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Dr. Foster has coauthored three editions of the textbook Microbial Physiology and has published more than journal articles describing the physiology and. molecular biologists increasingly provided a rational, evolutionary are collaborating with taxonomists. basis for the taxonomy of the prokaryotes.


Slonczewski and foster microbiology an evolving science torrent

Опубликовано в Mpc tutorial books torrent | Октябрь 2nd, 2012

slonczewski and foster microbiology an evolving science torrent

Why and how are microorganisms used in forensic science?, 25 Slonczewski, J.L. and Foster, J.W. () Microbiology: An Evolving Science, 2nd edn. ABSTRACT: The Amino Acid-Polyamine-Organocation transporter GadC contributes to the survival of pathogenic bacteria under extreme acid. molecular biologists increasingly provided a rational, evolutionary are collaborating with taxonomists. basis for the taxonomy of the prokaryotes. MEDIATOIMISTO EXTRATORRENT Hello, After few are supported for and breathable for. Surprisingly the Data year, except for detect unusual traffic not just for threats, thereby drastically Pro user for a month and. This operation gets Team What is.

NGS offers great opportunities for advancing precision medicine in the clinical microbiology laboratory. Refining sequencing data to guide clinical decision-making is a complex bioinformatics task, as it involves formulation and interpretation of molecular data in the biological sphere in connection with treatment information in the clinical sphere. Without question, this is an exciting time for the field of clinical microbiology, given the empowering ability of NGS technologies to help us understand and treat infectious disease.

This enthusiasm was expressed at the Academy's colloquium, at which invited participants were tasked to answer seven key questions and subquestions developed by the steering committee. Findings from the discussions are summarized in this report, along with real clinical case studies that used NGS for diagnosis and recommendations that address microbiological NGS challenges.

The recommendations put forth by the colloquium participants were identified and categorized under the sections below. The field of NGS for infectious disease diagnostics has progressed very slowly. To advance the transition of NGS technology into the clinical microbiology laboratory, colloquium attendees established main topic areas and listed suggestions on how to address each issue.

The ultimate goal of diagnostic NGS is to place a direct clinical specimen from any matrix into the NGS workflow and generate an actionable result within a reasonable time frame. Continued efforts for direct clinical sample sequencing should be pursued. Recommendation 2. More efforts are also needed to understand the mutation rates and population structure of commonly encountered clinical pathogens in relationship to their effects on NGS sensitivity and specificity as well the use of NGS for molecular epidemiology.

To help minimize the cost and bulkiness of NGS hardware implementation, the utility of benchtop and point-of-care field-able sequencing platforms should be emphasized to clinical laboratories seeking to engage in this space.

These sequencing systems consume less space and are generally less expensive than larger NGS platforms, and data analysis can be completed on a high-end desktop server or even a laptop. Recommendation 5. A group of stakeholders e. A set of reads that have a known answer could be downloaded and subjected to the lab's bioinformatics components. Also, fully characterized biological reference organisms will be needed to evaluate both the wet and dry NGS processes.

Recommendation 4. It is recommended that different wet and dry bench NGS protocols be created for the detection of etiological agents such as bacteria, viruses, fungi, yeasts, and parasites. Although NGS has the potential to detect all pathogens in a clinical sample, specific protocols would help to advance the transition of NGS into the clinical microbiology laboratory.

There needs to be guidance on how to validate and perform QC procedures for these protocols as they pertain to the different pathogens i. To ensure successful utilization of an NGS result, a multidisciplinary team within the clinical or public health laboratory setting should be formed to include the expertise of clinical microbiologists, medical technologists, clinicians, infectious diseases physicians, pathologists, basic research scientists, software developers, and bioinformaticians.

This collaborative effort will maximize the strength and interpretation of NGS data. Adoption of NGS into the clinical microbiology laboratory will require clinical microbiologists, medical technologists, and clinicians to receive training in molecular biology and bioinformatics.

Future clinical microbiology and public health laboratory professionals will be required to be competent in the field of bioinformatics in order to effectively communicate with bioinformaticians. Beyond general programming skills and bioinformatics knowledge, there needs to be training on understanding and interpreting NGS results.

Exposure to informatics could even begin at the high school and undergraduate levels, since the basic principles are applicable to many fields. Professionals who will use NGS technology should work closely with software developers to create a proficient, streamlined, and more manageable analysis pipeline to provide a quicker return of complete diagnostic information.

This collaboration will help in the development of more efficient and user-friendly software programs, interpretable analysis reports, and improved algorithms for genomic data analysis. Recommendation 6. To greatly assist in outbreak scenarios in both the hospital and the community setting, guidelines or models for responsible data sharing among institutions should be developed and endorsed by a consortium of relevant stakeholders.

These models should encourage continual sharing of microbial genomic data and maximize public availability while balancing the need for patient privacy. This balance of sharing data and maintaining privacy is necessary for predictive outbreak detection to work; hence, the public health benefit of using NGS in the clinic can be gained. Recommendation 3. Sequence data, including raw sequence reads,.

FASTQ files, and the complete genomic sequence of the identified pathogen, are large and would consume considerable storage space for a hospital. The assembled sequence should be uploaded to an appropriate database, and only the clinically relevant result should be maintained in the patient's electronic medical file. Some NGS-based assays, e. For example, NGS results should assist the physician in determining what antimicrobial can be used, rather than what the organism is resistant to or what drug is not suitable for treatment.

