A model system for assessing and comparing the ability of exon microarray and tag sequencing to detect genes specific for malignant B-cellsReportar como inadecuado




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BMC Genomics

, 13:596

Transcriptomic methods

Abstract

BackgroundMalignant cells in tumours of B-cell origin account for 0.1% to 98% of the total cell content, depending on disease entity. Recently, gene expression profiles GEPs of B-cell lymphomas based on microarray technologies have contributed significantly to improved sub-classification and diagnostics. However, the varying degrees of malignant B-cell frequencies in analysed samples influence the interpretation of the GEPs. Based on emerging next-generation sequencing technologies NGS like tag sequencing tag-seq for GEP, it is expected that the detection of mRNA transcripts from malignant B-cells can be supplemented. This study provides a quantitative assessment and comparison of the ability of microarrays and tag-seq to detect mRNA transcripts from malignant B-cells. A model system was established by eight serial dilutions of the malignant B-cell lymphoma cell line, OCI-Ly8, into the embryonic kidney cell line, HEK293, prior to parallel analysis by exon microarrays and tag-seq.

ResultsWe identified 123 and 117 differentially expressed genes between pure OCI-Ly8 and HEK293 cells by exon microarray and tag-seq, respectively. There were thirty genes in common, and of those, most were B-cell specific. Hierarchical clustering from all dilutions based on the differentially expressed genes showed that neither technology could distinguish between samples with less than 1% malignant B-cells from non-B-cells. A novel statistical concept was developed to assess the ability to detect single genes for both technologies, and used to demonstrate an inverse proportional relationship with the sample purity. Of the 30 common genes, the detection capability of a representative set of three B-cell specific genes - CD74, HLA-DRA, and BCL6 - was analysed. It was noticed that at least 5%, 13% and 22% sample purity respectively was required for detection of the three genes by exon microarray whereas at least 2%, 4% and 51% percent sample purity of malignant B-cells were required for tag-seq detection.

ConclusionA sample purity-dependent loss of the ability to detect genes for both technologies was demonstrated. Taq-seq, in comparison to exon microarray, required slightly less malignant B-cells in the samples analysed in order to detect the two most abundantly expressed of the selected genes. The results show that malignant cell frequency is an important variable, with fundamental impact when interpreting GEPs from both technologies.

KeywordsExon microarrayTag-seqGene expressionDetection limitSample purityElectronic supplementary materialThe online version of this article doi:10.1186-1471-2164-13-596 contains supplementary material, which is available to authorized users.

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Autor: MariaBroKloster - AndersEllernBilgrau - MariaRodrigo-Domingo - KimSteveBergkvist - AlexanderSchmitz - MadsSnderkr - JulieStveBdker

Fuente: https://link.springer.com/







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