Pool Whole Genome Sequencing

Question

[jaafari.omid]

Greeting and Salutation to everybody, I am involved in a project on fish and we are going to do pool whole genome sequencing but I don't know how many samples we can pool together. That should be 10, 12 or for example we can pool 30 samples for each group that we have? Actually what is the relationship between number of samples on each pool and the coverage yield? I am looking forward to hear your suggestions. Sincerely,

Answer 1

[Kevin]

Can I double check what do you mean by pool? There's making individual libraries for every sample but I am wondering if you mean to mix the DNA of the individual fishes in a pool for WGS to survey the genome. Fishes can have a rather large genome size. The nextseq can generate 120 gb of data Ion torrent can generate 15 30 50 gb of data (see https://www.thermofisher.com/sg/en/home/life-science/sequencing/next-generation-sequencing/ion-torrent-next-generation-sequencing-workflow/ion-torrent-next-generation-sequencing-run-sequence/ion-s5-ngs-targeted-sequencing.html )

Danio rerio has 1,674,207,132 bp genome size

so you can put only 2 Danio WGS if aiming for ~30x (assume 2 GB genome size) 1 Danio if you go for 550 chip on Ion Torrent.

If you are going for WGS for a de novo fish it's a different ball game already.

Cheers

[jaafari.omid]

For example each of my experimental groups totally have included by 30 fishes, and we are not going to do WGS individually, first we want to prepare 3 pool samples (each pool includes 10 fish in it with the same amount of input DNA from each fish) and then go for digestion and library preparation. Actually I want to know how we can understand how many samples we can consider in each pool? are there any rule? for example if first I want to mix all 30 samples (One pool sample) what will happen? Are there some problems with depth of coverage? The sequencing will be done on Illumina-Next Seq.

Cheers

[GenoMax]

(each pool includes 10 fish in it with the same amount of input DNA from each fish) and then go for digestion and library preparation

and

for example if first I want to mix all 30 samples (One pool sample) what will happen? Are there some problems with depth of coverage?

Are these fish of the same species? You would not be able to distinguish between those 30 fish since they would all get a single Illumina index. So you can't say anything about coverage among individuals. Just coverage as a pool (if they are all the same species) against a reference.

If you need to distinguish each fish the DNA comes from then either you will need to use UMI's or make each fish library independently before you pool the finished libraries for run.

[jaafari.omid]

Yes, all of them are one species

Answer 2

[ATpoint]

The relationship between number of samples and coverage is pretty simple. Given that you pool 12 on a flowcell, you'll get (very roughly) half the coverage than pooling 6. Have a look at the Illumina Coverage Calculator. You can enter the genome size of your organism, and the sequencing platform you plan to use, and get an idea on how many samples you can pool per flow cell, to get the desired coverage. Technically, you can pool as many samples as you want, as long as you have enough unique index combinations.

[jaafari.omid]

Thank you very much, that is practically I wanted to know.

Answer 3

[GenoMax]

Actually what is the relationship between number of samples on each pool and the coverage yield?

This depends on the amount of individual libraries you put in the pool. You will be making individual libraries for every sample. Quantitating them individually. Mixing them to make a pool (with variable amount of samples, so as to balance them, in theory) and then quantitate the pool again before loading it on the sequencer. While it is possible to get reasonably balanced number of reads (with practice), there is no guarantee that read numbers for each sample will be even (they never are).

We have sequenced thousands of samples in a pool so it is feasible to do so as long as you have enough unique index combinations available to label your samples.