DNA/RNA extraction and Sequencing solutions

Most of the work Biomcare does with analysis of microorganisms is based on DNA or RNA sequencing. We offer a range of solutions, including short and long-read platforms, and different types of sequencing solutions including amplicon (metabarcoding) and shotgun metagenomic sequencing.

Solutions

• DNA and RNA extraction
• Amplicon sequencing (metabarcoding, targeted analysis)
• Shotgun metagenomic sequencing (untargeted analysis)
• Metatranscriptomics (RNA sequencing)

We have experience with processing many types of samples. Below are some of the sample types we most often process. If your type of samples is not in the list please contact us for a discussion of experience and solutions.

Life science samples

Below are details of the different solutions. Please let us know if you have any further questions.

Targeted sequencing of a phylogenetic marker gene allows us to profile the taxonomic composition of a specific microbial kingdom, such as bacteria or fungi. With our amplicon sequencing services, we profile the bacterial microbiome by sequencing the 16S rRNA gene, and we most often profile the fungi microbiome by sequencing the internal transcribed spacer 2 (ITS2) sequence (the mycobiome). We can target many different marker regions and use both short and long read platforms for amplicon sequencing.

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 are 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 wast a lot of reads on sequencing other types of DNA in the sample.

Why chose amplicon sequencing?

• You are looking for taxonomic profiling and is satisfied with annotations being limited below genus level.
• You are interested in biodiversity (alpha diversity) and want to include unknown organisms in the diversity estimate (e.g. bacteria in soil that are not found in the existing reference databases).
• Your samples have low microbial biomass. In situations where there are 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 reagents to sequence other types of DNA in the sample. Can be a reason for amplicon sequencing of biopsies or ITS sequencing of soil.
• 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.

With shotgun metagenomic sequencing, we sequence the full DNA in the sample, without preselecting a genetic region or marker gene (as we do with amplicon sequencing). The method detects organisms across taxonomic kingdoms and allow for both taxonomic and functional profiling. The data generated can be processed in a number of ways depending on the aim, e.g., by relating to reference databases to measures abundance of known organisms, or building genomes based on the DNA sequences thereby allowing for de-novo detection and whole-genome evaluation of organisms in the samples. The method can be used for both community samples (samples with many organisms) and isolates (samples with one species).

Why chose metagenomic sequencing?

• You are looking for both taxonomic and functional profiling
• You are looking to profile at species or even strain level
• You are looking to detailed whole-genome analysis of a isolate
• You are looking to detect de-novo organisms’ by building their genomes without a reference genome

You get

• Support with study design and guidance on sample collection and shipment.
• DNA (or RNA) extraction*
• Library preparation and sequencing on a short- or long-read platform.
• The raw sequencing data as fastq files.

If you purchase our bioinformatic and biostatistical services, we perform

• Data quality evaluation and quality filtering to prepare the data for microbiome profiling
• Microbiome profiling: quantification and annotation of microbes and functional features (for metagenomes). The specific bioinformatic solutions applied are selected to meet your specific samples and project.
• Custom statistical analysis to answer the questions you like addressed.
• Reports describing the processes performed all customized to your samples and project.

* 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.

For each project, a private project folder is made available on the Biomcares 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 3 reports with descriptions of the data, the processing, analysis, and the results of the statistical analysis, together with illustrations and tables.

Sample collection, storage and shipment

When we start on a new project or collaboration, we will discuss your starting point and adjust our workflow to that. If you include Biomcare from the way beginning, the steps are:

• We guide on sample collection, storage and shipment id requested
• We send you a excel-table for filling out with your sample information (including the IDs on the tubes).
• We send you a guide for sample shipment
• We ask you to keep us updated and informed so we are ready to recive your samples when they arrive

Data

Different services result in different outputs. Some will lead to a single results report while others results in many different outputs. If you buy a sequencing service you can always get the raw data if you wish. If we are providing bioinformatic services you will also get quality filtered data and resulting microbiome profiles, along with reports describing the data and work we have performed. If we also support you with statistical analyses you will get many files with figures and tables and a statistical analyses report.

• Raw sequencing data in fastq format
• Quality filtered data in fastq format
• Microbiome taxonomic profiles (profiles are tables with identified and annotated microbes and their abundance)
• Microbiome functional profiles (profiles are tables with identified and annotated functional features such as genes and pathways, and their abundance)
• Figures
• Tables
• Reports

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 evaluations of the microbiome profiles
3. A report describing the statistical analysis performed and the results, including illustrations and tables.

Personal guidance and communication

A key factor for the successful outcome of every project is communication. Every project is different and good personal communication allows 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 when samples are being collected and shipped, and then again when statistical analysis is initiated and results start 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.

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

A leading solution to address the problem of relative abundance is to add a step in wet lab processing of the sample that provide additoinal information that can then be used later in data analyses to re-scale the data from relative to absolute values. The step added can be qPCR of a marker gene or spike-in of bacteria or bacterial DNA of known concentration to the sample. When the bacteria added with spike-in are rare and not found in the environment being investigated, this allows for statistical re-calculation of the data to obtain both relative and absolute abundance tables (absolute quantification). Both method is still at an early stage and has their limitations. For example, when the sequencing method used is amplicon sequencing (16S or ITS), absolute quantification is only available for bacterial profiling, as the spike-in organisms are bacteria and for qPCR the marker measures is most often the 16S rRNA gene. When using shotgun sequencing, all organisms are profiled in the same sequencing run and we can use the spike-in to quantify all detected organisms.

We have found that including absolute quantification can provide valuable information, especially if a shift in absolute abundance is expected e.g. in evacuations of antibiotic treatment effects, or if the sample size is low as discussed above.