We collected faecal samples from various species that we want to analyse their microbial communities' DNA via Next-Generation Sequencing. The problem is that due to budgetary constraints, we will have to pool the isolated DNA from each sample to be able to analyse them all. We will of course keep each species separated from the other ones, and we are planning to pool the samples from individuals which share enclosure only. Nevertheless, we do not know if there is a maximum number of samples that can be pooled together for NGS.
The number depends on the sample type, sequencing platforms and quantity of sequencing data per sample. If you want to study microbial communities samples, you can do 16s/18s/ITS amplicon sequencing, which is cheap and fast. Yun can also choose metagenome sequencing or meta-transcriptome sequencing, which need more data and the bioinformatics work are more complex. For amplion squencing, you can choose NGS or TGS. We now can pool more than 1500 samples per run at Novoseq 6000. For TGS, it's a little bit expensive, but you can get the full-length sequence of the amplificated area, which will gives you a resolution of species-level at diversity analysis.
There are kits for example with 384 different indexing primers. This means you could theoretically pool 384 samples. Depending on the read depth you need this would likely be unadvisable to pool this much. The largest constraint is the number of reads you need which will very greatly depending on the type of sequencing you are doing. Are you performing RNA-seq? What kind of sequencing is it?
@Joshua Beytebiere is right. We have worked with up to 96 samples pooled in one sequencing run at Illumina MiSeq. Again, it depends what analysis you require. I presume that you are performing metagenomics analysis. For 16S sequence based metagenomics, you can pool up to 96 samples in one sequencing run at MiSeq (V3 kit 300x2 cycles) to obtain good number of reads for each sample. If you are performing whole genome sequence based metagenomics analysis, you may pool up to 12 samples (~2Gb data/sample) for the same V3 kit at MiSeq. For pooling more number of samples, you may need to shift to other sequencing equipment with higher throughput.
The number depends on the sample type, sequencing platforms and quantity of sequencing data per sample. If you want to study microbial communities samples, you can do 16s/18s/ITS amplicon sequencing, which is cheap and fast. Yun can also choose metagenome sequencing or meta-transcriptome sequencing, which need more data and the bioinformatics work are more complex. For amplion squencing, you can choose NGS or TGS. We now can pool more than 1500 samples per run at Novoseq 6000. For TGS, it's a little bit expensive, but you can get the full-length sequence of the amplificated area, which will gives you a resolution of species-level at diversity analysis.
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