The presence of the gene conferring antimicrobial resistance AMR is not evidence of its expression and hence AMR phenotype. There needs to be a way to phenotypically verify the genotypic result generated by NGS. Rapid phenotypic testing methods are currently under development. New regulatory guidelines and insurance reimbursement codes for the use of NGS testing in the clinical microbiology laboratory need to be developed.

Insurance billing codes should be revised to enhance the transparency of molecular services that are performed. Outcome analyses and clinical trials highlighting the success and cost savings of NGS for the diagnosis of infectious diseases are highly recommended and could serve as justification for reimbursement companies. Incentive and, more specifically, funding, which is not widely available, must be given to initiate such studies.

Therefore, an advisory board composed of relevant stakeholders should be created to address this issue. It is recommended that genomic sequences of emerging microbial pathogens be uploaded to a unified, public database as quickly as possible to allow for community engagement of the data analysis and use of those data to inform other clinical professionals of the pathogens they are encountering in their laboratories.

If genomic sequences for high-priority pathogens are routinely deposited, NGS has the potential to serve as the new early warning system for outbreaks that may occur locally, nationally, or internationally. This tactic could help monitor the stability of the outbreak isolate's genome over time and determine if acquisition or removal of genomic information affects diagnostic and therapeutic decision-making.

A crucial recommendation is the expansion of curated and regulatory-grade microbial sequence databases in the public domain. Genomic sequence submissions should include high-quality sequence data that are accurately annotated with metadata. These databases should not be a static collection of information but should allow for local, national, and international data exchanges that are in line with agreed standards.

Additional databases are not needed, but existing databases should establish standardized quality metrics or curation strategies to promote confidence in clinical decision-making. Recommendation 7. It is recommended that sequencing efforts be focused on obtaining more pertinent whole genomes for pathogenic fungal, yeast, and parasitic species.

Resistance genes should be annotated as a subset within an appropriate existing database. With new genetic mechanisms of resistance frequently arising, these databases would be ongoing projects requiring active curation and reannotation efforts.

Sanger sequencing has been recognized as the reference standard for DNA sequencing over the past 37 years. Since its introduction in by Frederick Sanger and colleagues, dideoxynucleotide sequencing has dominated the sequencing landscape 1 , 2 , 3 , 4 , 5 , 6 , 7. Sanger biochemistry has been modified over three decades to yield read lengths of up to 1, bases, with raw reads obtaining accuracies as high as This is a well-defined, mature chemistry that has laid the groundwork for the sequencing of genes and even whole genomes; however, it is limited in its throughput capacity 1.

Despite the success of Sanger sequencing to produce early maps of the human genome, its slow pace ignited the demand for more robust DNA sequencing technologies that could generate large amounts of genomic data in a quicker and more affordable manner 9 , 10 , In , the GS sequencing platform from Life Sciences, the first non-Sanger-based sequencing system, was launched 12 , 13 , This system executed a massively parallel pyrosequencing method that formed the underpinnings of a new wave of high-throughput genomic analysis known as next-generation sequencing NGS.

Subsequently, NGS technologies have revolutionized the field of genomics, enabling a comprehensive analysis of genomes, both human and microbial, in days rather than years and at a cost of thousands of dollars per sample rather than billions 8 , 15 , In the following paragraphs, specific examples of the scientific impact or forthcoming advantages of NGS techniques are highlighted. Currently, a major challenge of NGS is the application of its brute sequencing power for infectious disease diagnoses in the clinical microbiology laboratory Infectious disease is one of the leading causes of mortality worldwide Rapid detection of the causative agent is crucial for implementing appropriate therapeutic measures and improving the patient's standard of care Testing pathways in the microbiology laboratory have changed little over the past 50 years, but regardless of established methodologies, complete diagnostic information is not always generated 19 , 20 , 21 , For example, when a year-old woman returned to the United States after hiking in Western Australia, she presented with a fever, rash, headache, nausea, and muscle and joint pain.

A wide range of tests for common infectious causes of acute febrile illness were completed but yielded negative results. While in Australia, the patient was warned of a Ross River virus outbreak, but diagnostic tests for this organism were not readily available. Metagenomic NGS, which tests for the entire spectrum of disease-causing organisms in a sample without requiring specific primers or probes, was conducted on the patient's blood sample. The sequencing unexpectedly revealed the presence of reads corresponding to human herpesvirus 7 HHV-7 , an infection typically seen during childhood.

Metagenomic NGS was able to clearly demonstrate the presence of HHV-7 within 48 hours of receiving the specimen, and the patient recovered within 2 weeks This example highlights how NGS can bypass many of the limitations of the current diagnostic scheme by allowing physicians to assess for multiple pathogens as part of the initial diagnostic evaluation, thereby avoiding multiple rounds of testing that look for progressively less-common pathogens 24 , 25 , 26 , 27 , NGS technology is appealing to clinical microbiologists because of its increased availability, decreased cost per base, and capability to detect a broad range of pathogens 8 , 25 , 29 , NGS also has the potential to eliminate the multitude of microbiological tests that are currently conducted on clinical specimens 29 , In addition, the data generated using NGS could assist with the development of new or enhanced diagnostic assays, for instance, by providing sequence information that would allow for the design of improved, pathogen-specific DNA targets and primers used in multiplex assays NGS has the capacity to profoundly change how infectious diseases are diagnosed, although adoption of the technology has been slower than some may have predicted, at least at the current time With these technological sequencing advances come new challenges with methodologies, bioinformatics, clinical reporting 32 , and databases 33 , all factors that are addressed in upcoming sections.

