Figure 1.
Experimental design.
Table 1.
Effect of globin depletion on RNA-Seq data quality.
Figure 2.
Globin depletion reduces the preponderance of reads mapping to globin transcript.
The RNA-Seq reads mapping to one of the globin gene transcripts, HBA1, are visualized in the Integrated Genome Viewer software for an exemplar RNA sample that was globin depleted (top) or not (middle). The gene model is shown at the bottom.
Figure 3.
Globin depletion has no impact on the correlation of technical replicates.
Correlation plot of the transcript FPKMs of lane technical replicate (top) or pooled technical replicate (bottom) exemplar pairs. Data is shown on a log scale. Spearman's rho is reported for all (rall) or reliably detectable transcripts (rFPKM≥1) only.
Figure 4.
Globin depletion yields 3500 additional robustly detectable transcripts from a single representative peripheral whole blood RNA-Seq experiment.
(A) Correlation plot of the transcript FPKMs of an exemplar biological sample, either globin depleted (y-axis) or not (x-axis). (B) Distribution of transcript FPKMs in the same exemplar biological sample, either globin depleted (blue) or not (red). Data is shown on a log scale. Significant FPKM cutoffs of 1, 2.5 and 1000 are marked by dashed line.
Figure 5.
Globin depletion reduces technical variability while increasing measurable biological variability.
Distribution of transcript coefficient of variation (CV) across pooled technical replicates (left) and biological replicates (right), either globin depleted (blue) or not (red).
Figure 6.
Globin depletion results in increased differential gene discovery.
(A) The results of differential gene expression analysis comparing NGD and GD samples are visualized for various experimental designs. The log2 fold-change of genes between NGD and GD is plotted against mean log2 counts per million mapped reads (CPM; a library-size normalized expression measure) across samples and significant genes (FDR≤0.01) are shown in red. Overlaps in significantly differentially expressed genes across the various designs are visualized in Venn diagrams for (B) up-regulated and (C) down-regulated genes.
Table 2.
Transcripts down-regulated as a result of globin depletion.
Table 3.
Enrichment of GO molecular function terms in transcripts down-regulated as a result of globin depletion.