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OnePot-seq and OPT-seq, highly multiplexed and targeted RNA-seq protocols for rapid, low-cost library generation

ABSTRACT: This SuperSeries is composed of the SubSeries listed below.

ORGANISM(S): Escherichia coli Bacillus subtilis

PROVIDER: GSE206425 | GEO | 2023/02/01

REPOSITORIES: GEO

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BaM-seq and TBaM-seq, highly multiplexed and targeted RNA-seq protocols for rapid, low-cost library generation from bacterial samples.

Project description:The ability to profile transcriptomes and characterize global gene expression changes has been greatly enabled by the development of RNA sequencing technologies (RNA-seq). However, the process of generating sequencing-compatible cDNA libraries from RNA samples can be time-consuming and expensive, especially for bacterial mRNAs which lack poly(A)-tails that are often used to streamline this process for eukaryotic samples. Compared to the increasing throughput and decreasing cost of sequencing, library preparation has had limited advances. Here, we describe bacterial-multiplexed-seq (BaM-seq), an approach that enables simple barcoding of many bacterial RNA samples that decreases the time and cost of library preparation. We also present targeted-bacterial-multiplexed-seq (TBaM-seq) that allows for differential expression analysis of specific gene panels with over 100-fold enrichment in read coverage. In addition, we introduce the concept of transcriptome redistribution based on TBaM-seq that dramatically reduces the required sequencing depth while still allowing for quantification of both highly and lowly abundant transcripts. These methods accurately measure gene expression changes with high technical reproducibility and agreement with gold standard, lower throughput approaches. Together, use of these library preparation protocols allows for fast, affordable generation of sequencing libraries.

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BaM-seq and TBaM-seq, highly multiplexed and targeted RNA-seq protocols for rapid, low-cost library generation from bacterial samples [BaM-seq]

Project description:Here, we report the development of two RNA-seq library preparation protocols that increase the throughput and decrease the cost of converting RNA to cDNA libraries compatible for sequencing on high-throughput platforms. BaM-seq allows for early barcoding of samples such that many biological samples can be processed simultaneously. TBaM-seq allows for enrichment of target RNAs to decrease the required sequencing depth. Both methods are able to accurately measure gene expression changes with high technical reproducibility and agreement with gold standard, lower throughput approaches.

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BaM-seq and TBaM-seq, highly multiplexed and targeted RNA-seq protocols for rapid, low-cost library generation from bacterial samples [TBaM-seq]

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OnePot-seq and OPT-seq, highly multiplexed and targeted RNA-seq protocols for rapid, low-cost library generation

Project description:OnePot-seq and OPT-seq, highly multiplexed and targeted RNA-seq protocols for rapid, low-cost library generation

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BaM-seq and TBaM-seq, highly multiplexed and targeted RNA-seq protocols for rapid, low-cost library generation from bacterial samples [TBaM-seq]

Project description:BaM-seq and TBaM-seq, highly multiplexed and targeted RNA-seq protocols for rapid, low-cost library generation from bacterial samples [TBaM-seq]

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BaM-seq and TBaM-seq, highly multiplexed and targeted RNA-seq protocols for rapid, low-cost library generation from bacterial samples [BaM-seq]

Project description:BaM-seq and TBaM-seq, highly multiplexed and targeted RNA-seq protocols for rapid, low-cost library generation from bacterial samples [BaM-seq]

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Low-cost, low-input RNA-seq protocols perform nearly as well as high-input protocols.

Project description:Recently, a number of protocols extending RNA-sequencing to the single-cell regime have been published. However, we were concerned that the additional steps to deal with such minute quantities of input sample would introduce serious biases that would make analysis of the data using existing approaches invalid. In this study, we performed a critical evaluation of several of these low-volume RNA-seq protocols, and found that they performed slightly less well in per-gene linearity of response, but with at least two orders of magnitude less sample required. We also explored a simple modification to one of these protocols that, for many samples, reduced the cost of library preparation to approximately $20/sample.

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Low-cost, low-input RNA-seq protocols perform nearly as well as high-input protocols

Project description:We sequenced mRNA according to several library prep protocols with known mixtures of two species of Drosophila in order to establish linear response in each protocol.

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Low-cost and High-throughput RNA-seq Library Preparation for Illumina Sequencing from Plant Tissue.

Project description:Transcriptome analysis can provide clues to biological processes affected in different genetic backgrounds or/and under various conditions. The price of RNA sequencing (RNA-seq) has decreased enough so that medium- to large-scale transcriptome analyses in a range of conditions are feasible. However, the price and variety of options for library preparation of RNA-seq can still be daunting to those who would like to use RNA-seq for their first time or for a single experiment. Among the criteria for selecting a library preparation protocol are the method of RNA isolation, nucleotide fragmentation to obtain desired size range, and library indexing to pool sequencing samples for multiplexing. Here, we present a high-quality and a high-throughput option for preparing libraries from polyadenylated mRNA for transcriptome analysis. Both high-quality and high-throughput protocol options include steps of mRNA enrichment through magnetic bead-enabled precipitation of the poly-A tail, cDNA synthesis, and then fragmentation and adapter addition simultaneously through Tn5-mediated 'tagmentation'. All steps of the protocols have been validated with Arabidopsis thaliana leaf and seedling tissues and streamlined to work together, with minimal cost in money and time, thus intended to provide a beginner-friendly start-to-finish RNA-seq library preparation for transcriptome analysis.

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