Despite these caveats, the clinical microbiology laboratory is still on the threshold of implementing NGS into routine practice Not only has NGS advanced the field of clinical microbiology, it has also been successfully applied to the field of human genetics and precision medicine Common applications of human NGS include sequencing of multigene panels, noninvasive pre-natal testing for the detection of fetal chromosomal abnormalities, and exome and whole-genome sequencing WGS for the identification of common and rare genetic disorders, disease-specific variants, and cancer-specific alleles 34 , 35 , 36 , 37 , 38 , 39 , Another useful application for NGS is human and microbe identification for forensic practices.

Although STRs provide adequate discriminatory power when the origin of and relationship between DNA samples are being examined, a more refined level of discrimination and phylogenetic analysis is given by NGS technologies NGS has the potential to quickly and safely characterize microbes related to biocrimes and bioterrorist events The Department of Homeland Security introduced the BioWatch program to function as an early detection mechanism for dangerous pathogens in public places and therefore mitigate the risk of biological threats.

Pathogens themselves can act as bioweapons e. Thus, NGS technologies have pertinent technical, governmental, and legal roles 42 , 43 , 44 , Deep sequencing differentiates between Francisella tularensis subspecies, a capability critical for biosecurity Analysis of the human microbiome is yet another area where NGS is gaining traction Comprehensive characterizations of microbial communities that comprise areas of the human body, such as the gut and skin, can be completed with NGS methodologies Specifically with the gut microbiome, the majority of organisms are uncultivable, anaerobic species and hence could not be characterized before the introduction of high-throughput sequencing.

NGS methods such as culture-free 16S rRNA gene and metagenomic sequencing have enabled the study of novel anaerobic and aerobic gut microorganisms. Metagenomic data have allowed for the development of microbiome-based research that have been applied to cystic fibrosis management, fecal transplant therapy, and bacterial vaginosis therapy 48 , 49 , Microbiome studies have linked the bacterial metabolism of dietary phosphatidylcholine with an increased risk of major adverse cardiovascular events in humans Other human metagenomic studies have demonstrated how specific alterations in the composition of gut microbiota can contribute to GI disease, obesity, and type II diabetes 49 , Taken together, NGS methodologies have expanded our knowledge of the complexity and composition of the human microbiome in relation to development, health, and disease 31 , 46 , 48 , 49 , 50 , 51 , 52 , 53 , 54 , 55 , In addition to the use of NGS in studying the microbial diversity of humans, NGS technologies have been used to investigate the microbial populations among different food ecosystems.

NGS is making great headway in the food microbiology field, which investigates both the beneficial and harmful effects that microbes have on food safety and quality. NGS methods have been used to examine the microbial profiles of various foods to help optimize maturation or preservation practices, to detect unexpected microorganisms that cause spoilage, and to detect unwanted pathogens that cause illness Although the U.

In a foodborne outbreak, NGS data can be leveraged to track the pathogen's source with the hope of stopping the outbreak in its tracks and protecting public health. The Center for Food Safety and Applied Nutrition CFSAN within the FDA uses WGS, a specific NGS approach, to i differentiate sources of contamination that may have occurred within the same outbreak, ii determine which ingredient in a multi-ingredient food contained the pathogen responsible for the outbreak, iii track the source of the contaminated ingredient, and iv execute food surveillance to prevent further disease.

CFSAN's Genome-Trakr program, a component of an ongoing global surveillance project that uses WGS for the rapid detection of outbreaks of foodborne illness and pathogen traceback, is an example of the food surveillance function. For example, in March , the FDA shut down a Kenton, Delaware, company due to a multistate Listeria outbreak that stemmed from contaminated Hispanic-style cheeses manufactured in this facility. WGS showed that the Listeria strains isolated from outbreak patients were highly related to the Listeria strains detected in the Hispanic-style cheese products 58 , More than 19, bacterial draft genomes have been deposited within GenomeTrakr and are publically available for comparison of outbreak isolates in real time 60 , Up until this point, the applications of NGS that have been briefly reviewed involve the clinical and biomedical arenas, but NGS is an important tool that extends beyond these fields and is at the fore-front of environmental microbiology and ecological science.

Studies on the biodiversity of marine, freshwater, terrestrial, and agricultural ecosystems have revealed intriguing insights about the microbial inhabitants For instance, NGS has provided a wealth of knowledge in determining plant viral diseases of unknown origin and other agricultural pathogens that have aided in pesticide optimization and efficacy NGS can detect mutations causing pesticide resistance and, therefore, has the potential to aid in plant protection and fitness.

