Impact of wet-lab protocols on quality of whole-genome short-read sequences from foodborne microbial pathogens
For successful elucidation of a food-borne infection chain, the availability of high-quality sequencing data from suspected microbial contaminants is a prerequisite. Commonly, those investigations are a joint effort undertaken by different laboratories and institutes. To analyze the extent of variab...
| Main Authors: | , , , , , , , , , , , , , , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Frontiers Media S.A.
2023-11-01
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| Series: | Frontiers in Microbiology |
| Subjects: | |
| Online Access: | https://www.frontiersin.org/articles/10.3389/fmicb.2023.1253362/full |
| _version_ | 1797448682396712960 |
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| author | Leonie F. Forth Erik Brinks Grégoire Denay Ahmad Fawzy Ahmad Fawzy Stefan Fiedler Jannika Fuchs Anne-Catrin Geuthner Thomas Hankeln Thomas Hankeln Ekkehard Hiller Larissa Murr Henning Petersen Ralf Reiting Christian Schäfers Claudia Schwab Kathrin Szabo Andrea Thürmer Anne Wöhlke Jennie Fischer Stefanie Lüth Michaela Projahn Kerstin Stingl Maria Borowiak Carlus Deneke Burkhard Malorny Laura Uelze |
| author_facet | Leonie F. Forth Erik Brinks Grégoire Denay Ahmad Fawzy Ahmad Fawzy Stefan Fiedler Jannika Fuchs Anne-Catrin Geuthner Thomas Hankeln Thomas Hankeln Ekkehard Hiller Larissa Murr Henning Petersen Ralf Reiting Christian Schäfers Claudia Schwab Kathrin Szabo Andrea Thürmer Anne Wöhlke Jennie Fischer Stefanie Lüth Michaela Projahn Kerstin Stingl Maria Borowiak Carlus Deneke Burkhard Malorny Laura Uelze |
| author_sort | Leonie F. Forth |
| collection | DOAJ |
| description | For successful elucidation of a food-borne infection chain, the availability of high-quality sequencing data from suspected microbial contaminants is a prerequisite. Commonly, those investigations are a joint effort undertaken by different laboratories and institutes. To analyze the extent of variability introduced by differing wet-lab procedures on the quality of the sequence data we conducted an interlaboratory study, involving four bacterial pathogens, which account for the majority of food-related bacterial infections: Campylobacter spp., Shiga toxin-producing Escherichia coli, Listeria monocytogenes, and Salmonella enterica. The participants, ranging from German federal research institutes, federal state laboratories to universities and companies, were asked to follow their routine in-house protocols for short-read sequencing of 10 cultures and one isolated bacterial DNA per species. Sequence and assembly quality were then analyzed centrally. Variations within isolate samples were detected with SNP and cgMLST calling. Overall, we found that the quality of Illumina raw sequence data was high with little overall variability, with one exception, attributed to a specific library preparation kit. The variability of Ion Torrent data was higher, independent of the investigated species. For cgMLST and SNP analysis results, we found that technological sequencing artefacts could be reduced by the use of filters, and that SNP analysis was more suited than cgMLST to compare data of different contributors. Regarding the four species, a minority of Campylobacter isolate data showed the in comparison highest divergence with regard to sequence type and cgMLST analysis. We additionally compared the assembler SPAdes and SKESA for their performance on the Illumina data sets of the different species and library preparation methods and found overall similar assembly quality metrics and cgMLST statistics. |
| first_indexed | 2024-03-09T14:13:52Z |
| format | Article |
| id | doaj.art-8f05a0d90403429f99d69bf819d24560 |
| institution | Directory Open Access Journal |
| issn | 1664-302X |
| language | English |
| last_indexed | 2024-03-09T14:13:52Z |
| publishDate | 2023-11-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Microbiology |
| spelling | doaj.art-8f05a0d90403429f99d69bf819d245602023-11-29T05:30:23ZengFrontiers Media S.A.Frontiers in Microbiology1664-302X2023-11-011410.3389/fmicb.2023.12533621253362Impact of wet-lab protocols on quality of whole-genome short-read sequences from foodborne microbial pathogensLeonie F. Forth0Erik Brinks1Grégoire Denay2Ahmad Fawzy3Ahmad Fawzy4Stefan Fiedler5Jannika Fuchs6Anne-Catrin Geuthner7Thomas Hankeln8Thomas Hankeln9Ekkehard Hiller10Larissa Murr11Henning Petersen12Ralf Reiting13Christian Schäfers14Claudia Schwab15Kathrin Szabo16Andrea Thürmer17Anne Wöhlke18Jennie Fischer19Stefanie Lüth20Michaela Projahn21Kerstin Stingl22Maria Borowiak23Carlus Deneke24Burkhard Malorny25Laura Uelze26Department of Biological Safety, German Federal Institute for Risk Assessment, Berlin, GermanyInstitute of Microbiology and Biotechnology, Max Rubner-Institut, Kiel, GermanyChemical and Veterinary Analytical Institute Rhein-Ruhr-Wupper (CVUA-RRW), Krefeld, GermanyDepartment of Veterinary Medicine, Hessian State Laboratory, Giessen, GermanyDepartment of Medicine and Infectious Diseases, Faculty of Veterinary Medicine, Cairo University, Giza, EgyptMethod Standardisation, Reference Laboratories, Resistance to Antibiotics, Federal Office of Consumer Protection and Food Safety, Berlin, GermanyChemical and Veterinary Analysis Agency Karlsruhe, Karlsruhe, GermanyDepartment of Food Safety, State Office for