Increased sensitivity of next generation sequencing-based expression profiling after globin reduction in human blood RNA

Abstract Background Transcriptome analysis is of great interest in clinical research, where significant differences between individuals can be translated into biomarkers of disease. Although next generation sequencing provides robust, comparable and highly informative expression profiling data, with several million of tags per blood sample, reticulocyte globin transcripts can constitute up to 76% of total mRNA compromising the detection of low abundant transcripts. We have removed globin transcripts from 6 human whole blood RNA samples with a human globin reduction kit and compared them with the same non-reduced samples using deep Serial Analysis of Gene Expression. Results Globin tags comprised 52-76% of total tags in our samples. Out of 21,633 genes only 87 genes were detected at significantly lower levels in the globin reduced samples. In contrast, 11,338 genes were detected at significantly higher levels in the globin reduced samples. Removing globin transcripts allowed us to also identify 2112 genes that could not be detected in the non-globin reduced samples, with roles in cell surface receptor signal transduction, G-protein coupled receptor protein signalling pathways and neurological processes. Conclusions The reduction of globin transcripts in whole blood samples constitutes a reproducible and reliable method that can enrich data obtained from next generation sequencing-based expression profiling.</p

Increased sensitivity of next generation sequencing-based expression profiling after globin reduction in human blood RNA

Mechanisms of disease, diagnostics and therap

The number of <i>cis-</i>regulated tags per gene.

<p>The percentages of cis-regulated tags mapping into the same gene are indicated (781 genes overall). For nearly half of the genes (48%) only one tag shows an eQTL effect. If multiple tags map within the same gene, only one eQTL tag should pass the FDR<0.05 significance threshold while the other tag could be less significant. For these eQTLs the allelic direction is shown: same allelic direction (multiple tags within the same gene are cis-regulated by a SNP in the same direction), significantly opposite allelic direction (multiple tags within the same gene are cis-regulated by a SNP but with opposite directions and the difference between the correlation coefficients is significant), or opposite allelic direction but not significant (if the difference between correlation coefficients is not significant).</p

Fraction of <i>cis-</i>regulated genes in bins by mean gene expression levels for DeepSAGE and Affymetrix data.

<p>For each dataset, all genes were sorted by their mean gene expression levels, and divided into ten equal bins. The X-axis reflects these bins, which are sorted by increasing mean gene expression levels. The Y-axis reflects the fraction of <i>cis</i>-regulated genes that fall into each bin.</p

Number of <i>cis-</i>regulated tags mapping to different genomic regions in tag-wise DeepSAGE eQTL mapping.

<p>Number of <i>cis-</i>regulated tags mapping to different genomic regions in tag-wise DeepSAGE eQTL mapping.</p

Description of RNA next generation sequencing datasets.

<p>Description of RNA next generation sequencing datasets.</p

<i>Cis-</i>regulating SNPs significantly^* affecting multiple tags of the same gene in opposite directions.

*<p>Only significant eQTLs with FDR<0.05 for both <i>cis-</i>regulated tags were used.</p