scholarly journals A reverse transcriptase-mediated ribosomal RNA depletion (RTR2D) strategy for the cost-effective construction of RNA sequencing libraries

2020 ◽  
Vol 24 ◽  
pp. 239-250 ◽  
Author(s):  
Zongyue Zeng ◽  
Bo Huang ◽  
Xi Wang ◽  
Jiaming Fan ◽  
Bo Zhang ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Rna Sequencing ◽  
Ribosomal Rna ◽  
Cost Effective ◽  
The Cost ◽  
Rna Depletion

scholarly journals Ribo-Pop: simple, cost-effective, and widely applicable ribosomal RNA depletion

RNA ◽  
2020 ◽  
Vol 26 (11) ◽  
pp. 1731-1742 ◽  
Author(s):  
Mary Kay Thompson ◽  
Maria Kiourlappou ◽  
Ilan Davis
Keyword(s):  
Ribosomal Rna ◽  
Cost Effective ◽  
Rna Depletion

scholarly journals Ribo-Pop: Simple, cost-effective, and widely applicable ribosomal RNA depletion

2020 ◽  
Author(s):  
Mary Kay Thompson ◽  
Maria Kiourlappou ◽  
Ilan Davis
Keyword(s):  
Ribosomal Rna ◽  
Massively Parallel Sequencing ◽  
Cost Effective ◽  
Design Strategy ◽  
Probe Selection ◽  
Sequencing Library ◽  
Input Range ◽  
Specific Species ◽  
Rna Depletion ◽  
Oligo Design

ABSTRACTThe measurement of RNA abundance derived from massively parallel sequencing experiments is an essential technique. Methods that reduce ribosomal RNA levels are usually required prior to sequencing library construction because ribosomal RNA typically comprises the vast majority of a total RNA sample. For some experiments, ribosomal RNA depletion is favored over poly(A) selection because it offers a more inclusive representation of the transcriptome. However, methods to deplete ribosomal RNA are generally proprietary, complex, inefficient, applicable to only specific species, or compatible with only a narrow range of RNA input levels. Here, we describe Ribo-Pop (ribosomal RNA depletion for popular use), a simple workflow and antisense oligo design strategy that we demonstrate works over a wide input range and can be easily adapted to any organism with a sequenced genome. We provide a computational pipeline for probe selection, a streamlined 20-minute protocol, and ready-to-use oligo sequences for several organisms. We anticipate that our simple and generalizable “open source” design strategy would enable virtually any lab to pursue full transcriptome sequencing in their organism of interest with minimal time and resource investment.

scholarly journals A simple, cost-effective, and robust method for rRNA depletion in RNA-sequencing studies

2020 ◽  
Author(s):  
Peter H. Culviner ◽  
Chantal K. Guegler ◽  
Michael T. Laub
Keyword(s):  
Gene Expression ◽  
Rna Sequencing ◽  
Ribosomal Rna ◽  
Bacterial Species ◽  
Cost Effective ◽  
Rna Seq ◽  
Robust Method ◽  
Total Rna ◽  
Metagenomic Sample ◽  
Rrna Depletion

AbstractThe profiling of gene expression by RNA-sequencing (RNA-seq) has enabled powerful studies of global transcriptional patterns in all organisms, including bacteria. Because the vast majority of RNA in bacteria is ribosomal RNA (rRNA), it is standard practice to deplete the rRNA from a total RNA sample such that the reads in an RNA-seq experiment derive predominantly from mRNA. One of the most commonly used commercial kits for rRNA depletion, the Ribo-Zero kit from Illumina, was recently discontinued. Here, we report the development a simple, cost-effective, and robust method for depleting rRNA that can be easily implemented by any lab or facility. We first developed an algorithm for designing biotinylated oligonucleotides that will hybridize tightly and specifically to the 23S, 16S, and 5S rRNAs from any species of interest. Precipitation of these oligonucleotides bound to rRNA by magnetic streptavidin beads then depletes rRNA from a complex, total RNA sample such that ~75-80% of reads in a typical RNA-seq experiment derive from mRNA. Importantly, we demonstrate a high correlation of RNA abundance or fold-change measurements in RNA-seq experiments between our method and the previously available Ribo-Zero kit. Complete details on the methodology are provided, including open-source software for designing oligonucleotides optimized for any bacterial species or metagenomic sample of interest.ImportanceThe ability to examine global patterns of gene expression in microbes through RNA-sequencing has fundamentally transformed microbiology. However, RNA-seq depends critically on the removal of ribosomal RNA from total RNA samples. Otherwise, rRNA would comprise upwards of 90% of the reads in a typical RNA-seq experiment, limiting the reads coming from messenger RNA or requiring high total read depth. A commonly used, kit for rRNA subtraction from Illumina was recently discontinued. Here, we report the development of a ‘do-it-yourself’ kit for rapid, cost-effective, and robust depletion of rRNA from total RNA. We present an algorithm for designing biotinylated oligonucleotides that will hybridize to the rRNAs from a target set of species. We then demonstrate that the designed oligos enable sufficient rRNA depletion to produce RNA-seq data with 75-80% of reads comming from mRNA. The methodology presented should enable RNA-seq studies on any species or metagenomic sample of interest.

