Name: Generating Whole Bacterial Genomes from Clinical Samples using a Target Enrichment Workflow (Video) | JoVE Journal
Uploaded: 2025-08-15T13:20:00.000+00:00
Duration: 10 min 44 s
Description: Learn target enrichment sequencing for bacterial STI genomics using clinical samples. This protocol enables complete genome recovery from Chlamydia trachomatis, Neisseria gonorrhoeae, and other pathogens for epidemiological surveillance.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

We sincerely thank all those who contributed to this project. From the University of Z&#252;rich, we acknowledge Dr Fanny Wegner, Srinithi Purushothaman, Dr Frank Imkamp, Dr Tim Roloff, Dr Dominique Braun, and Prof Dr Adrian Egli for their involvement in the development and optimization of the technique. We extend our gratitude to Daniel Gander, Val&#233;ria Pires, Arianita Asani, and Stefan Antener for their outstanding technical assistance with sequencing. From the University of Buenos Aires, we thank Dr Andrea Carolina Entrocassi, Dr Mar&#237;a Luc&#237;a Gallo Vaulet, Dr Deysi L&#243;pez Aquino, Dr Laura Svidler L&#243;pez, and Dr Luciana La Rosa for their valuable contributions. Finally, we are grateful to Dr Ibrahim Kisakesen and Dr Andreas Schreiber from Agilent for their support. This work was supported by the STIDirect Grant from the Gottfried und Julia Bangerter-Rhyner-Stiftung and by the STISeq Grant STWF-24-009 from the Stiftung fu&#776;r wissenschaftliche Forschung an der Universit&#228;t Zu&#776;rich. Additional unrestricted funds from the University of Zurich were provided.

<ol><li>Lefterova, M. I., Suarez, C. J., Banaei, N., Pinsky, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Next-generation+sequencing+for+infectious+disease+diagnosis+and+management%3A+a+report+of+the+Association+for+Molecular+Pathology%5BTitle%5D)+AND+%22J+Mol+Diagn%22%5BJournal%5D)">Next-generation sequencing for infectious disease diagnosis and management: a report of the Association for Molecular Pathology.</a> J Mol Diagn. 17 (6), 623-634 (2015).</li>
<li>Gwinn, M., Maccannell, D., Armstrong, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Next+generation+sequencing+of+infectious+pathogens%5BTitle%5D)+AND+%22JAMA%22%5BJournal%5D)">Next generation sequencing of infectious pathogens.</a> JAMA. 321 (9), 893-894 (2019).</li>
<li>Hasman, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rapid+whole-genome+sequencing+for+detection+and+characterization+of+microorganisms+directly+from+clinical+samples%5BTitle%5D)+AND+%22J+Clin+Microbiol%22%5BJournal%5D)">Rapid whole-genome sequencing for detection and characterization of microorganisms directly from clinical samples.</a> J Clin Microbiol. 52 (1), 139-146 (2014).</li>
<li>Gautam, S., KC, R., Leong, K. W. C., Mac Aogáin, M., O'Toole, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+step-by-step+beginner%27s+protocol+for+whole+genome+sequencing+of+human+bacterial+pathogens%5BTitle%5D)+AND+%22J+Biol+Methods%22%5BJournal%5D)">A step-by-step beginner's protocol for whole genome sequencing of human bacterial pathogens.</a> J Biol Methods. 6 (1), e110(2019).</li>
<li>Baert, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Guidance+document+on+the+use+of+whole+genome+sequencing+%28WGS%29+for+source+tracking+from+a+food+industry+perspective%5BTitle%5D)+AND+%22Food+Control%22%5BJournal%5D)">Guidance document on the use of whole genome sequencing (WGS) for source tracking from a food industry perspective.</a> Food Control. 130, 108148(2021).</li>
<li>Didelot, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transforming+clinical+microbiology+with+bacterial+genome+sequencing%5BTitle%5D)+AND+%22Nat+Rev+Genet%22%5BJournal%5D)">Transforming clinical microbiology with bacterial genome sequencing.</a> Nat Rev Genet. 13 (9), 601-612 (2012).</li>
<li>Scidmore, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cultivation+and+laboratory+maintenance+of+Chlamydia+trachomatis%5BTitle%5D)+AND+%22Curr+Protoc+Microbiol%22%5BJournal%5D)">Cultivation and laboratory maintenance of Chlamydia trachomatis.</a> Curr Protoc Microbiol. 11, (2006).</li>
<li>Edmondson, D. G., Norris, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vitro+cultivation+of+the+syphilis+spirochete+Treponema+pallidum%5BTitle%5D)+AND+%22Curr+Protoc%22%5BJournal%5D)">In vitro cultivation of the syphilis spirochete Treponema pallidum.</a> Curr Protoc. 1 (2), e44(2021).</li>
<li>Pitt, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Challenges+of+in+vitro+propagation+and+antimicrobial+susceptibility+testing+of+Mycoplasma+genitalium%5BTitle%5D)+AND+%22J+Antimicrob+Chemother%22%5BJournal%5D)">Challenges of in vitro propagation and antimicrobial susceptibility testing of Mycoplasma genitalium.</a> J Antimicrob Chemother. 77 (11), 2901-2907 (2022).</li>
<li>Büttner, K. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Evaluating+methods+for+genome+sequencing+of+Chlamydia+trachomatis+and+other+sexually+transmitted+bacteria+directly+from+clinical+swabs%5BTitle%5D)+AND+%22Microb+Genom%22%5BJournal%5D)">Evaluating methods for genome sequencing of Chlamydia trachomatis and other sexually transmitted bacteria directly from clinical swabs.</a> Microb Genom. 11 (2), 001353(2025).</li>
<li>Seth-Smith, H. M. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Whole-genome+sequences+of+Chlamydia+trachomatis+directly+from+clinical+samples+without+culture%5BTitle%5D)+AND+%22Genome+Res%22%5BJournal%5D)">Whole-genome sequences of Chlamydia trachomatis directly from clinical samples without culture.</a> Genome Res. 23 (5), 855-866 (2013).</li>
<li>SureSelect XT HS2 DNA kits: library preparation (+/-MBC) / fast-Hyb target enrichment / post-capture pooling workflow for Illumina platform NGS. , Agilent Technologies. 
 <a target="_blank" href="https://www.agilent.com/cs/library/usermanuals/public/G9983-90000.pdf">https://www.agilent.com/cs/library/usermanuals/public/G9983-90000.pdf</a> (2023).</li>
<li>Büttner, K. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chlamydia+trachomatis+genomes+from+rectal+samples%3A+description+of+a+new+clade+comprising+ompA-genotype+L4+from+Argentina%5BTitle%5D)+AND+%22Microb+Genom%22%5BJournal%5D)">Chlamydia trachomatis genomes from rectal samples: description of a new clade comprising ompA-genotype L4 from Argentina.</a> Microb Genom. 11 (2), 001350(2025).</li>
<li>Brown, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Norovirus+whole-genome+sequencing+by+SureSelect+target+enrichment%3A+a+robust+and+sensitive+method%5BTitle%5D)+AND+%22J+Clin+Microbiol%22%5BJournal%5D)">Norovirus whole-genome sequencing by SureSelect target enrichment: a robust and sensitive method.</a> J Clin Microbiol. 54 (10), 2530-2537 (2016).</li>
<li>Dao, T. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Application+of+the+SureSelect+target+enrichment+system+for+next-generation+sequencing+to+obtain+the+complete+genome+sequence+of+bovine+leukemia+virus%5BTitle%5D)+AND+%22Arch+Virol%22%5BJournal%5D)">Application of the SureSelect target enrichment system for next-generation sequencing to obtain the complete genome sequence of bovine leukemia virus.</a> Arch Virol. 163 (11), 3155-3159 (2018).</li>
<li>Cai, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((SureSelect+targeted+enrichment%2C+a+new+cost+effective+method+for+the+whole+genome+sequencing+of+Candidatus+Liberibacterasiaticus%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">SureSelect targeted enrichment, a new cost effective method for the whole genome sequencing of Candidatus Liberibacterasiaticus.</a> Sci Rep. 9 (1), 1-8 (2019).</li>
<li>Büttner, K. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((ompA+sequencing+and+multilocus+sequence+typing+of+lymphogranuloma+venereum+cases+in+Buenos+Aires+reveal+new+Chlamydia+trachomatis+genotypes%5BTitle%5D)+AND+%22Microorganisms%22%5BJournal%5D)">ompA sequencing and multilocus sequence typing of lymphogranuloma venereum cases in Buenos Aires reveal new Chlamydia trachomatis genotypes.</a> Microorganisms. 12 (3), 587(2024).</li>
<li>La Rosa, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chlamydia+trachomatis+anorectal+infections+by+LGV+%28L1%2C+L2+and+L2b%29+and+non-LGV+serotypes+in+symptomatic+patients+in+Buenos+Aires+Argentina%5BTitle%5D)+AND+%22Int+J+STD+AIDS%22%5BJournal%5D)">Chlamydia trachomatis anorectal infections by LGV (L1, L2 and L2b) and non-LGV serotypes in symptomatic patients in Buenos Aires Argentina.</a> Int J STD AIDS. 32 (14), 1270-1278 (2021).</li>
<li>Rodríguez Fermepin, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((More+than+L2b+in+rectal+lymphogranuloma+venereum%5BTitle%5D)+AND+%22Lancet+Microbe%22%5BJournal%5D)">More than L2b in rectal lymphogranuloma venereum.</a> Lancet Microbe. 2 (2), e55(2021).</li>
</ol>

