Peripheral blood is a highly accessible biofluid providing a rich source of information about human physiology and health status. However, for studies of the blood transcriptome with RNA sequencing (RNA-Seq) techniques, high levels of hemoglobin mRNAs (hgbRNA) present in blood can occupy valuable sequencing space, impacting detection and quantification of non-hgbRNAs.
In this study, researchers from the Oregon Health & Science University evaluated two methods for preparing ribosomal RNA (rRNA)-depleted sequencing libraries for RNA-Seq of whole blood, one of which is also designed to deplete hgbRNAs. Two experiments were performed: one evaluating library performance across 6 human blood samples and the other examining library reproducibility and performance in a two-subject subset. The researchers found that addition of hgbRNA depletion to the rRNA-depletion protocol for library preparation from blood RNA effectively reduces highly abundant hgbRNA reads; however, it does not result in a statistically significant increase in differentially expressed genes in our patient-control study. Bioinformatic removal of globin gene counts in non-hgbRNA depleted libraries provides improvement in overall performance of these libraries. They conclude that use of a standard ribosomal RNA depletion method for library preparation coupled with bioinformatic removal of globin gene counts is sufficient for reproducible and sensitive measurement of both coding and noncoding RNAs in the blood transcriptome.
Experimental Design and Analysis. (a) Blood RNA preparation steps from 6 human subjects: 3 controls (c) and 3 patients (P). RNA was used for preparing libraries with Ribo-Zero Gold (RZG) or Globin-Zero (GZ) kits as shown. In Experiment 1, a total of 33 libraries were prepared with either 250 ng total RNA for low (L) input or 900 ng for high (H) input. In Experiment 2 technical replicate libraries were prepared using 250 ng RNA from C1 and C2 with either RZG or Globin-Zero for a total of 12 libraries. Individual RNA-seq data files generated from the libraries shown in B and C were named according to the following convention: sample ID_RNA input_library method_replicate number, e.g., C1_L_GZ_1. (b) Fastq files were assessed for quality metrics, aligned and analyzed as shown.