do we clone sequences while performing shotgun sequencing on microbiome?

Question

[fyfes]

Hi,

I am reading about shotgun sequencing and I see that often it is written that: In shotgun method, the DNA is randomly broken into many small pieces, cloned into a bacterial host, and sequenced using chain termination.

I am wondering - if we are having a gut microbiome sample and we break those bacterial genomes into random pieces do we have to clone in into a bacterial host? I find it difficult to understand. And if we don't clone them what are we doing to amplify sequence fragments (do we need to have a template?) ?

thank you a lot (I will be so grateful for an article/open access book when sequecnign is well described in details)

Answer 1

[antonioggsousa]

Hi,

The need to cloning DNA fragments and sequence them by Sanger chain termination method refers to the beginning of the metagenomics field. The cloning was not only to amplify the DNA copy fragments but also to isolate them, since you can not sequence mixture of different templates by Sanger.

Nowadays, with next-generation sequencing technologies, such as Illumina, you don't need to do this. This is because these technologies can sequence multiple distinct template DNA fragments in one sequencing run (in Illumina, hypothetically each cluster will represent a different template). As far I understand, in a standard way, you don't need to amplify your DNA. Of course this requires great amounts of good-quality, purified DNA. If you amplify, and this can be done using whole genome amplification techniques. Although this can cause bias towards some particular fragments with higher/lower GC content, etc. Therefore, as far I understand, usually it is avoided.

Though, cloning-based metagenomics is still used nowadays, particularly for functional metagenomics. What this means is that, when you're interested in a specific functional trait of the metagenomes, let's say antibiotic resistance, you can extract the environmental DNA from mixed cultures or environmental samples, clone that DNA into vectors/BAC and transfect E. coli or yeast to express them; and then expose these colonies to some antibiotics. The ones that survive are resistant to antibiotics, and thus possess metagenomic DNA that encode antibiotic resistance genes. Then, these colonies can be picked, the DNA extracted from the vector/BAC and the cluster of genes sequenced by Sanger. This approach has been used particularly in the pharma industry to find new bioactive molecules.

Paper describing cloning-based metagenomics: https://mmbr.asm.org/content/mmbr/68/4/669.full.pdf

Paper describing shotgun NGS metagenomics (I did not read this paper yet, but it seems really good describing some topics that you're asking): https://www.nature.com/articles/nbt.3935

I hope this answers your questions,

António

[fyfes]

Hi, thank you so much Antonio. And the article is just yum :)

[antonioggsousa]

Glad that I helped.