The authors declare that there are no conflicts of interest.

Generating complete bacterial genome data from clinical samples is particularly challenging. Yet we need this data to gain information on circulating clones and to investigate antimicrobial resistance profiles, particularly in fastidious and difficult-to-culture bacteria. Using alternative methods such as deep sequencing or adaptive sequencing with Oxford Nanopore Technologies, sufficient data for whole-genome sequence analysis cannot be obtained. In cases such as STI samples, target enrichment is the optimum technique to access the full genome, and this method can be used across a panel of bacterial STI pathogens. While it is not a cheap procedure, the results it can offer cannot be obtained in any other way10.
Appropriate samples should be carefully selected, ideally to answer relevant and important questions in diagnostics and research. The medium used for collection does not necessarily play a critical role, with successful sequences obtained from both 2-SP and NAAT media. Sample storage is important, with the aim of maintaining DNA integrity. The most important factor was found to be diagnostic load, aiming for approximate Ct values under Ct30. For samples with Ct values over 30, full genome sequencing success is less likely, but partial data may provide relevant information in some cases.
The procedure is not trivial, involving many steps including PCR cycles, hybridization, and many bead washes. Careful attention to the procedure is required for optimal results. The critical steps of the protocol -- DNA fragmentation, hybridization with capture probes, and magnetic bead-based capture with washing -- require careful execution and proper handling. The protocol can be learned by researchers and technicians after a couple of attempts. In the first rounds, while learning, the 3-day protocol can be an advantage; with more experienced users, the 2-day protocol can speed up the results. As the reagents are expensive, it can be worth running a test run without the expensive components, for example, the probes.&#160;For alternative bait sets, the hybridization temperature can be adjusted, which may improve the sensitivity or specificity of the enrichment. If sufficient data is generated, and a considerable proportion of PCR duplicates are observed, the number of pre-capture and post-capture PCR cycles can also be adjusted.
Data obtained from these experiments can be used for typing, with MLST and other relevant targets extracted. It can further be analyzed in an epidemiological context, globally or locally, compared with relevant genomes for contemporary insights. Recombination and mobile element analysis across the genome also infers changes over time and under selective pressure, and possible co-infections. With sufficient data and associated metadata, temporal calculations can be made, and the inference of the ancestors of specific characteristics and lineages, as well as possible transmission routes and mechanisms. All of these advances in our knowledge of an important group of pathogens can inform public health strategies. Our protocol and representative results show the use of syndromic panel target probe sets, which are effective in bacterial STI samples and could be extended to additional sample types.

