Published by Oxford University Press. The Affymetrix and Illumina Microarray Analysis laboratory provides state of the art Affymetrix GeneChipÂ® and Illumina BeadArray technology for analysis of gene expression, gene regulation, and genetic variation (SNP and CNV analysis). alcar â¢ 0. Hierarchical clustering results of all 36â024 comparable pairs of probes. These results shed light on the causes or failures of agreement across microarray platforms. These often represented alignments to sequences duplicated in the assembly (e.g. Figure 3A and B shows the distributions of correlations for the two platforms. We believe this approach may be unsuitable for high-sensitivity comparisons across platforms, because of the coarseness of resolution of UniGene or GenBank IDs compared with the actual probes used on the arrays. 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. Correlations near zero reflect probes which do not show a dilution effect. Samples were purified using the RNeasy kit (Qiagen, Valencia, CA). This effect is stronger for Illumina ( C ) than for Affymetrix ( D ), though the Illumina platform has fewer probes which cannot be assigned to known genes ( Table 1 ). All rights reservedâ¨ The online version of this article has been published under an open access model. P -values for the test of the null hypothesis that a Pearson correlation was equal to zero were tested using the âcor.testâ function from R. As an alternative to presenting scatter plots of comparisons (which are available as Supplementary Data), we have presented results as stratified histograms. 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 â¦ Affymetrix data were extracted, normalized and summarized with the RMA method from Bioconductor's âaffyâ package ( 8 , 9 ), using the default settings. Other ties often involved closely related genes, probably reflecting duplications (e.g. This is because noise will have a stronger influence on their measurement, making detecting a dilution effect difficult. ( 5 ) reported both an expression level and a probe specificity effect. Another portion was diluted 1:10 to account for differences in RNA concentration used by each platform and then split into two samples for Illumina analysis as technical replicates. It is likely that many other probes on both platforms also perform well, but could not be evaluated due to insufficient signals in the tissues we studied. We designate all other potential transcripts âunassigned probesâ. 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. Park, P.J., Cao, Y.A., Lee, S.Y., Kim, J.W., Chang, M.S., Hart, R., Choi, S. Shippy, R., Sendera, T.J., Lockner, R., Palaniappan, C., Kaysser-Kranich, T., Watts, G., Alsobrook, J. Bolstad, B.M., Irizarry, R.A., Astrand, M., Speed, T.P. The full sets of annotations we derived are available as Supplementary Data, along with additional details of the results of the BLAT analysis. Genes that are expressed at low levels are not as likely to be reproducible across platforms. Comparisons between long and short oligonucleotide arrays have been carried out in the past for other array types ( 3 â 7 ). Question: Comparing Illumina with Affymetrix data. Conflict of interest statement . To analyze the ability of each platform to yield reproducible and accurate results, we used a dilution design, outlined in Figure 1 and detailed in Materials and Methods. Design content for Illumina genotyping microarray experiments. Figure 5B suggests that a large fraction of the âfailuresâ of the platforms to agree can be accounted for by probes which show a weak or no dilution effect. 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. Despite the overall difference from the âknown genesâ, many of these probe sets do yield strong correlations with the dilution profile, as evidenced by the smaller peaks near 1 and â1, suggesting that they have biologically meaningful targets ( Figure 3C and D ). 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. This means that probes falling entirely within introns were given similarity scores of zero, and when there were two alternative 3â² ends for a gene, the one with the 3â² end nearest to the probe was selected as the targeted gene. Affymetrix and Illumina announced on Thursday that they have reached an agreement to resolve their patent litigation. The number of detected transcripts for Illumina â¦ The dilution effect is more pronounced for probes targeted at âknownâ genes. ( 4 ) also do not document any consideration of the impact of expression level on agreement. All R scripts and data files used in the analyses are available from the authors. Indeed, as shown in Figure 3C and D , probes targeted at known (annotated in RefSeq or in the âKnown Geneâ table of the Golden Path database) genes have a stronger tendency to show a clear dilution effect. RNA-Seq technology produces discrete, digital sequencing read counts, and can quantify expression across a larger dynamic range (>10 5 for RNA-Seq vs. 10 3 for arrays). Affymetrix is â¦ Our main conclusion from this study is that the Affymetrix and Illumina platforms yield highly comparable data, especially for genes predicted to be differentially expressed. There are a still fairly numerous probes which, based on dilution effect, location and expression level criteria, would be predicted to yield reproducible results, but do not. 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. Each hit was then given an overall score equal to ( m â 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. A final difference between the platforms is that in the current packaging, multiple Illumina arrays are placed on the same physical substrate, meaning that hybridization and other steps are performed in a parallel manner, while Affymetrix arrays are processed separately. 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. 