ena-STUDY-UMCG Groningen-07-12-2017-18:45:03:997-8
Shotgun metagenomics for identification and typing of microorganisms in clinical samples
Faster methodologies leading to reliable and accurate detection of microorganisms are needed to take timely and appropriate measures in patient treatment and infection control. Shotgun metagenomics (SMg) provides not only information on the microorganisms present in patients' samples but can reveal additional information on the presence of virulence factors, antimicrobial resistance genes, as well as about the relatedness of the strains found. Here we applied SMg on different types of patients' samples, including body fluids and tissues. Prior to DNA extraction different methods were used to deplete human DNA. Libraries were prepared and sequenced with Illumina chemistry. Data was analyzed with CLC Genomics Workbench v10.0.1 (Qiagen), Taxonomer (IDbyDNA), CosmosID, BaseSpace and several Unix-based tools and compared to standard culturing and susceptibility testing methods. On average, 75% of the reads were mapped against the human genome. Most of the pathogens identified by culture were also identified through SMg, that also detected additional bacteria, mainly anaerobes. In three samples, we could infer the probable sequence type of the most abundant species. Moreover, several plasmids and antimicrobial resistance genes were found, compatible with the antimicrobial susceptibility profiles of the respective bacteria. Although most bacterial species identified by classical culturing were also identified by the tools used in this study, the high number of human reads resulted, in a few cases, in insufficient sequencing depth of bacterial DNA. However, identification of microorganisms, antimicrobial resistance genes, plasmids and typing data by a single method confirms how powerful SMg can be for clinical microbiology.
Faster methodologies leading to reliable and accurate detection of microorganisms are needed to take timely and appropriate measures in patient treatment and infection control. Shotgun metagenomics (SMg) provides not only information on the microorganisms present in patients' samples but can reveal additional information on the presence of virulence factors, antimicrobial resistance genes, as well as about the relatedness of the strains found. Here we applied SMg on different types of patients' samples, including body fluids and tissues. Prior to DNA extraction different methods were used to deplete human DNA. Libraries were prepared and sequenced with Illumina chemistry. Data was analyzed with CLC Genomics Workbench v10.0.1 (Qiagen), Taxonomer (IDbyDNA), CosmosID, BaseSpace and several Unix-based tools and compared to standard culturing and susceptibility testing methods. On average, 75% of the reads were mapped against the human genome. Most of the pathogens identified by culture were also identified through SMg, that also detected additional bacteria, mainly anaerobes. In three samples, we could infer the probable sequence type of the most abundant species. Moreover, several plasmids and antimicrobial resistance genes were found, compatible with the antimicrobial susceptibility profiles of the respective bacteria. Although most bacterial species identified by classical culturing were also identified by the tools used in this study, the high number of human reads resulted, in a few cases, in insufficient sequencing depth of bacterial DNA. However, identification of microorganisms, antimicrobial resistance genes, plasmids and typing data by a single method confirms how powerful SMg can be for clinical microbiology.