The advent of next-generation sequencing (NGS) techniques has facilitated bacterial whole-genome sequencing (WGS), allowing the sequencing of clinical isolates within many species of human bacterial pathogens. The technology has enabled numerous comparative studies through the generation of complete genomes at an unprecedented scale. This has had a large impact on clinical and public health microbiology, as accessing genome data from clinical samples through WGS enables typing and the detection of antimicrobial resistance genes. In addition, metagenomic approaches allow the direct detection of disease-associated pathogens from clinical samples. Such information creates new opportunities for personalized treatment strategies for patients. Additionally, WGS is a powerful tool that provides insights into the evolution of circulating strains of a specific pathogen, as well as their transmission patterns, enabling public health authorities to better understand and respond to pathogen outbreaks1,2,3.
To achieve sufficient sequencing quality for WGS, bacterial isolation through culture is typically needed to obtain the required microbial DNA amounts4,5,6. This represents a limiting factor for those organisms that are difficult to culture, those that cannot be cultivated due to non-viable diagnostic sample processing, or those exposed to antibiotics during patient treatment. Examples of such difficult-to-culture microorganisms include sexually transmitted bacterial pathogens, such as Chlamydia trachomatis, Neisseria gonorrhoeae, Treponema pallidum, and Mycoplasma genitalium7,8,9. These species are challenging to culture due to several factors, including their intracellular nature, fragility, fastidious growth requirements, and slow replication rates. For these reasons, developing and implementing techniques that enable WGS despite low DNA content -- as is often the case in patient samples -- is crucial for performing genomic analyses in clinical settings.
Target enrichment is the most promising method for genome sequencing when microbial DNA is scarce10. For instance, in the context of pathogens causing sexually transmitted infections (STIs), only a small fraction of the DNA present in a patient sample originates from the pathogen. In the case of C. trachomatis infections, pathogen DNA has been found to comprise up to a maximum of 0.6% of the total DNA, but values are typically much lower11. The target enrichment method consists of selectively capturing the microbial DNA of interest prior to NGS. This is achieved using specifically designed biotin-tagged RNA probes that are complementary to the target microbial DNA. Adapter ligation is performed to generate a genomic library, prior to hybridization, during which these probes bind to the complementary microbial DNA. Subsequently, streptavidin-coated magnetic beads are used to capture the biotin-labeled nucleic acid complexes, exploiting the strong biotin-streptavidin interaction. Finally, a polymerase chain reaction (PCR) amplifies the selected DNA fragments. These key conceptual steps are necessary for understanding the target enrichment strategy. However, additional procedural steps, such as washing and quality control, are also involved in the full protocol10,12.
Before initiating the sequencing protocol, it is important to evaluate the quantity of input DNA and ensure that conditions throughout the workflow preserve its integrity. According to the manufacturer&#39;s recommendations, the standard protocol requires 10-200 ng of DNA in a volume of 7 &#181;L, which corresponds to a concentration range of approximately 1.4 to 28.6 ng/&#181;L. When the DNA concentration is lower, a modified version of the protocol using 17 &#181;L is advised, allowing for concentrations between 0.6 and 11.8 ng/&#181;L while maintaining the required input amount12. Given the low concentrations often obtained from clinical specimens, we routinely implemented the 17 &#181;L protocol variant. Hybridization and sequencing performance are associated with DNA quality, underscoring the importance of appropriate sample storage and transport conditions to preserve nucleic acid integrity. Therefore, proper handling practices such as stabilizing collection media, rapid processing, maintaining cold-chain conditions, and minimizing freeze-thaw cycles are important measures to optimize DNA quality. As well as required DNA concentrations, DNA fragment length is an indicator of integrity. We recommend minimizing DNA degradation during sample handling and storage to obtain longer fragments and optimize sequencing outcomes. This is particularly relevant in clinical settings, where variability in collection methods, medium, and handling is common. We successfully applied this protocol to DNA extracted from both 2-SP medium and NAAT transport media, with mean fragment lengths down to 1kb, as detailed in the representative results. Finally, successful sequencing was defined as achieving more than 95% genome coverage with a mean read depth exceeding 10x10.
To date, target enrichment has been successfully applied by various research groups to a wide range of microorganisms and viruses, including pathogens causing STIs, noroviruses, livestock viruses, and plant pathogens10,13,14,15,16. However, so far, probe design has generally been restricted to single pathogens rather than a panel targeting complete genomes of multiple syndromically linked pathogens. Ideal targets are organisms with small, conserved genomes, which indeed include C. trachomatis, T. pallidum, and M. genitalium. While N.gonorrhoeae has a more diverse genome and larger pan-genome, the probes to cover it can still be rationalized in a design with the other three bacteria. There is some divergence in %G+C across these bacteria, and others that may be syndromically linked, which means that the conditions may need to be optimized.
The goal of this protocol is to obtain complete genomes directly from clinical samples to enable phylogenetic and epidemiological analyses. We demonstrate a novel and effective strategy using a probe panel tailored to C. trachomatis, N. gonorrhoeae, T. pallidum, and M. genitalium to recover complete genomes using extracted DNA from clinical samples such as urogenital and anorectal swabs. This approach has been shown to be successful and was applied in the characterization of the lymphogranuloma venereum (LGV) outbreak in Buenos Aires, enabling the description of a new lineage of C. trachomatis LGV10,13.
The target enrichment procedure will be described, including the use of a commercially available DNA capture method with post-capture pooling for next-generation sequencing (NGS) employed to obtain complete genomes directly from clinical samples from patients diagnosed with C. trachomatis, N. gonorrhoeae, T. pallidum, and/or M. genitalium infections. For optimal success, it is recommended to select samples with a Ct value below 30, as higher Ct values reflect lower pathogen loads and are less likely to yield complete genomes. DNA extract for the method can be obtained from clinical samples of various sample types and collection media. DNA extraction procedures can be performed either manually or with automated systems, using kits designed for the isolation of pathogen or viral DNA from human swab samples (see Table of Materials).
The probe set used in the target enrichment procedure (see Table of Materials) was specifically designed to capture the genomic diversity of C. trachomatis, N. gonorrhoeae, T. pallidum, and M. genitalium. Custom probes were generated using multiple complete genomes for each species, optimizing coverage of their known pan-genomes10. The design criteria included a minimum sequence homology of 90% to the reference sequences and a tiling frequency of 1x-2x to enhance target capture efficiency. To optimize cost-effectiveness, the algorithm used in probe design minimized the number of probes while maintaining comprehensive genomic representation. The final probe set consisted of 242,000 probes for the multiplex designs targeting C. trachomatis, N. gonorrhoeae, M. genitalium, and T. pallidum. The entire target enrichment protocol can be performed in 2 or 3 days. Clear indications of stopping points are provided for both the two-day and three-day workflow options.
ETHICS STATEMENT 
This study proposes methodological improvements based on previously published protocols10,13. The procedures described involve only de-identified samples and do not include patient data; therefore, no specific ethical approval was required for the methodological aspects. The representative results were obtained from two sets of previously studied samples: Argentinian samples collected under the approved protocol Detecci&#243;n de C. trachomatis en pacientes con rectitis infecciosa: prevalencia y tipificaci&#243;n (Gobierno de la Ciudad de Buenos Aires, approval no. 201723), with written informed consent obtained from all participants; and anonymized Finnish samples, for which no specific ethical approval was required. Prior to database submission of any resulting sequencing data, human reads need to be removed.

