Saptaparna Pal, Amity University Kolkata
Bacterial messenger RNA (mRNA) sequencing gives a snapshot of the genomic wide state of microbial population and hence provides a fundamental understanding of these varied phenotypes and microbial functions. Entire RNA isolated from the bacterial cells contains greater than 95% ribosomal RNA, and therefore it is cost-effective and high coverage sequencing of transcriptome requires the development of efficient strategies to deplete abundant 5s, 16s, 23s rRNA molecules. Several commercial kits had been developed to evolve bacterial RNA from total RNA sample, including the MICROBE express Bacterial mRNA enrichment unit, The Ribo-minus Transcriptome Isolation Unit, and the Ribo-Zero rRNA Depletion Unit.
Further, these commercial kits, are only effective on species targeted in the standard probe set. While the limitations of pre-designed kits had been overcome through the development of workflow to create custom subtractive hybridization probe sets for any species of interest. EMBR-seq is a fruitful and cost-effective Bacterial mRNA sequencing technology. It also allows mRNA sequencing from low input total RNA. EMBR-seq efficiently depletes RNA to sequence bacterial mRNA. EMBR-seq only requires primers at the 3’end of 5s, 16s, 23s rRNA followed by poly-adenylation with E Coli poly-A polymerase. To deplete, rRNA, EMBRseq only requires primers at the 3’end of the rRNA unlike recent methods tile the oligonucleotide along the entire length of rRNA molecules thereby reducing costs and making the approach more easily translated to other bacterial species. This EMBRseq provides a detailed view of the transcriptome without introducing technical issues.
The rRNA depletion efficiency of EMBRseq can be further improved through additional primers. EMBR sequencing effectively depleted rRNA derived reads compared to control samples from the 3’end of rRNA molecules, specific hotspots regions along the entire length of 16s and 23s rRNA were abundant in the rRNA capture profile. EMBRseq also gives a more in-depth view of the transcriptional landscape without initiation of technical artefacts.
Finally, EMBRseq effectively captures the transcriptome from 500-fold lower starting total RNA compared to the commercial kits thereby providing a powerful new approach to investigate gene expression pattern in rare and non-cultivable bacterial species.
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You may also read: Efficient and cost-effective bacterial mRNA sequencing from low input samples through ribosomal RNA depletion
Chatarin Wangsanuwat, Kellie A. Heom, Estella Liu, Michelle A. O’Malley, Siddharth S. DeybioRxiv 2020.06.19.162412; doi: https://doi.org/10.1101/2020.06.19.162412
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