Yes, you can use same index for multiple samples, conditionally if you have a way to separate the OTUs based on the gene type. And it would be useless computation to filter out 1 datasets for gaining three kind of paired/single end reads. Therefore, I would not say its logical. (based on what i understood at first with pooling same indexed but different amplicons)

However, by library preparation using custom primers (barcoded) you can pool several hundred samples together. But this custom primer library is much expensive than kits. If you are sequencing hundreds of samples frequently than using custom library prep kit would be good, for small/few sample sequencing are good with commercial kits.

Furthermore, if you want to pool several different amplicons, they must have approximately same size, or else the sequencing will not be equally efficient for individual amplicons.

• DegePrime, a Program for Degenerate Primer Design for Broad-Taxonomic-Range PCR in Microbial Ecology Studies; Luisa W. Hugerth, Hugo A. Wefer, Sverker Lundin, Hedvig E. Jakobsson, Mathilda Lindberg, SandraRodin, Lars Engstrand, Anders F. Andersson; Applied and Environmental Microbiology Jul 2014, 80 (16) 5116-5123; DOI: 10.1128/AEM.01403-14

• Can DNA-Based Ecosystem Assessments Quantify Species Abundance? Testing Primer Bias and Biomass—Sequence Relationships with an Innovative Metabarcoding Protocol; Elbrecht V, Leese F (2015) Can DNA-Based Ecosystem Assessments Quantify Species Abundance? Testing Primer Bias and Biomass—Sequence Relationships with an Innovative Metabarcoding Protocol. PLOS ONE 10(7): e0130324. https://doi.org/10.1371/journal.pone.0130324

Thanks Abhijeet for your answer. Can you please contribute with a kind of protocol. This will be helpful for many of us. As, I'm still convinced that the cheapest way to sequence is to order your own primers and perform a two-step PCR, even for few samples.

So please, if someone else is interested on the protocol feel free to contribute to the discussion.

Thanks

What do you mean by protocol?

I have already attached the references, please check it.

Thanks for the references, they give a nice overall view of what should be done. However, a Kind of SOP could more easy to fellow!! The design of the primers, their sequences. Index combination. PCR conditions, enzymes. The choice of nextera or Trueseq options. Some feedback or advises, etc. Sequencing experiment can be a bit expensive and more and more people are interested in doing some sequencing, for gene expression or genotyping or ..... So relying on article is not enough for newbies. That's why a nice detailed protocol could be really helpful.