Consumer Protection Saxony-Anhalt, Halle, GermanyInstitute of Organismic and Molecular Evolution, Johannes Gutenberg University Mainz, Mainz, Germany0StarSEQ GmbH, Mainz, Germany1Chemical and Veterinary Analysis Agency Stuttgart, Fellbach, Germany2Bavarian Health and Food Safety Authority, Oberschleißheim, Germany3Chemical and Veterinary Analytical Institute Ostwestfalen-Lippe, Detmold, Germany4Hessian State Laboratory, Kassel, Germany5Institute for Hygiene and Environment, Hamburg, Germany6Labor Kneißler GmbH & Co. KG, Burglengenfeld, GermanyMethod Standardisation, Reference Laboratories, Resistance to Antibiotics, Federal Office of Consumer Protection and Food Safety, Berlin, Germany7Methods Development and Research Infrastructure, Robert Koch-Institut, Berlin, Germany8Food and Veterinary Institute Braunschweig/Hannover, Lower Saxony State Office for Consumer Protection and Food Safety, Braunschweig, GermanyDepartment of Biological Safety, German Federal Institute for Risk Assessment, Berlin, GermanyDepartment of Biological Safety, German Federal Institute for Risk Assessment, Berlin, GermanyDepartment of Biological Safety, German Federal Institute for Risk Assessment, Berlin, GermanyDepartment of Biological Safety, German Federal Institute for Risk Assessment, Berlin, GermanyDepartment of Biological Safety, German Federal Institute for Risk Assessment, Berlin, GermanyDepartment of Biological Safety, German Federal Institute for Risk Assessment, Berlin, GermanyDepartment of Biological Safety, German Federal Institute for Risk Assessment, Berlin, GermanyDepartment of Biological Safety, German Federal Institute for Risk Assessment, Berlin, GermanyFor successful elucidation of a food-borne infection chain, the availability of high-quality sequencing data from suspected microbial contaminants is a prerequisite. Commonly, those investigations are a joint effort undertaken by different laboratories and institutes. To analyze the extent of variability introduced by differing wet-lab procedures on the quality of the sequence data we conducted an interlaboratory study, involving four bacterial pathogens, which account for the majority of food-related bacterial infections: Campylobacter spp., Shiga toxin-producing Escherichia coli, Listeria monocytogenes, and Salmonella enterica. The participants, ranging from German federal research institutes, federal state laboratories to universities and companies, were asked to follow their routine in-house protocols for short-read sequencing of 10 cultures and one isolated bacterial DNA per species. Sequence and assembly quality were then analyzed centrally. Variations within isolate samples were detected with SNP and cgMLST calling. Overall, we found that the quality of Illumina raw sequence data was high with little overall variability, with one exception, attributed to a specific library preparation kit. The variability of Ion Torrent data was higher, independent of the investigated species. For cgMLST and SNP analysis results, we found that technological sequencing artefacts could be reduced by the use of filters, and that SNP analysis was more suited than cgMLST to compare data of different contributors. Regarding the four species, a minority of Campylobacter isolate data showed the in comparison highest divergence with regard to sequence type and cgMLST analysis. We additionally compared the assembler SPAdes and SKESA for their performance on the Illumina data sets of the different species and library preparation methods and found overall similar assembly quality metrics and cgMLST statistics.https://www.frontiersin.org/articles/10.3389/fmicb.2023.1253362/fullinterlaboratory studywhole genome sequencingprotocolqualityCampylobacterEscherichia |
| spellingShingle | Leonie F. Forth Erik Brinks Grégoire Denay Ahmad Fawzy Ahmad Fawzy Stefan Fiedler Jannika Fuchs Anne-Catrin Geuthner Thomas Hankeln Thomas Hankeln Ekkehard Hiller Larissa Murr Henning Petersen Ralf Reiting Christian Schäfers Claudia Schwab Kathrin Szabo Andrea Thürmer Anne Wöhlke Jennie Fischer Stefanie Lüth Michaela Projahn Kerstin Stingl Maria Borowiak Carlus Deneke Burkhard Malorny Laura Uelze Impact of wet-lab protocols on quality of whole-genome short-read sequences from foodborne microbial pathogens Frontiers in Microbiology interlaboratory study whole genome sequencing protocol quality Campylobacter Escherichia |
| title | Impact of wet-lab protocols on quality of whole-genome short-read sequences from foodborne microbial pathogens |
| title_full | Impact of wet-lab protocols on quality of whole-genome short-read sequences from foodborne microbial pathogens |
| title_fullStr | Impact of wet-lab protocols on quality of whole-genome short-read sequences from foodborne microbial pathogens |
| title_full_unstemmed | Impact of wet-lab protocols on quality of whole-genome short-read sequences from foodborne microbial pathogens |
| title_short | Impact of wet-lab protocols on quality of whole-genome short-read sequences from foodborne microbial pathogens |
| title_sort | impact of wet lab protocols on quality of whole genome short read sequences from foodborne microbial pathogens |
| topic | interlaboratory study whole genome sequencing protocol quality Campylobacter Escherichia |
| url | https://www.frontiersin.org/articles/10.3389/fmicb.2023.1253362/full |
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