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

Area of Science:

• Molecular Biology
• Genomics
• Bioinformatics

Background:

• RNA sequencing (RNA-seq) enables transcriptome profiling and gene expression analysis.
• Bacterial mRNA library preparation for RNA-seq is challenging due to the lack of poly(A)-tails, making it time-consuming and expensive.
• Advances in library preparation have lagged behind sequencing technology improvements.

Purpose of the Study:

• To develop novel, efficient, and cost-effective methods for bacterial RNA sequencing library preparation.
• To introduce multiplexing and targeted approaches for enhanced bacterial transcriptomics.
• To reduce sequencing depth requirements while maintaining accurate gene expression quantification.

Main Methods:

• Bacterial-multiplexed-seq (BaM-seq): A method for simple barcoding of multiple bacterial RNA samples.
• Targeted-bacterial-multiplexed-seq (TBaM-seq): Enables differential expression analysis of specific gene panels with high read coverage enrichment.
• Transcriptome redistribution: A concept based on TBaM-seq to reduce sequencing depth for quantifying diverse transcript abundances.

Main Results:

• BaM-seq and TBaM-seq significantly decrease the time and cost of bacterial RNA library preparation.
• TBaM-seq achieves over 100-fold enrichment in read coverage for targeted gene panels.
• The developed methods demonstrate high technical reproducibility and agreement with gold standard approaches.
• Transcriptome redistribution effectively reduces sequencing depth requirements.

Conclusions:

• BaM-seq and TBaM-seq provide fast, affordable, and accurate solutions for bacterial RNA sequencing library preparation.
• These methods overcome limitations in current bacterial transcriptomic workflows.
• The developed protocols facilitate broader application of RNA-seq in bacterial research.