Efficient and cost-effective bacterial mRNA sequencing from low input samples through ribosomal RNA depletion

Abstract

Background RNA sequencing is a powerful approach to quantify the genome-wide distribution of mRNA molecules in a population to gain deeper understanding of cellular functions and phenotypes. However, unlike eukaryotic cells, mRNA sequencing of bacterial samples is more challenging due to the absence of a poly-A tail that typically enables efficient capture and enrichment of mRNA from the abundant rRNA molecules in a cell. Moreover, bacterial cells frequently contain 100-fold lower quantities of RNA compared to mammalian cells, which further complicates mRNA sequencing from non-cultivable and non-model bacterial species. To overcome these limitations, we report EMBR-seq (Enrichment of mRNA by Blocked rRNA), a method that efficiently depletes 5S, 16S and 23S rRNA using blocking primers to prevent their amplification. Results EMBR-seq results in 90% of the sequenced RNA molecules from an E. coli culture deriving from mRNA. We demonstrate that this increased efficiency provides a deeper view of the transcriptome without introducing technical amplification-induced biases. Moreover, compared to recent methods that employ a large array of oligonucleotides to deplete rRNA, EMBR-seq uses a single or a few oligonucleotides per rRNA, thereby making this new technology significantly more cost-effective, especially when applied to varied bacterial species. Finally, compared to existing commercial kits for bacterial rRNA depletion, we show that EMBR-seq can be used to successfully quantify the transcriptome from more than 500-fold lower starting total RNA. Conclusions EMBR-seq provides an efficient and cost-effective approach to quantify global gene expression profiles from low input bacterial samples.

Integrating Systems and Synthetic Biology to Understand and Engineer Microbiomes

“…Methods for prokaryotic rRNA depletion vary in efficacy on the basis of microbiome type (biofilm versus planktonic) and composition, and tool development is an active area of research (73). Methods for single-cell prokaryotic rRNA depletion are only beginning to show some success (74,75) and will be useful for interrogating the unique activities of low-abundance organisms as well as the cell-to-cell heterogeneity of gene expression within a given species that is not observable with bulk methods. The identification of optimal spatial locations and time points to discover ecological driver organisms or novel biochemical pathways mediating microbiome functions remains unresolved.…”

Single-Cell Technologies to Study Phenotypic Heterogeneity and Bacterial Persisters

“…Eukaryotic scRNA-seq methods leverage the poly(A) tail of mRNA transcripts for amplification; however, bacteria lack this RNA processing and require alternative enrichment strategies before sequencing. Without enrichment, the signals from abundant rRNAs and tRNAs will obscure the detection of rare transcripts; therefore, the utilization of exonucleases or Cas9 machinery has been implemented to degrade rRNA or tRNA [126,127]. Blocking primers that recognize and bind canonical rRNA sequences can also be used to prevent further reverse transcription [126][127][128].…”

“…Without enrichment, the signals from abundant rRNAs and tRNAs will obscure the detection of rare transcripts; therefore, the utilization of exonucleases or Cas9 machinery has been implemented to degrade rRNA or tRNA [126,127]. Blocking primers that recognize and bind canonical rRNA sequences can also be used to prevent further reverse transcription [126][127][128]. These depletion strategies can be complemented by exogenous E. coli poly(A) polymerase I that artificially adds poly(A) tails to facilitate subsequent amplification [127,129].…”

“…Blocking primers that recognize and bind canonical rRNA sequences can also be used to prevent further reverse transcription [126][127][128]. These depletion strategies can be complemented by exogenous E. coli poly(A) polymerase I that artificially adds poly(A) tails to facilitate subsequent amplification [127,129]. These steps enable relevant messenger transcript enrichment and amplification while decreasing the computational burden of sequence analysis in the end.…”

RiboRid: A low cost, advanced, and ultra-efficient method to remove ribosomal RNA for bacterial transcriptomics

“…However, this method has a relatively low efficiency and is limited by the fact that primary transcripts protected by triphosphate from 5 0phosphate-dependent terminator exonuclease (TEX) may not be a precise representation of mRNA levels in a cell, because a considerable amount of mRNA exists as a processed form. Several methods have been proposed for rRNA removal based on duplex-specific nucleasebased digestion, electrophoretic size selection, and sequence-specific blockage of reverse transcription, although they are not as efficient as commercial systems [9][10][11][12].…”