Large-scale RNA-seq series – Ep.1: RNA-seq helps uncover the genetic regulation of blood cells

The largest blood cell genotyping and transcriptomic study ever performed has shown that a much higher number of common changes in the DNA sequence affect gene expression in blood cells than previously thought (Võsa et al. 2021). In this article, we look at how the use of RNA-seq in this study contributed to these exciting new discoveries in the genetic regulation of blood cells.

Summary:

Genetics is one of the deciding factors of traits and disease susceptibility. When changes in DNA sequence can explain variation in gene expression levels for a specific trait or disease, they are called expression quantitative trait loci (eQTLs) (Nica and Dermitzakis 2013).

To detect eQTLs in blood cells, the eQTLGen Consortium analyzed a data set comprising high-throughput genomic and transcriptomic technologies, such as whole genome sequencing, RNA-seq and DNA variant and expression microarrays (Fig. 1) (Võsa et al. 2021). It included 37 studies with more than 31,000 study participants (Võsa et al. 2021).

Thanks to these high-throughput technologies including RNA-seq, and the large sample size, researchers discovered that genetic variants in close proximity to genes (cis-eQTLs) regulate 88% of all genes analyzed. This often includes a physical interaction between the variant and the gene (Võsa et al. 2021). In contrast, genomic variants over 5Mb away from genes (trans-eQTLs) regulate one third of all genes analyzed, likely due to transcription factor activity (Võsa et al. 2021). For example, one genetic variant possibly regulates the neuronal repressor REST and the expression of 88 neuronal genes in trans as a result (Võsa et al. 2021).

How RNA-seq was used:

eQTL detection relies firstly on genotyping to find genetic variations, followed by RNA-seq or microarray driven transcriptomics to detect gene expression variation for the whole genome of each individual (Fig. 1) (Nica and Dermitzakis 2013). Discovery of eQTLs are then made through the statistical association of these genetic variants with the expression level of the gene of interest. When the sample size is large enough, these statistics can even detect weak effects of genetic variants on gene expression.

Figure 1: Schematic showing high-throughput eQTL detection stages. Genotyping is first performed on each individual to identify genomic variants, followed by RNA-seq or microarray-based transcriptomics to identify gene expression changes associated with the genomic variant.

RNA-seq was used to investigate one fifth of the samples, whereas the remaining samples used expression microarrays (Võsa et al. 2021). To combine gene expression data from these different transcriptomic technologies, the authors used RNA-seq as the gold standard reference to calculate correlation scores for each gene with all other genes. Gene correlation profiles from the different microarrays were then correlated with profiles from RNA-seq data. This resulted in nearly 20,000 expressed genes tested for cis-eQTL or trans-eQTL associations in over 31,000 study participants.

How the large number of samples contributed to the results:

The authors discovered many weaker trans-eQTL effects thanks to the increased statistical power that such a large sample size provides. These weaker trans-eQTL effects are important because they are thought to have more impact on the trait or disease, compared to genes regulated by stronger cis-eQTL effects (Price et al. 2011; Westra et al. 2013).
Smaller studies may lack the statistical power needed to detect these important but weaker trans-eQTL effects. This was the case in the previous largest study investigating eQTLs in blood (Westra et al. 2013). It had six times fewer samples and found far less trans-eQTLs (Westra et al. 2013).

Sample size was also a problem when the eQTLGen Consortium tried to confirm their results with other smaller bulk RNA-seq eQTL data sets, such as GTex with just under 400 individuals (Võsa et al. 2021). The same issue arose in the analysis of a single-cell RNA-seq eQTL data set from over 1000 individuals, which the authors used to address concerns about cell composition in their original eQTL analyses (Võsa et al. 2021). In both cases, they could only confirm a small number of their initial results, likely due to reduced statistical power.

How novel higher-throughput transcriptomics could help in similar studies:

To address this, new technologies are available to boost sample sizes and therefore statistical power in transcriptomic studies by removing cost and hands-on-time as limiting factors. Large cohort studies reliant on transcriptomics are prohibitively expensive. Furthermore, if the eQTLGen Consortium had used the same transcriptomic method for all samples, study quality could have been improved by removing the need for independent pre-processing of each different microarray platform.

Novel 3’ mRNA-seq technologies such as Bulk RNA Barcoding and sequencing (BRB-seq) address these cost and throughput issues. This technology is based on barcoding and early multiplexing of hundreds of samples, requiring only one subsequent library preparation. It would enable a study of 31,000 samples to be performed at a fraction of the cost, while reducing both technical variation and turnaround times (Alpern et al. 2019).

Although BRB-seq does not allow for the analysis of full-length transcripts, this is not necessary for eQTL expression analyses. BRB-seq detects the same number of genes as the ‘gold standard’ Illumina TruSeq library preparation method and is highly accurate and cost-effective. It is reliable even for low quality RNA samples which may be a key concern when processing precious human samples.

For blood transcriptomic studies, the MERCURIUS™ Blood BRB-seq kits include proprietary globin blockers enabling efficient depletion of unwanted globin genes

To find out more about how BRB-seq could help your study, please contact us at info@stratech.co.uk

References:

• Alpern, D., Gardeux, V., Russeil, J., Mangeat, B., Meireles-Filho, A., Breysse, R., Hacker, D., Deplancke, B. 2019. BRB-seq: Ultra-affordable high-throughput transcriptomics enabled by bulk RNA barcoding and sequencing. Genome Biol, 20:71.
• Nica, A.C. and Dermitzakis, E.T., 2013. Expression quantitative trait loci: present and future. Philosophical Transactions of the Royal Society B: Biological Sciences, 368(1620).
• Price, A.L., Helgason, A., Thorleifsson, G., McCarroll, S.A., Kong, A. and Stefansson, K., 2011. Single-tissue and cross-tissue heritability of gene expression via identity-by-descent in related or unrelated individuals. PLoS genetics, 7(2).
• Võsa, U., Claringbould, A., Westra, H.J., Bonder, M.J., Deelen, P., Zeng, B., Kirsten, H., Saha, A., Kreuzhuber, R., Brugge, H. and Oelen, R., 2021. Large-scale cis-and trans-eQTL analyses identify thousands of genetic loci and polygenic scores that regulate blood gene expression. Nature genetics, 53(9).
• Westra, H.J., Peters, M.J., Esko, T., Yaghootkar, H., Schurmann, C., Kettunen, J., Christiansen, M.W., Fairfax, B.P., Schramm, K., Powell, J.E. and Zhernakova, A., 2013. Systematic identification of trans eQTLs as putative drivers of known disease associations. Nature genetics, 45(10).

Further Reading:

Large-scale RNA-seq series – Ep.2: Predicting cancer outcomes by estimation of tumor specific total mRNA levels in bulk RNA-seq

Large-scale RNA-seq series – EP.3: ‘Multi-omics’ improves the classification of 33 different cancer types using 10,000 tumor samples.

Large-scale RNA-seq series Ep. 4: BRB-seq uncovers novel features of fruit fly embryogenesis

Large-scale RNA-seq series – Ep. 5. – Gene expression networks control organ development across species

Large-scale RNA-seq series Ep. 6 – Sex Impacts Gene Expression, but not the way you think!