<table><tbody><tr><td>8-channel pipette 0.5-10 &amp;micro;L</td><td>Eppendorf</td><td>3125000010</td><td>Eppendorf Research&amp;nbsp;plus, 8-channel, 0.5-10 &amp;micro;L, medium gray, variable volume.</td></tr><tr><td>8-channel pipette 10-100 &amp;micro;L</td><td>Eppendorf</td><td>3125000036</td><td>Eppendorf Research&amp;nbsp;plus, 8-channel, 10-100 &amp;micro;L, yellow, variable volume.</td></tr><tr><td>Adapter Oligo Mix for MBC-tagged libraries</td><td>Agilent Technologies</td><td>5500-0147</td><td>Part of SureSelect XT HS2 Library Preparation Kit for ILM (Pre PCR).</td></tr><tr><td>Automated nucleic acid extraction system using a viral/pathogen DNA extraction kit</td><td>Qiagen</td><td>937036 (Kit) / 9001297 (Instrument)</td><td>QIAsymphony SP/AS instrument used with QIAamp DSP Virus/Pathogen Kit for automated DNA extraction.</td></tr><tr><td>Binding Buffer</td><td>Agilent Technologies</td><td>5190-9687</td><td>Part of SureSelect Target Enrichment Kit ILM Hyb Module Box 1 (Post PCR).</td></tr><tr><td>DNA extraction kit and automated instrument</td><td>Zymo Research Corporation</td><td>D3024/D3025</td><td>Quick-DNA Miniprep Kit used for total DNA extraction from clinical specimens. Manual or automated workflow compatible.</td></tr><tr><td>End Repair- A Tailing Enzyme Mix</td><td>Agilent Technologies</td><td>5500-0147</td><td>Part of SureSelect XT HS2 Library Preparation Kit for ILM (Pre PCR).</td></tr><tr><td>End Repair Tailing Buffer</td><td>Agilent Technologies</td><td>5500-0147</td><td>Part of SureSelect XT HS2 Library Preparation Kit for ILM (Pre PCR).</td></tr><tr><td>Ethanol</td><td>VWR Chemicals BDH</td><td>20816.298</td><td>Ethanol absolute &amp;ge;99.5% Ph. Eur., USP</td></tr><tr><td>Fluorometer with High Sensitivity dsDNA Assay / Fluorescence-based method to quantify DNA</td><td>Thermo Fisher Scientific (Invitrogen)</td><td>Q33238, Q33231</td><td>Qubit 4 Fluorometer with 1X dsDNA High Sensitivity (HS) Assay Kit.</td></tr><tr><td>Fragmentation Buffer</td><td>Agilent Technologies</td><td>5191-4080</td><td>Part of SureSelect Enzymetic Fragmentation Kit with the name &#039;&#039;5X Fragmentation Buffer&#039;&#039;.</td></tr><tr><td>Fragmentation Enzyme</td><td>Agilent Technologies</td><td>5191-4080</td><td>Part of SureSelect Enzymetic Fragmentation Kit.</td></tr><tr><td>High-fidelity DNA polymerase buffer</td><td>Agilent Technologies</td><td>5500-0147</td><td>Part of SureSelect XT HS2 Library Preparation Kit for ILM (Pre PCR) with the name &#039;&#039;Herculase II Buffer&#039;&#039;.</td></tr><tr><td>High-fidelity DNA polymerase buffer with dNTPs (5X)</td><td>Agilent Technologies</td><td>5190-9687</td><td>Part of SureSelect Target Enrichment Kit ILM Hyb Module Box 1 (Post PCR) with the name &#039;&#039;Herculase II Buffer&#039;&#039;.</td></tr><tr><td>High-fidelity fusion enzyme for PCR amplification</td><td>Agilent Technologies</td><td>5191-5688</td><td>Part of SureSelect Target Enrichment Kit ILM Hyb Module Box 1 (Post PCR) with the name &#039;&#039;Herculase II&#039;&#039;.</td></tr><tr><td>High-temperature Wash Buffer</td><td>Agilent Technologies</td><td>5190-9687</td><td>Part of SureSelect Target Enrichment Kit ILM Hyb Module Box 1 (Post PCR) with the name &#039;&#039;Wash buffer 2&#039;&#039;.</td></tr><tr><td>Hybridization Buffer&amp;nbsp;</td><td>Agilent Technologies</td><td>5500-0147</td><td>Part of SureSelect XT HS2 Library Preparation Kit for ILM (Pre PCR).</td></tr><tr><td>Indexed sequencing primers</td><td>Agilent Technologies</td><td>5500-0147</td><td>Part of SureSelect XT HS2 Index Primer Pairs 1-96 for ILM (Pre PCR).</td></tr><tr><td>In-house multiplex real-time PCR assay</td><td>Tib Molbiol</td><td>N/A</td><td>In-house developed and accredited assay based on Lightmix Kits for STI detection</td></tr><tr><td>Ligation Buffer</td><td>Agilent Technologies</td><td>5500-0147</td><td>Part of SureSelect XT HS2 Library Preparation Kit for ILM (Pre PCR).</td></tr><tr><td>Magnetic beads for clean up</td><td>Agilent Technologies</td><td>5191-5740</td><td>SureSelect DNA AMPure XP Beads.</td></tr><tr><td>Magnetic stand</td><td>Permagen Labware</td><td>MSRLV08</td><td>0.2mL PCR 8 Strip Magnetic Separator 5 &amp;micro;L ~0.2mL Volume.</td></tr><tr><td>Next-generation sequencing (NGS): High-throughput short-read sequencing system&amp;nbsp;</td><td>Illumina, Inc.</td><td>N/A</td><td>Paired-end 150 bp sequencing (PE150) performed on Illumina NextSeq1000 and MiSeq platforms.</td></tr><tr><td>Nuclease-free water</td><td>BioConcept</td><td>3-07F04-H</td><td>Water for Molecular Biology, DNA/DNAse/RNase free, sterile, PCR Inhibitor free.</td></tr><tr><td>Oligonucleotide Blocker Mix&amp;nbsp;</td><td>Agilent Technologies</td><td>5500-0147</td><td>Part of SureSelect XT HS2 Library Preparation Kit for ILM (Pre PCR).</td></tr><tr><td>PCR tube strip</td><td>avantor, delivered by VWR&amp;nbsp;&amp;nbsp;</td><td>732-3608</td><td>PCR tube strips with attached Flat Caps.</td></tr><tr><td>Post-Capture Primer Mix</td><td>Agilent Technologies</td><td>5190-9687</td><td>Part of SureSelect Target Enrichment Kit ILM Hyb Module Box 1 (Post PCR).</td></tr><tr><td>RNA Probe</td><td>Agilent Technologies</td><td>5191-6916&amp;nbsp;</td><td>SureSelect Custom Tier4 6Mb-11.9Mb Probe, eg Design ID S3465164).</td></tr><tr><td>RNase Blocker</td><td>Agilent Technologies</td><td>5500-0147</td><td>Part of SureSelect XT HS2 Library Preparation Kit for ILM (Pre PCR).</td></tr><tr><td>Room-temperature Wash Buffer</td><td>Agilent Technologies</td><td>5190-9687</td><td>SureSelect Target Enrichment Kit ILM Hyb Module Box 1 (Post PCR) with the name &#039;&#039;Wash buffer 1&#039;&#039;.</td></tr><tr><td>Streptavidin beads</td><td>Agilent Technologies</td><td>5191-5742</td><td>SureSelect Streptavidin Beads.</td></tr><tr><td>T4 DNA Ligase</td><td>Agilent Technologies</td><td>5500-0147</td><td>Part of SureSelect XT HS2 Library Preparation Kit for ILM (Pre PCR) .</td></tr><tr><td>Target enrichment method / DNA capture method</td><td>Agilent Technologies</td><td>G9983-90000</td><td>SureSelect XT HS2 DNA Kit with Library Prep, Fast-Hyb Target Enrichment, and Post-capture Pooling Workflow. Protocol Version F0, May 2023. For Illumina Platform NGS</td></tr><tr><td>Target enrichment probe set for &lt;em&gt;C. trachomatis&lt;/em&gt;, &lt;em&gt;N. gonorrhoeae&lt;/em&gt;, &lt;em&gt;T. pallidum&lt;/em&gt;, and &lt;em&gt;M. genitalium&lt;/em&gt;</td><td>Agilent Technologies / Enriching Microbiology Laboratory (IMM, UZH)</td><td>Design ID S3465164</td><td>Custom-designed probe set (~242,000 probes) for multiplex capture of four STI pathogens. Design based on multiple reference genomes.</td></tr><tr><td>Thermal cycler</td><td>Thermofischer</td><td>4375305</td><td>Veriti 96-Well Thermal Cycler.</td></tr></tbody></table>

