Amplicon metagenomic sequencing (16S, ITS)

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Targeted sequencing of a phylogenetic marker gene allows us to profile the taxonomic composition of a specific microbial kingdom. Sequencing of the 16S rRNA gene allow us to profile the community of bacteria, while sequencing of the internal transcribed spacer 2 (ITS2) sequence allow us to profile the community of fungi (the mycobiome).

The 16S rRNA gene encodes is a ribosomal subunit that comprises a number of conserved and variable regions. By sequencing a selected subset of variable regions, we can identify which bacteria are present in a sample and their relative abundance*, down to the species level. We use the software DADA2 to identify bacterial representative sequences and then use a reference database to annotate the detected bacteria.

The ITS2 sequence is situated between two ribosomal RNA genes (5.8S and 28S) and like the variable regions in the 16S rRNA gene, has highly conserved flanking sequences that allow the binding of primers for targeted amplicon sequencing. Due to its high degree of variation between even closely related species it is recommended as the universal barcode for fungi phylogenetic profiling. By sequencing the ITS2 sequence we can identify fungi-representative sequences in a sample and their relative abundance, and by mapping to reference databases we can annotate the identified organisms.

By applying our data processing and biostatistical platform, we can evaluate if a certain test condition, such as a new food ingredient, affects the overall composition or diversity of the microbiome, and identify the indicator microorganism or microorganisms.

Why chose amplicon sequencing?

  • Allow specific profiling of the bacterial or fungal microbiome
  • It is notably at a lower cost than shotgun sequencing

* Please note the option to use spike-in solutions for the calculation of absolute abundance. By default, sequencing methods provide information about the relative abundance of microorganisms in a sample. By using spike-in of known concentrations of microorganisms, we can calculate the actual number of organisms in a sample.

 

What we do

  • DNA extraction
  • PCR, library preparation and amplicon sequencing on a MiSeq Illumina platform
  • Data quality evaluation and quality filtering to prepare the data for microbiome profiling
  • Microbiome profiling – calculation of the relative abundance of specific organisms (and absolute bacterial abundances if spike-in solutions are used)
  • Statistical analysis

What you get

For each project, a private project folder is made available on the BIOMCARE server, where all resulting data, statistical results, illustrations, reports etc. are available for download.

Sample collection barcodes, tubes or kits

  • Depending on the type of sample you collect, we will ship barcode stickers or barcode-labeled tubes. For larger clinical projects, we can provide collection kits if requested.

Data

  • Raw sequencing data in fastq format
  • Quality filtered data in fastq format

Results reports

Three reports are generated with all necessary information on sequencing, data processing and statistical analysis, and all results, illustrations and tables.

  1. A report describing the performed sequencing, data evaluation and processing
  2. A report describing the generated microbiome profiles, and first microbiome evaluations
  3. A report describing the statistical analysis performed and the results, including illustrations and tables.

Personal guidance and communication

A key factor for successful outcome of every project is communication. Every project is different, and good personal communication allow us to understand exactly what our customers wish to achieve. This is especially essential when we design the statistical analysis and incorporate customer provided information (referred to as meta-data). We typically have the most active communication at the beginning where samples are being collected and shipped, and then again when statistical analysis are initiated and results starts to emerge. At the end of the project, we present the project results in an online or face-to-face meeting, depending on geographical circumstances and project size and complexity.

We used Biomcare to analyse paired human faecal samples from a clinical trial. The communication was swift and the samples rapidly analysed. ... All in all, making the data easily accessible to also less experienced in the field of 16S sequencing.

I can strongly recommend the company.

Sidsel Støy

M.D., Hepatology and Gastroenterology, Aarhus University Hospital