scholarly journals Comparative analysis of RNA enrichment methods for preparation of Cryptococcus neoformans RNA sequencing libraries

2021 ◽  
Author(s):  
Calla L Telzrow ◽  
Paul J Zwack ◽  
Shannon Esher Righi ◽  
Fred S Dietrich ◽  
Cliburn Chan ◽  
...  
Keyword(s):  
Cryptococcus Neoformans ◽  
Rna Sequencing ◽  
Rnase H ◽  
Rrna Genes ◽  
Rna Seq ◽  
Protein Coding ◽  
Mitochondrial Rrna ◽  
Non Coding Rna ◽  
Rrna Depletion ◽  
Enrichment Methods

Abstract RNA sequencing (RNA-Seq) experiments focused on gene expression involve removal of ribosomal RNA (rRNA) because it is the major RNA constituent of cells. This process, called RNA enrichment, is done primarily to reduce cost: without rRNA removal, deeper sequencing must be performed to compensate for the sequencing reads wasted on rRNA. The ideal RNA enrichment method removes all rRNA without affecting other RNA in the sample. We tested the performance of three RNA enrichment methods on RNA isolated from Cryptococcus neoformans, a fungal pathogen of humans. We find that the RNase H depletion method is more efficient in depleting rRNA and more specific in recapitulating non-rRNA levels present in unenriched controls than the commonly-used Poly(A) isolation method. The RNase H depletion method is also more effective than the Ribo-Zero depletion method as measured by rRNA depletion efficiency and recapitulation of protein-coding RNA levels present in unenriched controls, while the Ribo-Zero depletion method more closely recapitulates annotated non-coding RNA (ncRNA) levels. Finally, we leverage these data to accurately map the C. neoformans mitochondrial rRNA genes, and also demonstrate that RNA-Seq data generated with the RNase H and Ribo-Zero depletion methods can be used to explore novel C. neoformans long non-coding RNA genes.

scholarly journals Comparative analysis of RNA enrichment methods for preparation of Cryptococcus neoformans RNA sequencing libraries

2021 ◽  
Author(s):  
Calla L. Telzrow ◽  
Paul J. Zwack ◽  
Shannon Esher Righi ◽  
Fred S. Dietrich ◽  
Cliburn Chan ◽  
...  
Keyword(s):  
Cryptococcus Neoformans ◽  
Rna Sequencing ◽  
Rnase H ◽  
Rrna Genes ◽  
Rna Seq ◽  
Protein Coding ◽  
Expression Levels ◽  
Non Coding Rna ◽  
Long Non Coding Rna ◽  
Enrichment Methods

ABSTRACTRibosomal RNA (rRNA) is the major RNA constituent of cells, therefore most RNA sequencing (RNA-Seq) experiments involve removal of rRNA. This process, called RNA enrichment, is done primarily to reduce cost: without rRNA removal, deeper sequencing would need to be performed to balance the sequencing reads wasted on rRNA. The ideal RNA enrichment method would remove all rRNA without affecting other RNA in the sample. We have tested the performance of three RNA enrichment methods on RNA isolated from Cryptococcus neoformans, a fungal pathogen of humans. We show that the RNase H depletion method unambiguously outperforms the commonly used Poly(A) isolation method: the RNase H method more efficiently depletes rRNA while more accurately recapitulating the expression levels of other RNA observed in an unenriched “gold standard”. The RNase H depletion method is also superior to the Ribo-Zero depletion method as measured by rRNA depletion efficiency and recapitulation of protein-coding gene expression levels, while the Ribo-Zero depletion method performs moderately better in preserving non-coding RNA (ncRNA). Finally, we have leveraged this dataset to identify novel long non-coding RNA (lncRNA) genes and to accurately map the C. neoformans mitochondrial rRNA genes.ARTICLE SUMMARYWe compare the efficacy of three different RNA enrichment methods for RNA-Seq in Cryptococcus neoformans: RNase H depletion, Ribo-Zero depletion, and Poly(A) isolation. We show that the RNase H depletion method, which is evaluated in C. neoformans samples for the first time here, is highly efficient and specific in removing rRNA. Additionally, using data generated through these analyses, we identify novel long non-coding RNA genes in C. neoformans. We conclude that RNase H depletion is an effective and reliable method for preparation of C. neoformans RNA-Seq libraries.

scholarly journals Emerging Roles of Estrogen-Regulated Enhancer and Long Non-Coding RNAs