generating whole bacterial genomes from clinical samples using a target enrichment workflow

Generating Whole Bacterial Genomes from Clinical Samples using a Target Enrichment Workflow

Obtaining complete genomes of bacterial sexually transmitted infections (STIs) directly from clinical samples is challenging due to the presence of human DNA, microbiota, and very low bacterial pathogen loads. Culture is often not an option, as these species are fastidious, and most samples are taken in transport media that lyse microorganisms, rendering them non-viable for culture. To address these issues, we used a probe panel designed across four species to generate whole-genome sequences of bacterial STIs and performed target enrichment, a two- to three-day hybridization procedure prior to genome sequencing. Our initial results produced excellent genomic data using samples for which direct sequencing was not successful. Target enrichment proved to be highly effective for sequencing bacterial STI genomes, particularly when pathogen load was high (Ct &#60; 30). Tested samples containing Mycoplasma genitalium frequently had Ct values above 30, leading to lower genome recovery success rates; improvements have been observed after optimizing the hybridization temperature from 65 &#176;C to 62.5 &#176;C. In addition to enabling genomic surveillance, the availability of complete genomes can provide valuable insights into antimicrobial resistance, such as identifying acquired resistance determinants associated with Neisseria gonorrhoeae. This method has already led to the identification of a new lineage of Chlamydia trachomatis lymphogranuloma venereum in Buenos Aires, ompA-genotype L4, highlighting the potential of this approach for uncovering novel genomic diversity and improving STI surveillance.