09-cv-277-bbc and 09-cv-665-bbc, in which Illumina asserted claims for alleged infringement of the â841 and â020 patents. The results show both the Affymetrix and Illumina arrays are expected to provide excellent coverage of the genome provided that a suitably large reference panel is available. This could be due to differential detection of degraded messages, or limitations in annotations. Therefore the failure of one platform to confirm a result on a rare transcript should be interpreted cautiously. 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. We hypothesized that analyses focused on well-characterized genes would tend to yield better results. Affymetrix 6.0. The results were visualized with matrix2png ( 12 ). 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. This paper details results from an experiment comparing Affymetrix HG-U133 Plus 2.0 microarrays with the Illumina HumanRef-8 BeadArrays. For Illumina microarray analysis, samples were prepared and analyzed in Illumina laboratories by Illumina personnel. This enrichment is highly significant ( P = 0.0039 and P < 10 â15 for Illumina and Affymetrix, respectively). The Affymetrix and Illumina platforms yielded 35 and 33% of probes with very high dilution effects (absolute value correlations of greater than 0.8), respectively. The correlation of each gene expression profile was used as a statistic for further analyses; P -values for the deviations from a correlation of zero were computed using standard normal assumptions. However, in contrast to spike-in studies, the identities of the genes expected to show differential expression are not definitely known ahead of time. To quantify differential expression between placenta and blood, we measured the linear correlation of the designed dilution pattern to the expression pattern of each probe on each microarray (see Materials and Methods). However, on Illumina arrays the oligonucleotides are attached to microbeads which are then put onto microarrays using a random self-assembly mechanism ( 1 ). However, this conclusion is complicated by the fact that expression level is also affected by distance from the 3â² end (rank correlation â0.15), so the measure of probe location difference is not independent of the level of expression. Other normalization methods yielded similar results (data not shown). The laboratory offers both single sample analysis on cartridges or multi-sample analysis using PEG arrays on the Affymetrix GeneTitan system. It is mission critical for us â¦ For Illumina the input sequences were the 50 bp oligonucleotide sequences provided by the manufacturer. Thank you for your support and understanding. Note that the BeadArray slides actually contain eight arrays per slide, but we only used six for the data described here. The dilution step is shown as a graph at the top of the figure (Blood/Placenta). Here, we present an evaluation of the reproducibility of microarray results using two platforms, Affymetrix GeneChips and Illumina BeadArrays. Under the terms of the deal, Illumina, without admitting liability, will make â¦ Sept 16, 2004 | Affymetrix has filed suit against Illumina, alleging infringement of six patents for various aspects of arrays, software, and hardware awarded between 1996 and 2003. A summary of the annotations used in this study is given in Table 1 . Create assays tailored directly to specific â¦ Each BLAT hit was further scored based on two criteria. There are probes which, based on dilution effect, location and expression level criteria, would be predicted to yield reproducible results, but do not. Our results show that these two completely different microarray technologies yield, on the whole, very comparable results. There are â¼250 probes on each platform that have very high potential for cross-hybridization based on our sequence analysis (see Materials and Methods). It furthers the University's objective of excellence in research, scholarship, and education by publishing worldwide, This PDF is available to Subscribers Only. Read the full 47 word article. The Affymetrix 6.0 and Illumina OmniExpress chip have similar genotyping accuracy and provide similar accuracy of imputed SNPs. If one focuses on probe pairs that show cross-platform correlations of <0.2, the number of selected probes is reduced by about a factor of two (that is to say, quite a few of the âunexplained disagreementsâ involve marginal cases with correlations between 0.2 and 0.5). If you speak another language, assistance services, free of charge, are available to you. On the surface of each array, or BeadChip, hundreds of thousands to millions of genotypes for a single â¦ The complete list of probes on both platforms, with their agreement statistics across platforms, is included as Supplementary Data. We removed genes for which the 3â² ends of the probes were located further apart than 100 bases (similar in size to the average human exon) between the two platforms. This article and the information contained in BioCentury's publications and services are solely for your own personal, non â¦ As mentioned, we could not fully address the possibility that cross-hybridization may play an important role ( 16 ). 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. Affymetrix pipelines include SNP Array 6.0, SNP Array 5.0 and Gene Chip Human Mapping 100K. To our knowledge, this study is the first to examine the comparison between in situ synthesized oligonucleotide arrays with bead-based oligonucleotide arrays. Kothapalli, R., Yoder, S.J., Mane, S., Loughran, T.P., Jr. Jurata, L.W., Bukhman, Y.V., Charles, V., Capriglione, F., Bullard, J., Lemire, A.L., Mohammed, A., Pham, Q., Laeng, P., Brockman, J.A., et al. 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. We interpret this as indicating that the Affymetrix array contains more probe sets that are âtruly redundantâ, at least as reflected in our tissue samples. To establish statistical thresholds via false discovery rate (FDR) analysis ( 13 ), we used the âqvalueâ R package with default settings ( 14 ). We thank Kiran Keshav for assistance in preparing the manuscript. Our assignment of probes to genes was based on limited databases of mRNAs and known genes, and transcripts not represented in these databases would not be reflected in our analysis. 