A benchmark of hemoglobin blocking during library preparation for mRNA-Sequencing of human blood samples

Background: RNA-Sequencing (RNA-Seq) of peripheral blood can be a valuable source of information for investigating the status and mechanism of diseases. However, blood contains mostly unwanted hemoglobin (Hb) transcripts. A recent method for Illumina RNA-Seq features a 'Globin Block' (GB) module that depletes Hb transcripts during library preparation. Here, we aimed to assess GB's effectiveness, and we checked for technical biases attributable to GB. Results: We sequenced 91 pairs of GB and non-blocked samples (noGB) and 8 GB and noGB technical replicates from whole blood total RNA samples of 91 healthy individuals. GB reduced the fraction of Hb transcripts from 43 % to 8.0 %. From GB samples we detected 1,397 more expressed genes at approximately 11 million reads per RNA-isolate. Using correlation analyses we showed that GB and noGB samples could be assigned to the same RNA isolate with 89.8 % accuracy. Interestingly, lane and platform bias influenced accuracy stronger than GB. Gene expressions distinguishing between GB and noGB could not be enriched for sequence similarity or molecular function. Conclusions: Lexogen's GB RNA-Seq module is a valuable addition during mRNA-Seq library preparation. GB facilitates the detection of low abundant transcripts and yields more non-hemoglobin reads, while preserving biological information. Overall design: 91 healthy individuals' whole blood RNA samples were prepared with Lexogen's 3' mRNA-Seq Library Prep Kit, once with the optional Globin Block Module, once without. The effect of the Globin Block Module was investigated. 8 samples, once with the Globin Block were resequenced with Globin Block .