2020 ◽  
Vol 21 (10) ◽  
pp. 3711
Author(s):  
Melina J. Sedano ◽  
Alana L. Harrison ◽  
Mina Zilaie ◽  
Chandrima Das ◽  
Ramesh Choudhari ◽  
...  
Keyword(s):  
Rna Sequencing ◽  
Expression Patterns ◽  
Biological Significance ◽  
Rna Seq ◽  
Biological Functions ◽  
Protein Coding ◽  
Rna Molecules ◽  
Non Coding Rna ◽  
Genome Wide ◽  
Non Coding Rnas

Genome-wide RNA sequencing has shown that only a small fraction of the human genome is transcribed into protein-coding mRNAs. While once thought to be “junk” DNA, recent findings indicate that the rest of the genome encodes many types of non-coding RNA molecules with a myriad of functions still being determined. Among the non-coding RNAs, long non-coding RNAs (lncRNA) and enhancer RNAs (eRNA) are found to be most copious. While their exact biological functions and mechanisms of action are currently unknown, technologies such as next-generation RNA sequencing (RNA-seq) and global nuclear run-on sequencing (GRO-seq) have begun deciphering their expression patterns and biological significance. In addition to their identification, it has been shown that the expression of long non-coding RNAs and enhancer RNAs can vary due to spatial, temporal, developmental, or hormonal variations. In this review, we explore newly reported information on estrogen-regulated eRNAs and lncRNAs and their associated biological functions to help outline their markedly prominent roles in estrogen-dependent signaling.

scholarly journals A workflow for simultaneous detection of coding and non-coding transcripts by ribosomal RNA-depleted RNA-Seq.

2021 ◽  
Author(s):  
Joanna M. Williams ◽  
Nikita Potemkin ◽  
Sophie Cawood ◽  
Jackson Treece ◽  
Diane Guévremont ◽  
...  
Keyword(s):  
Simultaneous Detection ◽  
Rna Seq ◽  
Protein Coding ◽  
Biologically Relevant ◽  
High Loss ◽  
Highly Efficient ◽  
Non Coding Rna ◽  
Tissue Removal ◽  
Rrna Depletion ◽  
Unique Species

RNA sequencing offers unprecedented access to the transcriptome. Key to this is the identification and quantification of many different species of RNA from the same sample at the same time. In this study we describe a novel protocol for simultaneous detection of coding and non-coding transcripts using modifications to the Ion Total RNA-Seq kit v2 protocol, with integration of QIASeq FastSelect rRNA removal kit. We report highly consistent sequencing libraries can be produced from both frozen high integrity mouse hippocampal tissue and the more challenging post-mortem human tissue. Removal of rRNA using FastSelect was highly efficient, resulting in less than 1.5% rRNA content in the final library, significantly better than other reported rRNA removal techniques. We identified >30,000 unique transcripts from all samples, including protein-coding genes and many unique species of non-coding RNA, in biologically-relevant proportions. Furthermore, normalized sequencing read count for select genes significantly negatively correlated with Ct values from RT-qPCR analysis from the same samples. These results indicate that this protocol accurately and consistently identifies and quantifies a wide variety of transcripts simultaneously. The highly efficient rRNA depletion, coupled with minimized sample handling and without complicated and high-loss size selection protocols, makes this protocol useful to researchers wishing to investigate whole transcriptomes.

scholarly journals A method for simultaneous detection of small and long RNA biotypes by ribodepleted RNA-Seq

2022 ◽  
Vol 12 (1) ◽  
Author(s):  
Nikita Potemkin ◽  
Sophie M. F. Cawood ◽  
Jackson Treece ◽  
Diane Guévremont ◽  
Christy J. Rand ◽  
...  
Keyword(s):  
Simultaneous Detection ◽  
Pcr Analysis ◽  
Rna Seq ◽  
Protein Coding ◽  
Biologically Relevant ◽  
High Loss ◽  
Non Coding Rna ◽  
Tissue Removal ◽  
Rrna Depletion ◽  
High Integrity

