Now several scRNA-seq platforms are used in Bacteria. I can not get the point.
Firstly, Bacteria have Horizontal inheritance, meaning they can exchange the genome. How to get the ref genomes in the mapping process? Is the heterogeneity due to horizontal inheritance or regulation or genome mutation？
Secondly, most scRNA-seq platforms analyze the Bacteria in the culture but not the original. What's the obstacle here？ With culture, why not use bulk seq?
Thirdly, the RNA in Bacteria may be polycistronic mRNA, how to deal with them?
Why Not Single-Bacteria genome sequence?
Single cell sequencing does have advantages when processing environmental samples where not all species are able to be cultured.
The reference genomes are often constructed using other species in the same genus to help align the contigs.
Dera Hyde,
Single cell sequencing – of both DNA and RNA – address the question of whether bacterial populations are isogenic and are all responding to environmental conditions the same way. Long-term studies of chronic infections (e.g., of CF lungs with Pseudomonas aeruginosa) have shown conclusively that these 'single' species infections are actually comprised of different genetic lineages, often with small sequence differences, which nonetheless impact on host interaction and fitness. Similarly, bacterial populations are known to show heterogeneity in gene expression, even in homogenous liquid cultures, and it is highly likely that in a heterogeneous environment, such as the soil pore network, some cells will be responding in a different way to neighbours to environmental factors.
Andrew
1. This reference genome can either be from the same strain of bacteria or from a closely related strain. In terms of the source of heterogeneity, it can be due to various factors, including genetic regulation, genomic mutations, and horizontal gene transfer. Horizontal gene transfer is not the only source of genomic diversity in bacteria. Regulation and genome mutations can also contribute to heterogeneity within a bacterial population. Single-cell sequencing of bacteria can provide insights into the heterogeneity at the level of individual cells and can help distinguish between these factors.
2. Culture vs. original sample: Many scRNA-seq platforms require the bacteria to be cultured in the laboratory before sequencing. This can introduce biases and may not reflect the natural state of the bacteria in their original environment. In some cases, it may be possible to perform single-cell sequencing directly on environmental samples, but this can be technically challenging and may require specialized methods. Bulk sequencing can also be used to study bacterial communities without the need for single-cell isolation.
3. Polycistronic mRNA is a type of mRNA molecule that contains multiple coding regions, each encoding a different protein. These regions are separated by non-coding regions called intergenic regions. There are methods that can help to address this issue of polycistronic mRNA, such as using barcoding or unique molecular identifiers (UMIs) to label individual transcripts.
4. Single-bacteria genome sequencing can provide valuable information about the genetic makeup of individual bacteria, but it may not be suitable for all research questions.
When we describe “heterogeneity of cells”, do we mean the cells have the same genetic background? Both bacteria and cancer (they all have various genomes and high heterogeneity) studies need high-throughput platforms of scRNA-seq. However, the high-throughput platforms analyze sparse genes (1K-2K gene). Are the 1K-2K genes enough to resolve the sources of heterogeneity, and rule out the effect of the genome or horizontal gene transfer?
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