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Establishing a major cause of discrepancy in the calibration of Affymetrix GeneChips

BACKGROUND: Affymetrix GeneChips are a popular platform for performing whole-genome experiments on the transcriptome. There are a range of different calibration steps, and users are presented with choices of different background subtractions, normalisations and expression measures. We wished to esta...

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Autores principales: Harrison, Andrew P, Johnston, Caroline E, Orengo, Christine A
Formato: Texto
Lenguaje:English
Publicado: BioMed Central 2007
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1904248/
https://www.ncbi.nlm.nih.gov/pubmed/17562008
http://dx.doi.org/10.1186/1471-2105-8-195
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author Harrison, Andrew P
Johnston, Caroline E
Orengo, Christine A
author_facet Harrison, Andrew P
Johnston, Caroline E
Orengo, Christine A
author_sort Harrison, Andrew P
collection PubMed
description BACKGROUND: Affymetrix GeneChips are a popular platform for performing whole-genome experiments on the transcriptome. There are a range of different calibration steps, and users are presented with choices of different background subtractions, normalisations and expression measures. We wished to establish which of the calibration steps resulted in the biggest uncertainty in the sets of genes reported to be differentially expressed. RESULTS: Our results indicate that the sets of genes identified as being most significantly differentially expressed, as estimated by the z-score of fold change, is relatively insensitive to the choice of background subtraction and normalisation. However, the contents of the gene list are most sensitive to the choice of expression measure. This is irrespective of whether the experiment uses a rat, mouse or human chip and whether the chip definition is made using probe mappings from Unigene, RefSeq, Entrez Gene or the original Affymetrix definitions. It is also irrespective of whether both Present and Absent, or just Present, Calls from the MAS5 algorithm are used to filter genelists, and this conclusion holds for genes of differing intensities. We also reach the same conclusion after assigning genes to be differentially expressed using t-statistics, although this approach results in a large amount of false positives in the sets of genes identified due to the small numbers of replicates typically used in microarray experiments. CONCLUSION: The major calibration uncertainty that biologists need to consider when analysing Affymetrix data is how their multiple probe values are condensed into one expression measure.
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spelling pubmed-19042482007-06-29 Establishing a major cause of discrepancy in the calibration of Affymetrix GeneChips Harrison, Andrew P Johnston, Caroline E Orengo, Christine A BMC Bioinformatics Research Article BACKGROUND: Affymetrix GeneChips are a popular platform for performing whole-genome experiments on the transcriptome. There are a range of different calibration steps, and users are presented with choices of different background subtractions, normalisations and expression measures. We wished to establish which of the calibration steps resulted in the biggest uncertainty in the sets of genes reported to be differentially expressed. RESULTS: Our results indicate that the sets of genes identified as being most significantly differentially expressed, as estimated by the z-score of fold change, is relatively insensitive to the choice of background subtraction and normalisation. However, the contents of the gene list are most sensitive to the choice of expression measure. This is irrespective of whether the experiment uses a rat, mouse or human chip and whether the chip definition is made using probe mappings from Unigene, RefSeq, Entrez Gene or the original Affymetrix definitions. It is also irrespective of whether both Present and Absent, or just Present, Calls from the MAS5 algorithm are used to filter genelists, and this conclusion holds for genes of differing intensities. We also reach the same conclusion after assigning genes to be differentially expressed using t-statistics, although this approach results in a large amount of false positives in the sets of genes identified due to the small numbers of replicates typically used in microarray experiments. CONCLUSION: The major calibration uncertainty that biologists need to consider when analysing Affymetrix data is how their multiple probe values are condensed into one expression measure. BioMed Central 2007-06-11 /pmc/articles/PMC1904248/ /pubmed/17562008 http://dx.doi.org/10.1186/1471-2105-8-195 Text en Copyright © 2007 Harrison et al; licensee BioMed Central Ltd. http://creativecommons.org/licenses/by/2.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( (http://creativecommons.org/licenses/by/2.0) ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Research Article
Harrison, Andrew P
Johnston, Caroline E
Orengo, Christine A
Establishing a major cause of discrepancy in the calibration of Affymetrix GeneChips
title Establishing a major cause of discrepancy in the calibration of Affymetrix GeneChips
title_full Establishing a major cause of discrepancy in the calibration of Affymetrix GeneChips
title_fullStr Establishing a major cause of discrepancy in the calibration of Affymetrix GeneChips
title_full_unstemmed Establishing a major cause of discrepancy in the calibration of Affymetrix GeneChips
title_short Establishing a major cause of discrepancy in the calibration of Affymetrix GeneChips
title_sort establishing a major cause of discrepancy in the calibration of affymetrix genechips
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1904248/
https://www.ncbi.nlm.nih.gov/pubmed/17562008
http://dx.doi.org/10.1186/1471-2105-8-195
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