AbstractRNA sequencing offers unprecedented access to the transcriptome. Key to this is the identification and quantification of many different species of RNA from the same sample at the same time. In this study we describe a novel protocol for simultaneous detection of coding and non-coding transcripts using modifications to the Ion Total RNA-Seq kit v2 protocol, with integration of QIASeq FastSelect rRNA removal kit. We report highly consistent sequencing libraries can be produced from both frozen high integrity mouse hippocampal tissue and the more challenging post-mortem human tissue. Removal of rRNA using FastSelect was extremely efficient, resulting in less than 1.5% rRNA content in the final library. We identified > 30,000 unique transcripts from all samples, including protein-coding genes and many species of non-coding RNA, in biologically-relevant proportions. Furthermore, the normalized sequencing read count for select genes significantly negatively correlated with Ct values from qRT-PCR analysis from the same samples. These results indicate that this protocol accurately and consistently identifies and quantifies a wide variety of transcripts simultaneously. The highly efficient rRNA depletion, coupled with minimized sample handling and without complicated and high-loss size selection protocols, makes this protocol useful to researchers wishing to investigate whole transcriptomes.

scholarly journals Non-Coding RNA Signatures of B-Cell Acute Lymphoblastic Leukemia

2021 ◽  
Vol 22 (5) ◽  
pp. 2683
Author(s):  
Princess D. Rodriguez ◽  
Hana Paculova ◽  
Sophie Kogut ◽  
Jessica Heath ◽  
Hilde Schjerven ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
B Cell ◽  
Molecular Mechanisms ◽  
Lymphoblastic Leukemia ◽  
Transcriptome Profiling ◽  
Rna Seq ◽  
Protein Coding ◽  
Non Coding Rna ◽  
Technological Developments ◽  
Cell Acute Lymphoblastic Leukemia

Non-coding RNAs (ncRNAs) comprise a diverse class of non-protein coding transcripts that regulate critical cellular processes associated with cancer. Advances in RNA-sequencing (RNA-Seq) have led to the characterization of non-coding RNA expression across different types of human cancers. Through comprehensive RNA-Seq profiling, a growing number of studies demonstrate that ncRNAs, including long non-coding RNA (lncRNAs) and microRNAs (miRNA), play central roles in progenitor B-cell acute lymphoblastic leukemia (B-ALL) pathogenesis. Furthermore, due to their central roles in cellular homeostasis and their potential as biomarkers, the study of ncRNAs continues to provide new insight into the molecular mechanisms of B-ALL. This article reviews the ncRNA signatures reported for all B-ALL subtypes, focusing on technological developments in transcriptome profiling and recently discovered examples of ncRNAs with biologic and therapeutic relevance in B-ALL.

Abstract 5686: Long RNA sequencing of human plasma exosomes reveals full coverage of diverse protein coding and long non coding RNA

2017 ◽  
Author(s):  
Sudipto K. Chakrabortty ◽  
Lisa Bedford ◽  
Hidefumi Uchiyama ◽  
Vasisht Tadigotla ◽  
Michael D. Valentino ◽  
...  
Keyword(s):  
Human Plasma ◽  
Rna Sequencing ◽  
Protein Coding ◽  
Full Coverage ◽  
Non Coding Rna ◽  
Long Non Coding Rna

scholarly journals Functional Conservation of Mitochondrial RNA Levels Despite Divergent mtDNA Organization

2020 ◽  
Author(s):  
James P Held ◽  
Maulik R Patel
Keyword(s):  
Mitochondrial Genome ◽  
Digital Pcr ◽  
Rrna Genes ◽  
Mitochondrial Rna ◽  
Protein Coding ◽  
Promoter Sequences ◽  
C Elegans ◽  
Functional Conservation ◽  
Mitochondrial Rrna ◽  
Mrna Ratio

Abstract Objective: Mitochondria-encoded ribosomal RNA (rRNA) genes in humans are expressed at a higher rate than protein coding genes of the mitochondria. The organization of the human mitochondrial genome (mtDNA) is amenable to differential expression of rRNAs as it contains a promoter specific to the transcription of the two rRNAs. However, mtDNA is not organized in the same way as humans in all metazoans. In the nematode, Caenorhabditis elegans, the rRNA genes are on opposite sides of the mtDNA molecule and there are no obvious promoter sequences specific to the rRNA genes. Thus, we asked whether rRNA levels are higher relative to mRNAs in mitochondria of C. elegans as they are in humans.Results: Using droplet digital PCR, we discovered that steady-state mitochondrial rRNA transcript levels are approximately 120 times higher than the levels of mitochondrial mRNAs. These data demonstrate that despite the lack of conservation in mitochondrial genome organization, a high mitochondrial rRNA-to-mRNA ratio is a conserved feature of metazoans.

