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

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE133758 | GEO | 2020/04/14

REPOSITORIES: GEO

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Project description:Hypoxia, a naturally occurring phenomenon, results in tremendous losses in the growth of plants and in their productivity. On the physiological level oxygen deprivation results in distinct metabolic rearrangements, is accompanied by dehydration, and oxidative and nitrosative stresses. Six to seven core hypoxia-responsive genes, which are always induced under oxygen deprivation, have been shown to include hemoglobin 1 (Hb1) across a wide range of plant species. However, the function of this induced Hb1 is not completely understood .The importance of elevated Hb expression has been attributed mainly to its nitric oxide (NO) scavenging properties, and hence, to the elimination of this free-radical and signaling species. NO is known to accumulate under hypoxia and its level is in reciprocal relationship with the Hb expression levels. However, evidence has accumulated on Hbs as essential for normal plant development and they take part in the regulation of e.g. flowering, germination and lateral and adventitious root emergence. The physiological role of induced hemoglobin (Hb) expression and its role in NO scavenging were assessed by introducing bacterial (Vitreoscilla stercoraria) hemoglobin (VHb) to the cytoplasm and mitochondria of Arabidopsis thaliana. Heterologous expression was chosen to avoid interference with developmental functions of endogenous Hbs. Sixteen day old transformants were subjected to hypoxia or to NO donor treatment (DETA, 500 µM) for 2h and 24h and global changes in gene expression were assessed using a microarray approach. Both hypoxic and NO-treatment experiments were aimed also to reveal novel functions for Hb under abiotic stress conditions, exemplified by oxygen deprivation and NO/oxidative stresses and during normal development of Arabidopsis transformants on the background of normally expressed endogenous Hbs Hypoxia, a naturally occurring phenomenon, results in tremendous losses in the growth of plants and in their productivity. On the physiological level oxygen deprivation results in distinct metabolic rearrangements, is accompanied by dehydration, and oxidative and nitrosative stresses. Six to seven core hypoxia-responsive genes, which are always induced under oxygen deprivation, have been shown to include hemoglobin 1 (Hb1) across a wide range of plant species. However, the function of this induced Hb1 is not completely understood .The importance of elevated Hb expression has been attributed mainly to its nitric oxide (NO) scavenging properties, and hence, to the elimination of this free-radical and signaling species. NO is known to accumulate under hypoxia and its level is in reciprocal relationship with the Hb expression levels. However, evidence has accumulated on Hbs as essential for normal plant development and they take part in the regulation of e.g. flowering, germination and lateral and adventitious root emergence. The physiological role of induced hemoglobin (Hb) expression and its role in NO scavenging were assessed by introducing bacterial (Vitreoscilla stercoraria) hemoglobin (VHb) to the cytoplasm and mitochondria of Arabidopsis thaliana. Heterologous expression was chosen to avoid interference with developmental functions of endogenous Hbs. Sixteen day old transformants were subjected to hypoxia or to NO donor treatment (DETA, 500 µM) for 2h and 24h and global changes in gene expression were assessed using a microarray approach. Both hypoxic and NO-treatment experiments were aimed also to reveal novel functions for Hb under abiotic stress conditions, exemplified by oxygen deprivation and NO/oxidative stresses and during normal development of Arabidopsis transformants on the background of normally expressed endogenous Hbs

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

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