scholarly journals A simple, cost-effective and automation-friendly direct PCR approach for bacterial community analysis

2021 ◽  
Author(s):  
Fangchao Song ◽  
Jennifer V. Kuehl ◽  
Arjun Chandran ◽  
Adam P. Arkin
Keyword(s):  
Microbial Communities ◽  
Rna Sequencing ◽  
Ribosomal Rna ◽  
Cost Effective ◽  
Community Analysis ◽  
Extraction Methods ◽  
16S Ribosomal Rna ◽  
Direct Pcr ◽  
Microbial Composition ◽  
Pcr Method

ABSTRACTBacterial communities in water, soil, and humans play an essential role in environmental ecology and human health. PCR-based amplicon analysis, such as 16s ribosomal RNA sequencing, is a fundamental tool for quantifying and studying microbial composition, dynamics, and interactions. However, given the complexity of microbial communities, a substantial amount of samples becomes necessary to analyses that parse the factors that determine microbial composition. A common bottleneck in performing these kinds of experiments is genomic DNA (gDNA) extraction, which can be biased on the types of species, time-consuming and expensive. Direct PCR methods are a potentially simpler and more accurate alternative to gDNA extraction methods that do not require the intervening purification step. In this study, we evaluated three variations of direct PCR methods using diverse heterogeneous bacterial cultures, ZymoBIOMICS Microbial Community Standards, and groundwater. By comparing direct PCR methods with DNeasy blood and tissue kits and DNeasy Powersoil kits, we found a specific variant of the direct PCR method exhibits a comparable overall accuracy to the conventional DNeasy Powersoil protocol. We also found the method showed higher efficiency for extracting gDNA from the gram negative strains compared to DNeasy blood and tissue protocol. This direct PCR method is 1600 times cheaper ($0.34 for 96 samples), 10 times simpler (15 min hands-on time for 96 samples) than DNeasy Powersoil protocol. The direct PCR method can also be fully automated, and is compatible with small volume samples, thereby permitting scaling of samples and replicates needed to support high-throughput large-scale bacterial community analysis.IMPORTANCEUnderstanding bacterial interaction and assembling in complex microbial communities using 16s ribosomal RNA sequencing normally requires a large experimental load. However, the current DNA extraction methods including cell disruption and genome DNA purification are normally biased, costly, time and labor consuming, and not amenable to miniaturization by droplets or 1536 well plates due to the significant DNA loss during purification step for tiny volume and low cell density samples. Direct PCR method could potentially solve these problems. In this study, we demonstrate a direct PCR method which exhibits similar accuracy as the widely used method – DNeasy Powersoil protocol, while 1600 times cheaper and 10 times faster to execute. This simple, cost-effective, and automation friendly direct PCR based 16s ribosomal RNA sequencing method allows us to study the dynamics, microbial interaction and assembly of varying microbial communities in a high throughput fashion.

scholarly journals Effective ribosomal RNA depletion for single-cell total RNA-seq by scDASH

PeerJ ◽  
2021 ◽  
Vol 9 ◽  
pp. e10717
Author(s):  
Danson S.C. Loi ◽  
Lei Yu ◽  
Angela R. Wu
Keyword(s):  
Single Cell ◽  
Ribosomal Rna ◽  
Single Cell Analysis ◽  
Cell Analysis ◽  
Rna Seq ◽  
Total Rna ◽  
The Cost ◽  
Rna Depletion ◽  
Polyadenylated Mrna ◽  
Rrna Sequences

A decade since its invention, single-cell RNA sequencing (scRNA-seq) has become a mainstay technology for profiling transcriptional heterogeneity in individual cells. Yet, most existing scRNA-seq methods capture only polyadenylated mRNA to avoid the cost of sequencing non-messenger transcripts, such as ribosomal RNA (rRNA), that are usually not of-interest. Hence, there are not very many protocols that enable single-cell analysis of total RNA. We adapted a method called DASH (Depletion of Abundant Sequences by Hybridisation) to make it suitable for depleting rRNA sequences from single-cell total RNA-seq libraries. Our analyses show that our single-cell DASH (scDASH) method can effectively deplete rRNAs from sequencing libraries with minimal off-target non-specificity. Importantly, as a result of depleting the rRNA, the rest of the transcriptome is significantly enriched for detection.

