16S data processing and analysis

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16S data processing and analysis

Sequencing of the 16S rRNA gene is commonly used for profiling of the microbiome community in a complex biological sample, such as a fecal sample or an environmental samples (e.g. marine water or soil).

 

Independent of the origin, the generated sequencing data needs extensive processing and analysis before it provides biologically meaningful information. BiomCare offer to perform the necessary quality-control of reads, microbiome profiling and subsequent analyses.

Microbiome profiling (taxonomic analysis)

A package offer that include:

  • Reads quality filtering
  • Read clustering
  • Taxonomic assignment (phylum to genus)
  • Processing report

See details on the software used for microbiome profiling here.

Basic processing and analysis

Microbiome profiling results in abundance tables (rows of samples, columns of taxa). These tables are input for further processing and analysis, such as calculation of alpha diversity, beta diversity, and visualization (e.g. stacked barplots and ordination plots). You may wish to do this yourself. Alternatively we are happy to help. Here are an overview of the analysis that are provided with “Basic processing and analysis”. If you wish other/additional analysis we are happy to discuss.

Analysis & processing:

  • Normalization
  • Filtering of samples (e.g. removal based on low abundance)
  • Filtering of taxa (identifying a core of more abundant taxa for further statistical analyses)
  • Calculation of alpha diversity indices (see details here)
  • Calculation of beta diversity indices (see details here)

Visualization:

  • Stacked barplots of reads per sample (raw and/or normalized)
  • Ordination or tree based visualization of sample relations

Advanced bioinformatic analysis

After the basic processing and analysis is performed, it is time to start asking statistical questions related to the meta data (including phenotypic or environmental information, or experimental conditions). With 16S rRNA gene data, the questions are often:

  • Is there community level differences between groups of samples?
  • Does alpha diversity differ significantly between groups of samples?
  • Are specific taxa showing different abundance between groups of samples?

And when we ask these questions we need to consider: Are there confounders we need to adjust for? How do we handle the multiple-analysis burden? What tools can handle the non-normal distribution of taxa abundance data? And so forth.

 

The analysis at this level is increasingly project specific and require considerations of details related to the project design and samples. It is also here we start to get the interesting results that motivated design and execution of the project.

 

We offer to perform the necessary analyses to answer the questions you wish to ask of the data. However, we cannot do so without you! We need to understand your questions, the details of the project and to access the meta data. Therefore, our Advanced Bioinformatic Service include time for communication scaled to the size of the project.