A study on the C. trachomatis LGV outbreak in Buenos Aires, Argentina, showed some novel features in the samples, namely, 10 nucleotide substitutions in the ompA gene compared to ompA-genotype L1, and an unusual Multi-Locus Sequence Typing (MLST) sequence type 217,18,19. We aimed to investigate the genomes from these clinical samples, placing them within the context of global data. To achieve this, we compared the performance of a target enrichment approach with direct metagenomic sequencing for WGS of bacterial STIs from clinical samples, in an attempt to overcome the challenges posed by low bacterial loads and abundant human and microbial DNA.
DNA used was either from rectal swab samples collected between 2017 and 2023 in Buenos Aires, Argentina, placed in 2-SP medium (sucrose-phosphate medium) supplemented with 2% fetal calf serum (FCS) and antibiotics (gentamicin 25 µg/mL, vancomycin 0.5 mg/mL, and amphotericin B 2.5 µg/mL) and stored at −80 °C, or pooled urethral, anorectal, and pharyngeal swabs collected at the University Hospital Zurich (USZ) in NAATs (Nucleic Acid Amplification Tests) buffer. DNA from the Buenos Aires samples was extracted using a silica-based manual extraction kit, while DNA from USZ samples was extracted using an automated magnetic bead-based system.
All clinical samples included in this study were confirmed positive by an in-house developed multiplex real-time PCR assay (see Table of Materials) with qPCR cycle threshold (Ct) values ranging as follows: C. trachomatis: 17-34, N. gonorrhoeae: 24-31, T. pallidum: 27-36, and M. genitalium: 30-37. DNA concentration was quantified using a high-sensitivity fluorometric assay prior to sequencing to characterize the input material of the clinical samples (see Table of Materials).
To compare direct metagenomic sequencing to target enrichment, we used 32 samples, of which 29 were positive for C. trachomatis, nine for N. gonorrhoeae, five for T. pallidum, and three for M. genitalium. Direct metagenomic sequencing did not result in any complete genomes and indeed barely achieved target data above the baseline seen in negative samples. Target enrichment with hybridization at 65 °C resulted in vastly better results for each pathogen, and successful genomes were obtained in many cases from this method (Figure 1). 
To assess the optimum sample selection for target enrichment, we evaluated the relationship between diagnostic qPCR Ct values and the efficiency of genome recovery, as measured by the percentage of on-target reads (%OTR). This metric reflects the proportion of sequencing reads that map to the target organism's genome and is commonly used to assess enrichment performance. A significant correlation was found between pathogen load and sequencing success for C. trachomatis, with a Ct cut-off for success around Ct30. Although data for the other target pathogens were limited, a similar trend was observed, where genome sequencing success occurred predominantly in samples with pathogen load below Ct30 (Figure 2).
This experiment with a hybridization temperature of 65 °C gave genome sequencing success rates with target enrichment of 75%-80% for C. trachomatis, N. gonorrhoeae, and T. pallidum, based on samples with Ct values below 30. The three M. genitalium positive samples had Ct values of 30-37 and achieved lower genome coverage and mean read depth. In order to improve these results, we experimented with reducing the hybridization temperature, which improves hybridization due to the low %G+C content in the M. genitalium genome. Using lower hybridization temperatures, such as the now-recommended temperature of 62.5 °C, we can improve recovery of M. genitalium genomes without compromising recovery of other genomes (Figure 3). These results demonstrate that target enrichment is an effective approach for obtaining valuable genomic information from clinical samples and can be adapted to a wide range of genomes, also across a panel-based design.
Phylogenetic analysis of the successfully enriched C. trachomatis genomes showed that the majority of samples from Buenos Aires cluster within the ompA-genotype L2b clade, suggesting a successful lineage with global circulation. The analysis also revealed the presence of a novel, distinct lineage in Argentina, causing LGV. The samples from this clade differ by approximately 600 SNPs from the other LGV lineages, and this lineage is proposed as ompA-genotype L4 (Figure 4). A detailed description of the samples used in Figure 1, Figure 2, and Figure 4 is provided in Supplementary Table 1.
These findings underscore the value of target enrichment in studying pathogen diversity and demonstrate a high degree of genome sequencing success from clinical samples.
<img alt="Genome sequencing result chart, showing %OTR via Direct sequencing vs. SureSelect methods." class="xfigimg" src="/files/ftp_upload/68506/68506fig1.jpg" data-altupdatedat="1762509930000" data-alt="Figure 1"> 
Figure 1: Enrichment of genomes using target enrichment compared to direct metagenomic sequencing. Percentage of on-target reads (%OTR), defined as the percentage of sequencing reads mapping to the genome of the target organism, shows the degree of enrichment achieved with target enrichment. The line represents the median of all data points, and the box indicates the 50% interquartile range (IQR). Data point colors correspond to the sample source: pale blue for Argentina and red for Switzerland. Genome sequencing success, defined as coverage &gt;95% and mean read depth &gt;10, is represented by large points, while failure is indicated by small points. Note the different scales on the y-axis. Abbreviations: CT = C. trachomatis, NG = N. gonorrhoeae, TP = T. pallidum, MG = M. genitalium. This figure has been modified from10. <a href="https://www.jove.com/files/ftp_upload/68506/68506fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Genome sequencing scatter plots; %OTR vs. Ct; success/fail analysis; data visualization." class="xfigimg" src="/files/ftp_upload/68506/68506fig2.jpg" data-altupdatedat="1762509930000" data-alt="Figure 2"> 
Figure 2: Correlation of %OTR from target enrichment with diagnostic qPCR Ct values. Data point colors represent the sample source: pale blue for Argentina and red for Switzerland. The genome sequencing success, defined as coverage &gt;95% and mean read depth &gt;10, is indicated by large points, while failure is represented by small points. Note the different scales on the y-axis. Abbreviations: CT = C. trachomatis, NG = N. gonorrhoeae, TP = T. pallidum, MG = M. genitalium. This figure has been modified from10. <a href="https://www.jove.com/files/ftp_upload/68506/68506fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Hybridization temperature vs. coverage graph; microorganisms differentiation; data points analyzed." class="xfigimg" src="/files/ftp_upload/68506/68506fig3.jpg" data-altupdatedat="1762509930000" data-alt="Figure 3"> 
Figure 3: Effect of hybridization temperature on recovery of complete genomes across all STI bacterial pathogens in ten samples. Human DNA in the range of that in clinical samples was spiked with known concentrations of target bacterial DNA separately (single) and also altogether to represent a sample with multiple infections (pooled). Two experiments, each with the five pathogens/combinations, were performed: 0.01% and 0.03% to represent the approximate percentage of DNA from each microorganism relative to the total DNA in the sample (the remaining DNA corresponds to human DNA). Recovery of M. genitalium is improved at lower temperatures. C. trachomatis DNA was spiked at a low concentration; further experiments show that the temperatures did not affect the recovery of C. trachomatis genomes (data not shown). Abbreviations: CT = C. trachomatis, MG = M. genitalium, NG = N. gonorrhoeae, TP = T. pallidum. <a href="https://www.jove.com/files/ftp_upload/68506/68506fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Phylogenetic analysis diagram of bacterial genotypes using SNP data, showcasing diversity by country." class="xfigimg" src="/files/ftp_upload/68506/68506fig4.jpg" data-altupdatedat="1762509930000" data-alt="Figure 4"> 
Figure 4: Recombination-adjusted phylogeny of LGV strains from Argentina. The analysis includes all available LGV genomes with published data. Metadata displayed to the right of the phylogeny includes duplicate patient samples, Bayesian Analysis of Population Structure (BAPS; cluster = clade, with the polyphyletic BAPS cluster 6 representing the genetic diversity of the population), genome sequencing method (data from this study), ompA genotype, country, and year, as indicated in the key at the top left. Missing data is shown in white. Genome names from this study are highlighted in light blue for Argentina and dark blue for Finland (not obtained by target enrichment). These colors are used solely to distinguish the genomes generated in this study. Bootstrap values from 1000 ultrafast bootstraps are displayed as percentages on major branches. This figure has been modified from13. <a href="https://www.jove.com/files/ftp_upload/68506/68506fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Table 1: Description of materials, reagents, instruments, and equipment used in the study. The table includes detailed information on the commercial name, manufacturer, and catalogue number of the components used for DNA extraction, library preparation, target enrichment, and sequencing. It also includes items used for in-house qPCR assays and general molecular biology procedures. Equivalent alternatives may be used for items marked with an asterisk (*). <a href="https://www.jove.com/files/ftp_upload/68506/Supplementary_Table_1_RE.xlsx" target="_blank">Please click here to download this Table.</a>