scholarly journals Paired rRNA-depleted and polyA-selected RNA sequencing data and supporting multi-omics data from human T cells

2020 ◽  
Vol 7 (1) ◽  
Author(s):  
Li Chen ◽  
Ruirui Yang ◽  
Tony Kwan ◽  
Chao Tang ◽  
Stephen Watt ◽  
...  
Keyword(s):  
Rna Sequencing ◽  
Ribosomal Rna ◽  
Rna Seq ◽  
Healthy Individuals ◽  
Sequencing Data ◽  
Advantages And Disadvantages ◽  
Human Primary Cells ◽  
Human T Cells ◽  
Differential Gene ◽  
Rna Depletion

Abstract Both poly(A) enrichment and ribosomal RNA depletion are commonly used for RNA sequencing. Either has its advantages and disadvantages that may lead to biases in the downstream analyses. To better access these effects, we carried out both ribosomal RNA-depleted and poly(A)-selected RNA-seq for CD4+ T naive cells isolated from 40 healthy individuals from the Blueprint Project. For these 40 individuals, the genomic and epigenetic data were also available. This dataset offers a unique opportunity to understand how library construction influences differential gene expression, alternative splicing and molecular QTL (quantitative loci) analyses for human primary cells.

The IBM-PC in electron microscopy

1996 ◽  
Vol 54 ◽  
pp. 612-613
Author(s):  
James F. Mancuso
Keyword(s):  
Head Start ◽  
Image Acquisition ◽  
Cost Effective ◽  
Financial Applications ◽  
Number Of Factors ◽  
Instrument Control ◽  
The Cost ◽  
Training And Support ◽  
Control Image ◽  
Ibm Pc

IBM PC compatible computers are widely used in microscopy for applications ranging from control to image acquisition and analysis. The choice of IBM-PC based systems over competing computer platforms can be based on technical merit alone or on a number of factors relating to economics, availability of peripherals, management dictum, or simple personal preference.IBM-PC got a strong “head start” by first dominating clerical, document processing and financial applications. The use of these computers spilled into the laboratory where the DOS based IBM-PC replaced mini-computers. Compared to minicomputer, the PC provided a more for cost-effective platform for applications in numerical analysis, engineering and design, instrument control, image acquisition and image processing. In addition, the sitewide use of a common PC platform could reduce the cost of training and support services relative to cases where many different computer platforms were used. This could be especially true for the microscopists who must use computers in both the laboratory and the office.

Sonographically guided, transcatheter foam sclerotherapy of the great saphenous vein

Phlebologie ◽  
2007 ◽  
Vol 36 (06) ◽  
pp. 309-312 ◽  
Author(s):  
T. Schulz ◽  
M. Jünger ◽  
M. Hahn
Keyword(s):  
Saphenous Vein ◽  
Great Saphenous Vein ◽  
Clinical Stage ◽  
Clinical Signs ◽  
Cost Effective ◽  
Duplex Sonography ◽  
Foam Sclerotherapy ◽  
Average Loss ◽  
One Year ◽  
The Cost

Summary Objective: The goal of the study was to assess the effectiveness and patient tolerability of single-session, sonographically guided, transcatheter foam sclerotherapy and to evaluate its economic impact. Patients, methods: We treated 20 patients with a total of 22 varicoses of the great saphenous vein (GSV) in Hach stage III-IV, clinical stage C2-C5 and a mean GSV diameter of 9 mm (range: 7 to 13 mm). We used 10 ml 3% Aethoxysklerol®. Additional varicoses of the auxiliary veins of the GSV were sclerosed immediately afterwards. Results: The occlusion rate in the treated GSVs was 100% one week after therapy as demonstrated with duplex sonography. The cost of the procedure was 207.91 E including follow-up visit, with an average loss of working time of 0.6 days. After one year one patient showed clinical signs of recurrent varicosis in the GSV; duplex sonography showed reflux in the region of the saphenofemoral junction in a total of seven patients (32% of the treated GSVs). Conclusion: Transcatheter foam sclerotherapy of the GSV is a cost-effective, safe method of treating varicoses of GSV and broadens the spectrum of therapeutic options. Relapses can be re-treated inexpensively with sclerotherapy.

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