Next Generation Sequencing (NGS) and Bioinformatics

NGS analyses

Several different NGS-based analyses are offered within the Agency's specialised diagnostics.

Metagenomics for detection of an unknown pathogen

Metagenomic sequencing is offered in cases where infection is suspected but other methods have failed to identify a pathogen. All nucleic acid in the clinical sample is sequenced and the resulting data is analysed by experienced bioinformaticians. The result is a comprehensive technical report, including a detailed list of database matches and an interpretation of potential clinical relevance following consultation with a clinical microbiologist. This allows for the detection and characterisation of pathogens with high precision.

Metagenomic sequencing engables a thorough mapping of the genetic material in the clinical sample, providing a broader understanding of the microbiological profile. The result of of metagenomic sequencing is the outcome of a collaborative effort between bioinformaticians and clinical microbiologists, where the clinical interpretation confirms and complements the technical analysis.

16S metagenomic sequencing

16S sequencing is performed to determine which bacterial species are present in a clinical sample or isolate. In contrast to metagenomic detection of unknown pathogens which sequences all available genetic material, 16S sequencing specifically targets the small ribosomal subunit. This allows for the identification and classification of bacteria with high precision. The 16S rRNA gene is highly conserved with certain variable regions. Sequence variations between different bacteria provide a unique identity, and the analysis of these sequence enables taxonomic placement. The laboratory procedure is based on amplifying and sequencing four variable regions of the 16S rRNA gene with two amplicons. These amplicons include V1V2 and V3V4 with the approximate sizes 350 bp and 460 bp, respectively. The procedure's high sequence depth allows for these amplicons to be analysed separetely so different bacteria from the same sample can be identified. The method uses an internal control sequence (spike) which is amplified and sequenced simultaneously with the sample to facilitate the assessment of the bacterial content and to detect amplification-inhibiting factors in the sample that may lead to a negative result. The result is a list of database hits and an estimated quantity compared with an internal standard.

Whole genome sequencing of bacterial isolates

For contact tracing or inquiries related to antibiotic resistance, whole genome sequencing (WGS) of bacterial isolates is offered. By sequencing the entire genome, a comprehensive understanding of the genetic composition is obtained. In epidemiological contact tracing, this technology enables the mapping of infection spread and identification of sources of infection through the comparison of genetic sequences. Whole genome sequencing can also provide insights into the specific genes or mutations that may be linked to antibiotic resistance.

NGS bioinformatics

The bioinformaticians at the Public Health Agency play a central role within the Department of Microbiology by analysing extensive biological data and developing sophisticated software. Their focus is creating pipelines to enhance and automate analyses. Pipelines are series-connected data processes that utilise software tools to structure, process, and interpret genetic information.

GENSAM

The basis of the GENSAM project is the joint national management of clinical microbial sequencing data. It is a platform and database for the automated analysis of whole genome sequencing data. Clinical microbiology laboratories across Sweden can submit data from independent sequencing, which is then centrally analysed and shared with regional infection protection units and other relevant health organisations. Currently, GENSAM manages SARS-CoV-2, but analysis of other pathogens are also under implementation. GENSAM has been funded with the assistance of Vinnova and the European Commission. For inquiries about GENSAM, please send an email to gensam@folkhalsomyndigheten.se.