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, such as bacteria or fungi. In our amplicon sequencing services, we profile the bacterial microbiome by sequencing of the 16S rRNA gene, and we profile the fungi microbiome by sequencing of the internal transcribed spacer 2 (ITS2) sequence (the mycobiome).

A cost-effective approach

Amplicon sequencing is a cost-effective approach for profiling a specific part of the microbiome and can be used for most any sample – including soil, water, skin and stool – to detect the different species found in the sample. In situations where there is expected to be relatively few microorganisms in the samples, amplicon sequencing has an advantage over shotgun sequencing as it targets the organism of interest and therefore does not use a lot of reagent to sequence other types of DNA in the sample.

Please see below for some more information about the 16S rRNA gene and the ITS sequence. These are the two marker regions most often purchased via our amplicon sequencing service.

Why chose amplicon sequencing?

  • Allow specific profiling of the bacterial or fungal microbiome
  • It is cheaper than shotgun sequencing although the cost difference continues to reduce, and mostly depends on the desired sequencing depth and the sample type.
  • The data is easier to process and analyse, and data storage is less challenges do to smalle amounts of data.
  • It has some advantages over shotgun sequencing for samples with low microbiome concentration.

What we do

  • Support with study design and guidance on sample collection and shipment.
  • DNA extraction*
  • Library preparation and amplicon sequencing on an Illumina platform
  • If you purchase our biostatistical service, we also perform
    • Data quality evaluation and quality filtering to prepare the data for microbiome profiling
    • Microbiome profiling – calculation of the abundance of specific organisms (including absolute bacterial abundances if spike-in solutions are used*)
    • Statistical analysis

* 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 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.

For projects where you have purchased both sequencing and data analysis, we deliver three reports with descriptions of the data, the processing, analysis and the results of the statistical analysis, together with illustrations and tables.

Sample collection barcodes, tubes or kits

  • Depending on the type of sample you collect, we will send a table for filling in your sample information, or we will ship barcode stickers or barcode-labelled 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.

About the 16S rRNA gene and ITS sequences

Figure: Concept of amplicon sequencing here illustrated for the variable region V1V2 of the 16S rRNA gene for bacterial profiling.

The 16S rRNA gene encodes a ribosomal subunit. The gene sequence comprises a number of conserved and variable regions. The conserved regions are consistent across bacteria and allow us to use a single set of primers to target the bacteria in a sample. We then sequence a subset of variable regions between the targeted conserved regions. By sequencing the variable regions, which are specific for each species of bacteria, we can identify which bacteria are present in a sample and calculate their relative abundance*. This is possible with a resolution 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(s).

* 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.

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