Here, we present a protocol to enable whole-genome sequencing of bacterial sexually transmitted infections from clinical samples using target enrichment. This novel, syndromic panel-based method overcomes challenges of abundant human DNA and low bacterial loads, facilitating genomic surveillance for Chlamydia trachomatis, Neisseria gonorrhoeae, Treponema pallidum, and Mycoplasma genitalium.

Obtaining complete genomes of bacterial sexually transmitted infections (STIs) directly from clinical samples is challenging due to the presence of human ...

Genomes can provide information on the ancestry, transmission, and capabilities of a pathogen. For unculturable bacteria, this information can only be obtained through target enrichment. Sequencing bacterial pathogens directly from clinical samples is challenging because of the overwhelming human DNA, the diverse microbiota and extremely low bacteria loads.In many cases, culture is not an option, due to the sampling method and fastidious bacteria. While applying this technology to sexual transmitted bacteria, we discovered a novel Chlamydia trachomatis LGV lineage called ompA-genotype L4, and confirmed global expansion of the dominant LGV lineage, delivering key insight for molecular diagnostic and epidemiological surveillance. This progress enrichment protocol enables complete genome recovery from positive diagnosed STI clinical samples.We found that this technique outperforms metagenomic deep sequencing, host DNA depletion, and nanopore adaptive sequencing. We're investigating the circulating strains of these pathogens across Europe and in Argentina. This has the potential to inform regarding transmission and antimicrobial stewardship.To begin, measure the DNA concentration of the DNA extracts from clinical samples using a sensitive and accurate fluorescence-based method. Dilute each DNA sample using nuclease-free water to 10 to 200 nanograms in a final volume of 17 microliters in each PCR tube strip. Add 3 microliters of fragmentation master mix to each well containing the 17 microliters of diluted DNA.Pipette up and down 20 times to mix thoroughly. Then, seal the PCR tube strip. Vortex the strip at high speed for 10 seconds and spin briefly to collect the contents.Then, place the PCR tube strip in the thermal cycler and start the pre-programmed protocol for fragmentation and incubation. Next, prepare the end repair and dA-tailing master mix as shown in the table. Vortex and spin the tube as shown earlier and keep it on ice.Then, pipette 20 microliters of the end repair and dA-tailing master mix into each well containing 50 microliters of DNA fragments. Seal the strip and vortex at high speed for eight seconds. After briefly spinning the strip, place it back into the thermal cycler.Once the thermal cycler program finishes, place the PCR tube strip on ice. Next, add 25 microliters of room temperature ligation master mix, which was prepared 30 to 45 minutes in advance to each well containing the DNA fragments. Add 5 microliters of adapter oligo mix for NBC-tagged libraries to each well.Immediately place the PCR tube strip back into the thermal cycler and continue the program. To begin DNA purification, retrieve the magnetic beads from 4 degrees Celsius and allow them to come to room temperature before use. Then, add 80 microliters to each sample well.Place the PCR tube strip on the magnetic stand and wash each well twice with 200 microliters of 70%ethanol. Wait for one minute during each wash and carefully remove the ethanol without disturbing the magnetic beads. After mixing and incubating the beads with 35 microliters of nuclease-free water for five minutes, carefully aspirate the cleared supernatant from each well and transfer it into a corresponding well in a new PCR tube strip.Place the new PCR tube strip on ice and discard the magnetic beads. Take the prepared pre-recapture PCR reaction mix and add 11 microliters of it into each sample well containing the purified DNA library. Add 5 microliters of the selected index prime repair to each reaction and seal the PCR tube strip.Start the thermal cycler program for PCR amplification as specified in the table to perform the pre-capture PCR. Only add the PCR tube strip once the thermal cycler has reached 98 degrees Celsius. After completing the pre-capture PCR, perform another bead-based cleanup to purify the library.Incubate the PCR product with magnetic beads. Then, place the PCR strip tube on a magnetic separation device and wait 5 to 10 minutes until the solution becomes clear. While the strip remains on the magnetic stand, carefully remove and discard the clear supernatant from each well.Then, elute the DNA in 15 microliters of nuclease-free water and measure the DNA concentration of the purified DNA library. For sample hybridization, program the thermal cycler according to the protocol specified here. Start the program and immediately pause the run.Dilute 500 to 1, 000 nanograms of the prepared DNA libraries to a final volume of 12 microliters using nuclease-free water. After vortexing and spinning down the oligonucleotide blocker mix, add 5 microliters to each sample tube. Seal the PCR tube strip and place it in the thermal cycler.Pause the thermal cycler when it reaches step three of the protocol. Add 13 microliters of the prepared room temperature probe hybridization mix to each sample while keeping the strip inside the thermal cycler. Pipette slowly up and down 8 to 10 times to mix.Ensure no air bubbles are present, and place the strip back into the thermal cycler. Then, confirm that all tubes are sealed tightly. To capture the hybridized library, transfer the entire volume from each tube to the corresponding tubes containing 200 microliters of pre-washed streptavidin beads.Collect the streptavidin beads before washing them with room temperature wash buffer. Once the PCR tube strip is removed from the magnetic separator, add 200 microliters of high temperature wash buffer pre-warmed to 70 degrees Celsius and re-suspend the beads by pipetting up and down. Seal the tube strip, vortex at high speed, and spin quickly as demonstrated earlier.Place the sealed tube strip in the thermal cycler block at 70 degrees Celsius and incubate for 5 minutes. Transfer the PCR tube strip from the thermal cycler back to the magnetic separator at room temperature. Wait for one minute until the solution becomes clear.Then, carefully remove and discard the supernatant. Then, add 25 microliters of nuclease-free water to each tube containing the beads. Program the thermal cycler as specified in the table shown here.Add 25 microliters of the prepared post-capture PCR reaction mix to each sample tube. Mix the reaction until the suspension is uniform, and securely seal the PCR tube strip without spinning it down. Place the sealed PCR tube strip in the thermal cycler.After transferring the PCR product to a fresh tube, add 50 microliters of bead suspension to each sample tube and perform the DNA cleanup as shown earlier. Measure the DNA concentration of the final purified library DNA using a fluorescence-based quantification method. Target enrichment resulted in significantly higher percentages of on-target reads, and therefore complete genomes compared to direct sequencing across all pathogens tested, including Chlamydia trachomatis, Neisseria gonorrhoeae, Treponema pallidum, and Mycoplasma Genitalium.Genome sequencing success was predominantly observed in samples with qPCR cycle threshold values below 30 for all four pathogens. With a clear inverse correlation between CT values and percentage of on-target reads for Chlamydia trachomatis. Using a hybridization temperature of 62.5 degrees Celsius compared to the previous 65 degrees Celsius, improved genome coverage for mycoplasma genitalium without reducing performance for the other target pathogens.Phylogenetic analysis of Chlamydia trachomatis genomes from Argentina showed many samples clustering within the L2b clade and revealed a novel lineage proposed as ompA-genotype L4 separated from all previous LGV genomes by approximately 600 single nucleotide polymorphisms.

