DNA vs RNA seq for microbiome

Question

[MaxF]

Hey everyone,

I usually study eukaryotes but I've started working on a microbiome project. I am wondering about the utility of DNA sequencing vs RNA sequencing.

I know that RNA sequencing tells me about expression levels of specific genes while DNA sequencing simply reveals the presence of those genes in the microbiome. I am familiar with analysis of eukaryotic cells, where the presence of a specific gene is basically a given and, ultimately, expression has to be investigated by RNAseq. Expression levels between any two genes can vary nearly infinitely, so DNAseq tells you almost nothing about expression.

So, I am wondering if it is the same situation for the microbiome. If I do DNA sequencing and find one million copies of gene A and only 20 copies of gene B. that means there are more species/organisms that contain gene A. Can I assume that gene A is more highly expressed in my sample? Are there some genes that are "silenced" in the bacterial genome?

Any advice or papers that deal with this would be greatly appreciated!

[Zhilong Jia]

DNA-Seq of microbes: metagenomes

RNA-Seq of microbes: metatranscriptomes.

While metagenomics focuses on studying the genomic content and on identifying which microbes are present within a community, metatranscriptomics can be used to study the diversity of the active genes within such community, to quantify their expression levels and to monitor how these levels change in different conditions (e.g., physiological vs. pathological conditions in an organism). from Wiki

The gene expression of a gene in a species is different from the abundance of species.

Papers:

Abu-Ali GS, Mehta RS, Lloyd-Price J, Mallick H, Branck T, Ivey KL, Drew DA, DuLong C, Rimm E, Izard J, Chan AT, Huttenhower C. Metatranscriptome of human faecal microbial communities in a cohort of adult men. Nat Microbiol. 2018 Mar;3(3):356-366. doi: 10.1038/s41564-017-0084-4. Epub 2018 Jan 15. PMID: 29335555; PMCID: PMC6557121.

Answer 1

[Mensur Dlakic]

I think by number of copies you actually mean the depth of sequencing. It tells you about the relative abundance of the organism to which that gene is assigned. That means you are not simply assigning reads to features as you would with RNAseq. First, you have to bin the contigs into groups, usually by using tetra-nucleotide frequencies.

If I do DNA sequencing and find one million copies of gene A and only 20 copies of gene B. that means there are more species/organisms that contain gene A.

It means that the group to which gene A is assigned is more abundant in population.

Can I assume that gene A is more highly expressed in my sample?

No. In fact, you will have zero information about gene expression from metagenomic DNA sequencing. To get that information, you will need RNAseq just like in your previous research work.

Are there some genes that are "silenced" in the bacterial genome?

Many genes are silenced in bacterial genomes just like they are in other organisms. If you mean "silenced" in terms of not being amenable to DNA sequencing, the answer is no. The caveat is that repetitive DNA sequences will cause assembly problems, as will near-repetitive sequences such as 16S rRNA from various species. They are similar enough even between unrelated bacterial species to make it difficult to assemble 16S rRNAs from metagenomes. It is possible, though.