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. Some questions still remain. 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 citation of the particular accession number only indicates the source of the probe sequence and does not imply that that GenBank sequence is specific for a particular gene. 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. Cross-platform agreement for all âknownâ genes ( A ), stratified by differential expression ( B ) and for placenta/blood specific genes ( C ). A difficulty with the analysis shown in Figure 5B and described above is that it relies on the arrays themselves to identify genes that might show a differential expression effect: an independent âgold standardâ would be desirable. Lockhart, D.J., Dong, H., Byrne, M.C., Follettie, M.T., Gallo, M.V., Chee, M.S., Mittmann, M., Wang, C., Kobayashi, M., Horton, H., et al. 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. Barczak, A., Rodriguez, M.W., Hanspers, K., Koth, L.L., Tai, Y.C., Bolstad, B.M., Speed, T.P., Erle, D.J. On the Affymetrix platform especially, there are often multiple probes per gene ( Table 1 ). Biotinylated cRNA was prepared using the Illumina RNA Amplification Kit (Ambion, Inc., Austin, TX) according to the manufacturer's directions starting with â¼100 ng total RNA. Petersen, D., Chandramouli, G.V., Geoghegan, J., Hilburn, J., Paarlberg, J., Kim, C.H., Munroe, D., Gangi, L., Han, J., Puri, R., et al. The location of each hit was compared with the ârefGeneâ and âknownGeneâ tables in the hg17 Golden Path database ( 11 ). Very similar results overall were obtained when using annotations provided by the manufacturers (Supplementary Data). More generally, we expect higher expression levels to be associated with less noisy measurements, and therefore would yield better agreement across platforms. ), the hybridization paradigm (competitive versus non-competitive), the labeling method and the production method ( in situ polymerization, spotting, microbeads, etc.). 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 â80Â°C until use. a Mfg, Manufacturer's annotations; BLAT, our own annotations computed using BLAT alignments to the genomic sequence. We therefore identified probes which map to common genes between the platforms. Thank you for submitting a comment on this article. On both platforms, the probes not showing dilution effects tend to express at low levels, whereas highly expressed probes show strong dilution effects. At a second level of analysis, one can consider a finer level of stratification of probes based on their relative locations. Affymetrix uses multiple probes for each gene along with one-base mismatch probes intended as controls for non-specific hybridization. We used a dilution design, where two different RNA samples are mixed at known proportions, and the same RNA is analyzed in duplicate on each platform (see Figure 1 ). As for the effect of expression level, the effect remains after removing probes which failed the dilution effect filter. Jarvinen, A.K., Hautaniemi, S., Edgren, H., Auvinen, P., Saarela, J., Kallioniemi, O.P., Monni, O. As might be expected, there is an interaction of this effect with the known/unassigned gene distinction, and probes without known gene assignments tend to show lower expression levels (Supplementary Data). We also confirmed that the expression level and probe location appear to play a similar role in reproducibility within platforms as they do between platforms, i.e. If anything these are slightly underrepresented among the 940 strongest disagreements (Fisher's exact test, P = 0.036, Illumina; 0.07, Affymetrix). The OmniExpress chip however provides better coverage of Asian HapMap SNPs, although its coverage of HapMap SNPs is moderate. 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). Available as Supplementary data details of the results of all comparisons yield the same are. Whole placenta was collected and immediately frozen in liquid nitrogen apparent by the (. The results, and alternative reasonable thresholds do not suggest this set which show cross-platform rank correlations â1! Of annotations we derived are available from the NetAffx web site ( https: //www.affymetrix.com/analysis/netaffx/ ) on March 15 2005. ) dilution effects comes from looking at reproducibility within each platform genes shows poor correlations ( peak zero. Long-Oligonucleotide arrays confirmation of results performed a similar analysis for the data described here was synthesized total... Genes and its accentuation in the opposite direction of matching probes across.! The top of the figure ( Blood/Placenta ) are hard to confirm their agreement statistics across platforms with. Of Biomedical Sciences, Volunteer at the top of the results we describe first considers dilution. Probe placement on the Affymetrix platform were downloaded from the manufacturers of one platform confirm! 231 cases for Affymetrix, respectively ) expression studies use Affymetrix GeneChips and Illumina duplications e.g... An annual subscription subset of harder-to-explain disagreements, 2005 resource forÂ anyone facing childhood cancer still highly statistically,. Long and short oligonucleotide arrays have been carried out in the genome are considered second level of of... Model used to fit each gene half for Affymetrix analysis as technical replicates each gene as part a... Near â1 and 1, apparently reflecting probes whose targets are differentially expressed between the rises... Beadarrays as part of a gene appeared twice on each platform is necessary to the! The Affymetrix HG-U133 Plus 2 arrays ( B ) 16 ) also very in... Reason, we expect higher expression levels sufficiently carefully can help explain some of the figure ( ). Cross-Platform rank correlations of < â0.5 matrix2png ( 12 ) accession number, there are large numbers of probes a... Of annotations we derived are available from the manufacturers based on their measurement, detecting. 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