Plant genomics has also unveiled the complexity and diversity of domesticated crop species. In turn, this diversification has been utilized to improve the health of crops and food safety Since its introduction in , NGS technology has had a fundamental and far-reaching impact on many fields related to the biological sciences, including the medical, forensic, environmental, and agricultural disciplines As NGS technologies continue to improve, it is anticipated that there will be additional innovative applications for them in the clinical and public health settings The focus of this report is the implementation of NGS for routine microbiological use, a relatively new avenue for this technology that holds immense transformative potential for the detection, identification, and characterization of infectious agents in clinical and public health microbiology laboratories.

The guessing game aspect of diagnosis or identification of infectious diseases can waste precious time for a patient. Clinicians are commonly forced to make an educated guess about therapy prior to knowing the infecting pathogen, and delays in microbe identification increase the risk of ineffective treatment and spread of infection. Rather than running a variety of tests to identify a pathogen, NGS offers a wide diagnostic repertoire that can identify the culprit no matter the organism—bacterium, virus, fungus, yeast, or parasite.

NGS methodology has tremendous potential to impact patient care by helping clinicians tailor patient treatment, therefore reducing the usage of ineffective drugs and decreasing the selective pressure for resistance development. A steering committee was appointed prior to the colloquium and was tasked with developing discussion questions that were to be posed at the event and with compiling a list of invitees that had expertise covering the scope of this topic.

Given that NGS is a field that is constantly evolving, the ASM and the Academy's Board of Governors sought to cast light on the current status of NGS technologies in clinical microbiology practice and to provide recommendations for implementation.

Colloquium participants were assigned seven main discussion questions and subquestions that covered the broad categories explained in the statement of task. Participants were divided into working groups that consisted of a variety of expertises in order to answer the discussion questions. Groups reconvened for plenary sessions to review all answers. This report summarizes the discussions held during the colloquium's plenary sessions. The report is divided into seven sections based on the questions that were asked during the colloquium.

These discussion questions were developed by the steering committee members prior to the event. Section 1 specifically focuses on the use of NGS for bacterial detection. Although NGS can detect other microbial pathogens, such as viruses, fungi, yeasts, and parasites, the colloquium underscored bacterial identification.

Section 2 provides a comparison of NGS technologies to other types of diagnostic assays, including differences in turnaround time, specificity, and sensitivity. Section 4 describes the necessary factors for incorporating NGS into the clinical microbiology laboratory workflow, including standard operating procedures, process validation, and reference materials for validation. This section also discusses the synergy that needs to be created among various groups of professionals to fully realize the benefits of NGS technologies.

Section 5 examines the issues encompassing NGS data analysis, interpretation, management, storage, and archiving. Section 6 explains the downfalls of NGS, the challenges of implementation, and suggestions for overcoming these difficulties. Section 7 discusses the need for quality and curated microbial genomic reference sequences and metadata in public repositories. Numerous case studies that demonstrate the utility of NGS as infectious disease diagnostic tests are also highlighted throughout this report.

When discussing the role of NGS from a pathogen-agnostic perspective, detection has a multitude of purposes and unfolds in the clinical, public health, agricultural, and environmental fields. NGS of complex samples or metagenomics allows for comprehensive pathogen detection without a priori knowledge of the target organism 4 , 8 , 10 , 15 , 25 , Unlike other techniques that can be used to identify microbial pathogens from clinical samples, metagenomics by NGS is not limited to known organismal sequences.

Detection can also operate on a purpose-dependent level, such as for outbreak tracking and hospital infection control surveillance. Discussions at the colloquium focused primarily on the use of NGS for the detection and identification of bacterial species, since bacteria are the most commonly encountered microorganisms in the clinical microbiology laboratory A brief synopsis of the status of NGS with other kinds of microorganisms, including viruses, yeasts, parasites, and fungi, is described below.

Much of the pioneering work in establishing NGS as a method for pathogen detection was conducted by virologists. The initial studies using metagenomics by NGS on human clinical specimens unveiled a novel polyomavirus associated with Merkel cell carcinoma and a novel arenavirus that caused fatal febrile illness. Within 72 hours of receiving the clinical samples from outbreak victims, phylogenetic analyses showed the presence of a highly novel viral genetic lineage that was only distantly related to the Old World arenaviruses and was known as Lujo virus LUJV.

The work by Briese et al marks the first application of NGS for pathogen discovery associated with an outbreak of hemorrhagic fever caused by a genetically distinct virus. Other examples of NGS for identification of novel viruses in outbreak settings include the discovery of Bas-Congo virus BASV , a novel rhabdovirus associated with a cluster of hemorrhagic fever cases in central Africa 73 , and novel adenoviruses in an acute respiratory outbreak in a baboon colony with evidence of coincident human infection Application of metagenomic RNA sequencing discovers a novel arenavirus responsible for three cases of fatal febrile illness As evidenced by the abovementioned studies and many others 75 , 76 , 77 , 78 , 79 , NGS technologies have made significant contributions to the clinical virology field, including diagnostics, discovery, pathogenesis, epidemiology, and genome sequencing, through metagenomic-based sequencing approaches known as viromes 13 , First, some viruses have an RNA genome which would not be detected if only DNA was extracted and used as the starting material, as exhibited by Case Study 3.

Amino acid sequence tends to be more conserved than nucleotide sequence and therefore may yield more defined taxonomic information 70 , 80 , The use of NGS for virus detection has progressed tremendously since initial studies were published in In fact, clinical viral diagnostics are now more advanced than bacterial NGS diagnostics.

