Metagenome sequencing vs Bacterial whole genome sequencing protocols

Question

[anwesh023]

How are the sequencing protocols different for a single bacterial genome and a metagenome, say if we are using same platform? Are there any special steps necessary while preparing the library? Any differences in the sequencing depth? Are these two procedures really different?

Usually, for sequencing the bacterial whole genome, we go for 100x coverage with 250bp PE reads on MiSeq platform. Now we want to sequence metagenome samples, so what precautions are needed to get good quality reads and lose as fewer data as possible?

Please provide the link to the respective topic, if it was already asked...

Thank you,

With regards, Anwesh

Answer 1

[tokeemdtareq]

I am relatively new to the field of library preparation. If you are talking about 16S metagenomics, there is a whole different protocol from Illumina. If you are talking about deep sequencing, I would like the answer from someone else, because I have very limited experience in it. All I can say for now is, The DNA isolation is quite different, as eukaryotic genomes are removed from the sample. It usually varies based on what type of sample are you using (like environmental water, saliva or wound swab etc.).

[anwesh023]

Well, thank you... We have done 16S metagenomics, no problem with that protocol, it is straightforward. But, deep sequencing (say stool sample) is the one I'm getting confused. Whether the library preparation and other downstream steps are really different from those used for sequencing the whole genome of a single bacterium OR pretty much the same?

Just curious, removing the eukaryotic sequences is done during the analysis using in silico tools, isn't it?

Came across this article, I think it may clear some of my doubts...

[tokeemdtareq]

Thanks for sharing the article, I found another one. Hope this helps.

I really have no specific experience in handling metagenomics deep sequencing library preparation. But, we are also planing for such a library preparation and will probably follow the WGS protocol for the trial run.

Yep, eukaryotic genomes will be easily separated in metagenomics analysis pipe. But, in a previous run in nextseq machine, we sequenced just two fungus with some bacteria using nexteraXT kit. The result was devastating like low data amount, low data quality and lots of unassigned fastq reads (~80%) etc. Due to our previous experiences, we are very cautious of sample preparation.

[anwesh023]

A much useful article... Thank you...

Please keep updating the status of your future work on library preparation, if possible...