scholarly journals Differential Expression of Long Non-Coding RNA (lncRNA) in Mediterranean Mussel (Mytilus galloprovincialis) Hemocytes under Immune Stimuli

Genes ◽  
2021 ◽  
Vol 12 (9) ◽  
pp. 1393
Author(s):  
Patricia Pereiro ◽  
Rebeca Moreira ◽  
Beatriz Novoa ◽  
Antonio Figueras
Keyword(s):  
Regulation Of Gene Expression ◽  
Poly I:C ◽  
Rna Seq ◽  
Protein Coding ◽  
Non Coding Rna ◽  
Discrete Response ◽  
Mediterranean Mussel ◽  
Long Non Coding Rna ◽  
Mollusk Species

The Mediterranean mussel is one of the most economically relevant bivalve mollusk species in Europe and China. The absence of massive mortalities and their resistance to pathogens affecting other cultured bivalves has been under study in recent years. The transcriptome response of this species to different immune stimuli has been extensively studied, and even the complexity of its genome, which has recently been sequenced, has been suggested as one of the factors contributing to this resistance. However, studies concerning the non-coding RNA profiles remain practically unexplored—especially those corresponding to the lncRNAs. To the best of our knowledge, this is the second characterization and study of lncRNAs in this bivalve species. In this work, we identified the potential repertoire of lncRNAs expressed in mussel hemocytes, and using RNA-Seq we analyzed the lncRNA profile of mussel hemocytes stimulated in vitro with three different immune stimuli: LPS, poly I:C, and β-glucans. Compared to unstimulated hemocytes, LPS induced the highest modulation of lncRNAs, whereas poly I:C and β-glucans induced a similar discrete response. Based on the potential cis-regulatory activity of the lncRNAs, we identified the neighboring protein-coding genes of the regulated lncRNAs to estimate—at least partially—the processes in which they are implicated. After applying correlation analyses, it seems that—especially for LPS—the lncRNAs could participate in the regulation of gene expression, and substantially contribute to the immune response.

scholarly journals Evaluation of two main RNA-seq approaches for gene quantification in clinical RNA sequencing: polyA+ selection versus rRNA depletion

2018 ◽  
Vol 8 (1) ◽  
Author(s):  
Shanrong Zhao ◽  
Ying Zhang ◽  
Ramya Gamini ◽  
Baohong Zhang ◽  
David von Schack
Keyword(s):  
Rna Sequencing ◽  
Rna Seq ◽  
Gene Quantification ◽  
Rrna Depletion

scholarly journals Combining DGE and RNA-sequencing data to identify new polyA+ non-coding transcripts in the human genome

2013 ◽  
Vol 42 (5) ◽  
pp. 2820-2832 ◽  
Author(s):  
Nicolas Philippe ◽  
Elias Bou Samra ◽  
Anthony Boureux ◽  
Alban Mancheron ◽  
Florence Rufflé ◽  
...  
Keyword(s):  
Human Genome ◽  
Rna Sequencing ◽  
Dynamic Range ◽  
Tiling Array ◽  
Expression Data ◽  
Rna Seq ◽  
Sequencing Data ◽  
Data Set ◽  
Protein Coding ◽  
Protein Coding Genes

Abstract Recent sequencing technologies that allow massive parallel production of short reads are the method of choice for transcriptome analysis. Particularly, digital gene expression (DGE) technologies produce a large dynamic range of expression data by generating short tag signatures for each cell transcript. These tags can be mapped back to a reference genome to identify new transcribed regions that can be further covered by RNA-sequencing (RNA-Seq) reads. Here, we applied an integrated bioinformatics approach that combines DGE tags, RNA-Seq, tiling array expression data and species-comparison to explore new transcriptional regions and their specific biological features, particularly tissue expression or conservation. We analysed tags from a large DGE data set (designated as ‘TranscriRef’). We then annotated 750 000 tags that were uniquely mapped to the human genome according to Ensembl. We retained transcripts originating from both DNA strands and categorized tags corresponding to protein-coding genes, antisense, intronic- or intergenic-transcribed regions and computed their overlap with annotated non-coding transcripts. Using this bioinformatics approach, we identified ∼34 000 novel transcribed regions located outside the boundaries of known protein-coding genes. As demonstrated using sequencing data from human pluripotent stem cells for biological validation, the method could be easily applied for the selection of tissue-specific candidate transcripts. DigitagCT is available at http://cractools.gforge.inria.fr/softwares/digitagct.

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