Given the high specificity of viral sequence data, presently it is more practical for a viral diagnostic test to be CLIA Clinical Laboratory Improvement Amendments of approved than a bacterial NGS assay. Applying NGS to bacterial analysis to make a specific diagnosis can be complicated when performing a metagenomic NGS application, simply because large amounts of commensal bacterial microorganisms can colonize the patient, making determination of the causative organism more difficult.

Furthermore, it has been demonstrated that contamination of laboratory reagents that are used for NGS, such as DNA extraction kits or molecular biology-grade water a common contaminant is Bradyrhizobium sp. In contrast, while certain viruses can occasionally constitute part of the normal microbial flora e. This high detection rate cannot be said for bacteria unless targeted methods such as 16S rRNA gene sequencing are used. The identification of reads corresponding to a virus is likely indicative of ongoing disease.

For example, if influenza virus is detected in a bronchoalveolar lavage BAL fluid specimen of a patient with pneumonia, then the infection was likely caused by this virus. On the other hand, if Staphylococcus aureus is detected in a BAL specimen, it is not guaranteed that S. Despite the advancements made with NGS viral diagnostics, there are challenges that viral genera and other nonbacterial organisms face when subjected to the technology.

Briefly, there are studies being conducted using NGS to detect viral quasispecies with human immunodeficiency virus HIV , Ebola virus, and influenza virus 56 , 79 , 87 , 88 , These small minor variant viral populations can be clinically important but are often challenging to detect, since viral nucleic acid integrity can be compromised during extraction. Furthermore, parasites, yeasts, and fungi are eukaryotic microorganisms that pose bioinformatics challenges.

These organisms possess larger genomes than viruses and bacteria, and their reads can be difficult to distinguish from human reads. More discovery efforts are necessary for parasite, yeast, and fungus NGS in addition to reference databases for these nonbacterial organisms 69 , 91 , 92 , 93 , There are various medically important parasites, yeasts, and fungi that do not have high-quality reference genomes, and therefore NGS applications involving these organisms will have a tough time advancing without more concrete and curated reference databases.

NGS reveals a Coccidioides immitis cluster in three organ transplant patients As the Precision Medicine Initiative moves forward, NGS has the potential to be incorporated into routine clinical microbiology workups to direct diagnostic and therapeutic decision-making that is specific for each patient. Since this colloquium focused mainly on the clinical bacterial applications of NGS, participants identified five main areas that they believe could benefit from the capabilities of NGS.

These include i clinical identification from primary samples or a pure culture, ii infection control actions, iii antimicrobial stewardship, iv outbreak investigation in community and hospital settings to guide measures for containment, and v pathogen discovery 1 , 70 , 95 , 96 , Competition among sequencing vendors has resulted in sustained improvements to NGS platforms from streamlined setup to sequencing chemistry adjustments and user-friendly data analysis Several commercial NGS platforms are currently available in the sequencing marketplace, and newer systems are on the horizon 3 , 7 , 22 , 25 , 33 , 38 Figure 2.

The basic functionalities of these platforms include WGS, whole-exome sequencing WES , metagenomic sequencing, and targeted gene sequencing Table 1 1 , 3 , 31 , The optimal sequencing platform for a particular laboratory is highly dependent on the purpose for which it will be used. In the clinical microbiology laboratory, NGS platforms are primarily used for WGS and metagenomic sequencing, of which the latter task does not require knowledge of the possible causative agent.

Targeted sequencing is also being used in the clinical laboratory if an etiological agent is suspected at the time of clinical sample collection. Regardless of the application, the capital outlay to purchase and maintain NGS equipment is considerable, and smaller hospitals and institutions may not have adequate funding, particularly without a well-established billing infrastructure in place Although these platforms differ in their sequencing chemistries and engineering principles, they all perform massively parallel sequencing which yields terabytes of data This common technological feature involves the sequencing of either spatially separated, clonally amplified DNA or spatially separated, single DNA molecules on a flow cell 1 , 3 , 5 , 8 , 15 , 22 , Hundreds of megabases millions to gigabases billions of DNA sequence data can be produced from a single NGS run, which is in stark contrast to the hundreds of bases produced from an individual sequencing reaction of a targeted region by the Sanger method.

Also, the amount of input DNA is dependent on the NGS platform and the intended application, along with the library preparation protocol used to extract the DNA to be sequenced. All of these factors might affect expected results and data output 1. Newer, single-molecule, real-time sequencing e. There was a paradigm shift in sequencing design when the Roche system previously Life Sciences entered the market in Although the Roche instrument had a higher throughput capacity and a lower sequencing cost per base than the Sanger methodology, interest in this next-generation technology among the scientific community was muted.

Critics of the technology identified read length, fidelity, infrastructure requirements, and cost of operation as the main issues; however, these same concerns were also voiced when Sanger sequencing was introduced. Currently, the Roche is considered a legacy platform and has paved the way for more advanced and higher-throughput sequencing platforms as described below The Illumina platforms employ bridge or cluster PCR to amplify adapter-ligated DNA libraries on the surface of a proprietary flow cell 5 , 8.

