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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]

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

PROVIDER: PRJNA850640 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR19744900.fastq.gz	Fastqsanger.gz
	SRR19744901.fastq.gz	Fastqsanger.gz
	SRR19744902.fastq.gz	Fastqsanger.gz
	SRR19744882.fastq.gz	Fastqsanger.gz
	SRR19744883.fastq.gz	Fastqsanger.gz

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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.

2023-02-01 | GSE206423 | GEO

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

2023-02-01 | GSE206424 | GEO

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.

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

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

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Project description:Bulk and single-cell RNA-seq are powerful tools for transcriptomic analysis, providing insights into many aspects of molecular and cellular phenotypes. Costs constrain the amount of biological insight obtainable within a given budget, and as sequencing prices decline, efficient library protocols have become a decisive factor. In this study, we introduce an approach to systematically optimize the number of usable reads that RNA-seq protocols generate. We applied this ”funnel strategy” to prime-seq, an early-barcoding bulk RNA-seq protocol, by systematically testing critical protocol steps totaling 1080 samples in 49 libraries. This resulted in the optimized prime-seq2 protocol that increases the number of usable reads by 60 % and improves one of the most cost-efficient bulk RNA-seq protocols available. Our study also suggests that monitoring the filtering of usable reads can serve as a valuable quality control for many RNA-seq protocols and sheds light on the complexity of the conditions and interactions that shape RNA-seq library composition and their interpretation.

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Sequencing coliphage Bam

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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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