generating-whole-bacterial-genomes-from-clinical-samples-using-target

Research

JoVE Journal

Bioengineering

We describe a methodology to perform genetic analysis in Chlamydia based on chemical mutagenesis and whole genome sequencing. In addition, a system for DNA exchange within infected cells is described that can be used for genetic mapping. This method may be broadly applicable to microbial systems lacking transformation systems and molecular genetic tools.

Forward Genetic Approaches in Chlamydia trachomatis

gDNA enrichment for NGS sequencing is an easy and powerful tool for the study of constitutional mutations. In this article, we present the procedure to analyse simply the complete sequence of 11 genes involved in DNA damage repair.

gDNA Enrichment by a Transposase-based Technology for NGS Analysis of the Whole Sequence of BRCA1, BRCA2, and 9 Genes Involved in DNA Damage Repair

An integrated system for targeted next-generation sequencing of oncology specimens is described. This cross-platform system is optimized for low-quality and low-quantity tumor biopsies, accommodates low DNA inputs, includes well-characterized multi-variant controls, and features a novel variant caller that is informed by quantitative pre-analytical quality control measures.

Integration of Wet and Dry Bench Processes Optimizes Targeted Next-generation Sequencing of Low-quality and Low-quantity Tumor Biopsies

We describe an efficient, robust, and cost effective method for extracting nucleic acid from swabs for characterization of bacterial communities using 16S rRNA gene amplicon sequencing. The method allows for a common processing approach for multiple sample types and accommodates a number of downstream analytic processes.

Efficient Nucleic Acid Extraction and 16S rRNA Gene Sequencing for Bacterial Community Characterization

Here we present a protocol to investigate genome wide DNA methylation in large scale clinical patient screening studies using the Methyl-Binding DNA Capture sequencing (MBDCap-seq or MBD-seq) technology and the subsequent bioinformatics analysis pipeline.

Methyl-binding DNA capture Sequencing for Patient Tissues

This protocol is to recover and prepare rare target cells from a mixture with non-target background cells for molecular genetic characterization at the single-cell level. DNA quality is equal to non-treated single cells and allows for single-cell application (both screening based and targeted analysis).

Target Cell Pre-enrichment and Whole Genome Amplification for Single Cell Downstream Characterization

This manuscript describes a detailed standardized protocol of high-throughput 16S rRNA-amplicon sequencing. The protocol introduces an integrated, uniformed, feasible, and inexpensive protocol starting from fecal sample collection through data analyses. This protocol enables analysis of large numbers of samples with rigorous standards and several controls.

Guided Protocol for Fecal Microbial Characterization by 16S rRNA-Amplicon Sequencing

This protocol details a comprehensive approach for the culturing, sequencing, and de novo hybrid genome assembly of urinary bacteria. It provides a reproducible procedure for the generation of complete, circular genome sequences useful in studying both chromosomal and extrachromosomal genetic elements contributing to urinary colonization, pathogenesis, and antimicrobial resistance dissemination.

Hybrid De Novo Genome Assembly for the Generation of Complete Genomes of Urinary Bacteria using Short- and Long-read Sequencing Technologies

The feasibility of whole-genome sequencing (WGS) strategies using benchtop instruments has simplified the genome interrogation of every microbe of public health relevance in a lab setting. A methodological adaptation of the workflow for bacterial WGS is described and a bioinformatics pipeline for analysis is also presented.

Characterization of a Pathogenic Escherichia coli Strain Derived from Oreochromis spp. Farms Using Whole-Genome Sequencing

Clinical metaproteomics offers insights into the human microbiome and its contributions to disease. We harnessed the computational power of the Galaxy platform to develop a modular bioinformatics workflow that facilitates complex mass spectrometry-based metaproteomic analysis and characterization of diverse clinical sample types relevant to studies of disease.

A Clinical Metaproteomics Workflow Implemented within Galaxy Bioinformatics Platform to Analyze Host-Microbiome Interactions Underlying Human Disease

The current study describes a workflow for nanopore sequencing of plasmids when the chromosomal sequence is already known or of no interest. An initial plasmid DNA enrichment step ensures optimal plasmid sequence depth and maximizes the number of samples that can be sequenced per flow cell.

Long-Read Plasmid Sequencing and Assembly Using Nanopore Sequencing-Based Workflows

Learn target enrichment sequencing for bacterial STI genomics using clinical samples. This protocol enables complete genome recovery from Chlamydia trachomatis, Neisseria gonorrhoeae, and other pathogens for epidemiological surveillance.

Generating Whole Bacterial Genomes from Clinical Samples using a Target Enrichment Workflow

Explore More Videos