Illumina technology uses reversible dye terminator sequencing by synthesis chemistry that involves repetitive cycles of single base incorporation, imaging, and dye chemistry termination Figure 4 5. Sequencing by synthesis is the most widely adopted NGS technology. Illumina sequencers offer the option of paired-end sequencing that enables reads to be generated from both ends of a single clonal fragment. The forward and reverse reads are aligned as read pairs, which allows for more accurate read alignment and superior indel detection than that provided by a single end of the pair.

In recent years, Illumina has dominated the sequencing market for both microorganisms and larger organisms because of the platform's high sequence throughput, low error rate, and low sequencing cost per base. Illumina has a line of machines serving a multitude of purposes and sequencing power on all scales Illumina Genome Analyzer sequencing, the first of the Illumina platforms to be commercially launched.

The SOLiD Sequencing by Oligonucleotide Ligation Detection platforms by Life Technologies, including the W series genetic analysis systems, use a sequencing approach known as oligonucleotide ligation detection. The sequencing by ligation method utilizes dinucleotide encoding, which measures every base twice. Because of a two-base sequencing method, the SOLiD sequencing systems can obtain base call accuracies as high as Although SOLiD sequencing is well suited for high-accuracy applications such as genome resequencing and polymorphism detection, these systems are fading platforms Figure 5 5.

Of all available NGS platforms, the SOLiD sequencing method produces the shortest read lengths 35 to 50 bp and has the longest run times 1 to 2 weeks. Although similar to other platforms in their sequencing by synthesis methodology and amplification by emulsion PCR, Ion Torrent platforms differ from other technologies in the detection step.

Both the PGM and the Ion Proton have flexible reagent chips that generate different scales of sequencing output 10 Mb, Mb, 1 Gb, 10 to Gb depending on the user's desired sequencing coverage The entire workflow, from DNA extraction to annotation, can be completed in less than 24 hours, making the Ion Torrent sequencers most suitable for targeted sequencing or smaller genome sequencing projects Nucleotides are labeled with dye and continuously added to the growing DNA strand by use of a highly processive DNA polymerase.

Attached to this enzyme is a waveguide detector that permits the constant monitoring of the incorporated dye-labeled nucleotides. The sequencing reaction takes place on zero-mode waveguide nanostructure arrays Because of its long reads, PacBio is suited to a variety of applications such as de novo genome assembly and, hence, the characterization of new organisms along with targeted sequencing to detect base modifications Consensus assembly and long reads are providing new reference sequences in regions of genomes which the short-read sequencers fail to uncover, and longer-read PCR sequencing shows promise for phasing alleles and in viral quasispecies.

Additionally, this quantity may not be obtainable when analyzing clinical samples However, it is well suited for the creation of high-quality reference genomes and produces the most accurate and complete genomes of the current NGS platforms The first in a new breed of ultrafast DNA sequencing technologies is Oxford Nanopore , with nanopore sequencing platforms.

A single DNA molecule is guided through a protein nanopore, which results in changes in electrical current across a lipid membrane. Nanopore technology reads native DNA and is predicted to be able to sequence directly from clinical samples with a low abundance of DNA With Nanopore sequencing, DNA sequence is produced in real time, which enables analysis to be conducted on a continuous stream of long reads Since the MinION sequences a single DNA molecule per pore, it eliminates the library amplification phase and its associated biases, which is a common mechanism used by many NGS technologies.

The MinION has a low capital cost, and its flow cells that contain the nanopores are to be used only once, which eliminates the tribulations of installation and instrument maintenance. However, there is a lot of uncertainty around Nanopore technology. As with any emerging technology, it is difficult to separate promises from the true capabilities of the instrument 15 , 22 , There are many sequencing manufacturers that are competing for sequencing supremacy As more companies enter the NGS race, it is probable that instrument purchase costs will decline and democratization of NGS will occur.

Currently, the purchase of NGS instrumentation is a significant financial obligation and commitment 9. It is paramount to assess the needs and demands placed on a laboratory to determine which platform is most suitable and fits within the specified budget If the goal is to incorporate NGS into routine use in the clinical microbiology laboratory, the machine must be able to handle the projected daily volume of isolates and to yield cost-effective and actionable results in a clinically relevant time frame 9 , 10 , 27 , 28 , Even though the technical specifics of NGS technologies differ, there are several common steps that are shared among the majority of high-throughput sequencing methods, with the exception of single-molecule real-time NGS as described below 1 , 5.

The clinical sample, e. Clinical specimen types for NGS evaluation will differ depending on the patient's clinical syndrome but should be collected from appropriate sources during disease progression. The type of transport device, storage time, and temperature can influence the quantity and integrity of the nucleic acids present. Many transport mechanisms are not microbial DNA free, raising the possibility of false positives and the introduction of background contaminants.

Nucleic acids can be prepared from clinical samples by using a variety of methodologies, some of which are dependent on the NGS system being used. Suitable extraction methods are essential for a successful result and thereby help to lessen the introduction of biases and false negatives. Once the input nucleic acid e. These fragments are then ligated to platform-specific oligonucleotide adapters to create a library of overlapping sequences 7.

The library is then hybridized to beads or a flow cell, which is followed by clonal amplification, such as emulsion PCR or bridge amplification. Not all platforms require the clonal amplification phase or preparation of a DNA library 8. Enrichment procedures can also be completed at this stage to help select for a specific type of DNA if an organism is suspected. Depending upon the NGS platform, the clonally amplified templates are sequenced by various chemistries, such as pyrosequencing, reversible dye terminators, oligonucleotide probe ligation, and phospholinked fluorescent nucleotides.

