Sequencing bacterial 16S amplicons and Fungal ITS amplicons from same soil samples using same indices

General Discussion
1

I am planning to do a soil microbiome study (for both bacteria and fungi). I have 96 soil samples. The ideal approach is that I prepare 96 bacterial 16S amplicon libraries using 96 combinations of indexes, and 96 fungal ITS amplicon libraries using 96 other combinations of indexes (total192 libraries). It would require me to use 2 flowcells (v2, 2 X 250).

However, to save the cost of using 2 flowcells, I want to use same combination of indices for the 16S and ITS libraries of the same soil sample, and then based on the sequence similarity, separate the bacterial and fungal reads having same indexes.

Is it a good approach? Is some tool available for this purpose? Or will the 16S and ITS reads be automatically filtered out while using UNITE and SILVA databases, respectively?

The second concern is of the coverage. Illumina recommends having 100,000 reads per sample. In this, I should ideally be getting around 156,250 reads per sample i.e. technically 78,125 reads for the 16S amplicons and 78,125 reads for the ITS amplicons per sample.

I am looking forward to getting expert advice, and I am open to any suggestions.

Thanks!

2

Hi @ahsankaleem,

The bioinformatics on this is relatively easy; I will leave off target to people who do a little more combined sequencing that I do.

My recomendation for demultiplexing here would be

  1. Demultiplex by sample (same participant index) using whatever method is used by your sequencing center. Do not remove the primer pair.
  2. Use cutadapt to with primer trimming to retrieve data with your 16S primers or ITS primers, discarding untrimmed sequences.

Best,
Justine

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3

Thank you very much. It will be of much help.

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