Sequence data are then analyzed to determine the composition of the DNA sequences for pathogen identification. The final components of the NGS workflow are data release and dissemination of a clinically actionable report. Key Finding 1. However, this is not to say that bacterial and viral diagnostic applications do not have challenges. The key issues surrounding viral NGS diagnostics are maintaining viral nucleic acid integrity during the extraction phase and utilizing an extraction method that is capable of isolating quasispecies that may be clinically important.

Fungi, yeasts, and parasites possess large genomes that complicate data analysis, can be confused with human host reads, and may be present in low titers in the clinical specimen, all of which challenge the NGS process. There are limited databases with clinically important fungi, yeast, and parasite species, further causing these NGS applications to fall behind bacterial and viral NGS testing.

NGS has undeniably reinvigorated the DNA sequencing field, yet it faces competition from other molecular and immunological diagnostic tests that are currently on the market. Each technology, including NGS, has its own benefits and pitfalls. Nonetheless, a recurring theme with NGS, unlike with many of the molecular approaches presented in this section, is that prior knowledge of the suspected organism or genome annotation is not required for pathogen identification.

Thereby, pathogen-specific primers are not needed to perform NGS, but high-quality microbial genomic reference sequences are necessary to successfully identify the pathogen 9 , Some of these applications are geared more toward research or human genetic testing laboratories rather than the clinical microbiology laboratory.

Nevertheless, some of the available technologies that are currently competing with NGS are described in more detail below Table 2. The 16S rRNA gene is found in all bacterial genomes and is composed of both highly conserved and divergent regions. The dissimilar regions of the 16S rRNA genes create distinct microbial signatures that allow for molecular identification of bacteria A combination of PCR amplification and sequence analysis of the 16S gene is the accepted reference standard for identifying unknown bacterial species for a single isolate 1 , , NGS, however, is the only method for use in mixed-sample sequencing.

There is no accepted cutoff value of 16S rRNA sequence similarity for species definition. In addition, this method is highly dependent on the sequences provided in a database for correct bacterial identification. If a bacterial species is not listed or is incorrectly labeled in a database, the 16S rRNA sequence may not be able to correctly identify the organism 1.

Similar to 16S rRNA sequencing, the use of microarrays such as Affymetrix tiling arrays also requires knowledge of the query genome or genomic features. Therefore, discovery and metagenomic analyses cannot be performed with microarrays As stated previously, metagenomic sequencing interrogates all DNA present in a sample at one time and does not target specific genes or pathogens Another intrinsic limitation of microarrays is probe cross-hybridization to similar sequences within a genome.

Cross-hybridization is not an issue for NGS, as single-nucleotide resolution can distinguish allelic differences within one nucleotide, provided there is sufficient read coverage Hybridization arrays do not provide the richness of data that NGS can produce.

The dynamic range and analytical sensitivity are scalable for NGS, a feature that is not applicable to microarrays. NGS measures digital sequencing read counts that can be adjusted based on optimal throughput; however, microarrays measure continuous signal, which limits the detection range due to signal saturation and noise.

Even though it is highly probable that NGS platforms will outstrip the applications of microarrays, specific niches in the clinical microbiology laboratory will still be fulfilled by microarrays 10 , MALDI-TOF MS matrix-assisted laser desorption ionization—time of flight mass spectrometry can rapidly identify organisms by comparing proteomic profiles of highly conserved proteins to a database of reference protein profiles Figure 6A.

Species-specific spectral signatures that can be used to identify microorganisms are produced 9 Figure 6B. NGS analysis offers de novo assembly which does not require foreknowledge of a sample's composition but needs very-high-quality sequence data i.

Taken as a whole, MALDI-TOF MS is considered useful for culture-based identification but involves large capital expenses and is limited in its potential to identify organisms from direct clinical specimens and in its capacity to provide information regarding antimicrobial resistance AMR or virulence 7 , General schematic for MS analysis of ionized microbiological isolates and clinical material.

WGS identified a novel genomospecies of Bacteroides Currently, BioFire offers clinical diagnostic detection for more than bacterial, viral, yeast, and parasitic pathogens. BioFire has three FDA-cleared panels for the detection of pathogens causing respiratory, gastrointestinal, or bloodstream infections.

This technology applies multiplex PCR and melting curve analysis to an unprocessed sample, without need for culture. Results are delivered within an hour in a simple, easy-to-read format, but AMR profiles are not included in the data output It may be necessary to perform susceptibility testing on specific isolates if this information is needed for clinical management.

Hence, BioFire does not necessarily free a clinical laboratory from culturing. In comparison to the quick turnaround time offered by BioFire panels, the abovementioned Luminex assays have slightly longer run times of 4 to 6 hours, which consumes an entire shift in the clinical laboratory , Similar to BioFire, Luminex assays are not all-encompassing but use specific primer sets to detect the pathogens represented in the panel.

The respiratory virus panel offered by GenMark has been cleared by the FDA and is able to detect 14 respiratory virus types and subtypes in approximately 4. In contrast to GenMark, Luminex, and BioFire multiplex PCR assays, there is no need to develop specific primers to amplify target sequences nor is there a need to continuously alter the primer design to detect new variants with NGS technology Furthermore, there are diagnostic singleplex PCR and real-time PCR assays that are available with turnaround times of 2 to 3 hours.

These assays detect and amplify only a single target and therefore are extremely limited in utility compared to the multiplexing capabilities of NGS. Additionally, detection of conventional PCR products requires agarose gel electrophoresis, a technique that is laborious and not suitable for high throughput.

Similar to singleplex assays, enzyme-linked immunosorbent assays ELISAs typically target a single organism or category of organisms due to the specificity of the antibody-antigen interaction. With the high-throughput and automated ELISA systems that are now available for the detection of particular pathogens, the turnaround time for a result is relatively quick within 2 hours However, it is likely that NGS assays will initially enter the clinical landscape via a batch processing approach until all issues are addressed for immediate and daily use.

Compared to existing technologies, NGS generates a wealth of data and enables detection of a broader scope of targets 7 , These technologies can be applied to a variety of potential applications. For example, NGS facilitates more precise genotyping and allows for better characterization of organisms discovered in clinical samples.

More recently, it has been found that RNA-seq analyses from NGS data may allow for host profiling in response to a specific type of infection. It is hypothesized that the host immune response may differentiate between various types of infections, such as viral versus bacterial infections.

Determination of an anthrax outbreak among European heroin users by WGS technologies In addition, BioFire technologies can be applied directly to a clinical sample, eliminating the need for isolated and purified microorganisms. Initially, the application of NGS required the isolation of a pure bacterial species from culture in order to deliver key diagnostic information; however, many groups ventured away from this approach and explored the use of NGS for direct detection from clinical samples If feasible, direct clinical specimen sequencing or metagenomic sequencing could theoretically reduce turnaround times from days or weeks to only a few hours, making NGS a procedure that could be completed within an average clinical laboratory workday Figure 7.

Although studies that applied NGS to crude clinical specimens have been published, there are still hurdles, including contaminating normal human microbiota and low-copy-number pathogens that require further evaluation 29 , 83 , 95 , , , Because NGS technologies sequence both viable and nonviable organisms in a sample, more efforts are needed to establish a normal baseline versus contaminants versus infectious agents.

Moreover, the application of NGS to determine a clinical answer does not require knowledge of the infecting pathogen s. NGS can establish a cause of infection and provide a potential answer in cases where other technologies may not provide an actionable finding 21 , 36 , 70 , 71 , 96 , Principals of current processing of bacterial pathogens in the clinical microbiology laboratory.

In principle, NGS data should not only detect the invading pathogen but also predict phenotypic resistance through the examination of genetic determinants of AMR. Traditional antimicrobial susceptibility testing requires an extra day of laboratory workup, extending the turnaround time to 3 or more days.

Genetic information obtained from an NGS approach would ideally prompt rapid antibiotic treatment decision-making for the clinician and the patient. However, NGS is not at the stage where phenotypic susceptibility data can be extracted regularly or reliably from the genotype, although recent works of Pecora et al and Tyson et al are optimistic about this possibility.

Genotypic data do not necessarily correlate to a clinical phenotype, and some types of AMR have nothing to do with genotype, such as intrinsically resistant microorganisms. For example, most Gram-negative rod bacteria are intrinsically resistant to vancomycin because the large-polypeptide antibiotic cannot penetrate the outer membranes of these organisms. Furthermore, detection of antibiotic resistance genes in some organisms, such as mecA in methicillin-resistant Staphylococcus aureus MRSA , is more reliable than resistance gene detection in more challenging organisms, such as Pseudomonas aeruginosa.

Hence, standardized growth-based susceptibility testing and perhaps newer, rapid phenotypic testing methods will likely be necessary to confirm an NGS result in the foreseeable future. A transcriptome-proteome combination could also assist in extrapolating the genotypic and phenotypic connections. Overall, NGS can provide more information than is achievable by other methods. Sequencing run times continue to progressively decrease as technology evolves.

However, compared to existing molecular diagnostic assays that can take minutes to a few hours, current NGS tests are tremendously slow. Although NGS technologies have longer run times, the trade-off is the comprehensive genomic data that are produced. With this barrage of sequencing data comes the challenge of elucidating tangible information that would be desired by the clinician.

Just as there are differences in the volume of genomic data generated, there are also differences in the quality of NGS data that are generated from the various platforms. The same confidence in base calling cannot be applied to all technologies. Performance metrics such as read length, accuracy, and sequence output coverage vary between platforms and dictate the type of applications that can be performed. Laboratories will have different uses for these platforms, and therefore a comparison of the base calling and error rates among the different NGS systems is not feasible or useful.

Because there are systematic biases between NGS platforms, it is difficult to assess the difference in specificity and sensitivity of the platforms in their abilities to detect etiological agents when compared to the detection abilities of other DNA technologies.

With each successive generation of sequencing platforms, performance continues to improve along with overall sensitivity. Better-targeted enrichment procedures are needed, and elimination of contaminating host DNA is critical for extracting higher-quality DNA. In general, there is a higher error rate associated with NGS than with traditional Sanger sequencing, mainly due to ambiguity in short read sequence alignment, resulting in inadequate coverage 14 , If you have this book go ahead and post it here and your listing will appear for all students at your school who have classes requiring this specific book.

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