The transformation of pathogen diagnostics through next generation sequencing

July 20, 2016

Clinical laboratories have multiple methods for diagnosing infectious disease, but the fact remains that despite culture, microscopy, antibody-based testing, and molecular testing such as PCR, up to 75 percent of infections are not diagnosed in a timely manner, if at all.1,2 One of the biggest issues is that a target is needed to know which test(s) can lead to an accurate diagnosis; the laboratorian must know what pathogen is suspected. And since many pathogens exhibit common symptoms, it is often difficult to determine an appropriate target.

The diagnostic challenges have resulted in a reliance on differential diagnosis and can result in spread of infectious diseases, longer hospital stays, more critical illnesses, longer recoveries, and compromised outcomes related to delayed or ineffective treatment. In addition, partly due to ineffective diagnostics, antibiotics are significantly overused in the United States. Such misuse of antibiotics is creating drug-resistant pathogens. According to the Centers for Disease Control  and Prevention (CDC), “Each year in the United States, at least two million people become infected with bacteria that are resistant to antibiotics, and at least 23,000 people die each year as a direct result of these infections.”3

In the midst of these concerns is the reality that infectious disease outbreaks are no longer “local.” As large populations travel internationally, local authorities are often under-resourced in coping with identified outbreaks, and inadequate diagnostics and treatments leave us all more vulnerable. News headlines alert us to the Lyme disease season and to the risk of the Zika virus, while not so long ago people in Africa were dying by the thousands from Ebola. The confluence of all these factors makes it more important than ever to develop accurate and timely diagnostics for infectious diseases.

NGS: consider the possibilities

Next generation sequencing (NGS) has been effectively implemented in the diagnosis of cancers and genetic disorders, including neonatal testing, but it is much easier to evaluate the human genome alone as opposed to more than 19,000 known pathogen genomes. Now a significant advancement in NGS is capable of yielding the most accurate and comprehensive pathogen diagnoses of any method available. In theory, it is possible to diagnose all possible pathogens utilizing the power of NGS.

From a single sample, it is possible to simultaneously screen any clinical sample for all pathogens (whether bacteria, virus, fungus or parasite). The sample can be tissue, blood, swab, stool, or any sample that contains the pathogen being sought. This method not only can identify known pathogens, but is also capable of revealing the presence of unknown pathogens. In this new application of NGS, unbiased genetic sequencing provides a “genetic blueprint” from all detectable microorganisms in the sample. Sophisticated bioinformatics can make sense of the raw data by matching it against all sequenced pathogen genomes to identify the pathogens present in the sample. This system can detect the presence of multiple pathogens.

Other applications of NGS include pathogen surveillance (such as in animal populations, water supplies, or food processing) and can discover previously unrecognized or never-before seen pathogens. Since all genetic information from the sample is sequenced, it is not necessary to know which microbes are suspected; a target is not necessary. Moreover, whole genome/ whole transcriptome sequencing methods can provide higher sensitivity and specificity than other diagnostic technologies in use today. NGS also has the ability to identify drug-resistance genes in identified pathogens, study the interaction between host and pathogens, and characterize the host’s immune response through gene regulation exploration.

NGS: be aware of the challenges

Many experts see sequencing as the future for clinical laboratory diagnostics, but while this breakthrough technology holds exciting potential, there are some important cautions to consider before adopting NGS for pathogen diagnosis. Current challenges for broad implementation of NGS for pathogens include turnaround time (TAT) and costs—not inconsiderable real-world obstacles. The outlook is improving considerably, however. The cost for sequencing a single human genome has dropped from ~$14 million in 2006 to ~$1,500 in 2016 (it should be noted that the fabled “$1000 Genome” requires substantial instrumentation and human capital, which is not attainable by most clinical laboratories). And, while TAT is currently slowed by the time required for sample preparation and sequencing, advanced protocols for sample preparation are being introduced that can reduce it, as well as the cost of sequencing. One example of such improvements is the use of barcoding to allow multiplexing of up to 96 samples in a single sequencing run.

An additional challenge is in collecting samples that include the pathogen being sought. Sample collection and stabilization is crucial for identification of pathogens; the pathogen can only be identified if it is present in the sample. Samples must be extracted and processed with robust protocols that should be conducted under appropriate quality guidelines. Further, when conducting whole genome sequencing from human-derived samples (particularly tissue and blood), it can be challenging to obtain adequate pathogen DNA/RNA due to the overabundance of host genetic material. Host cell depletion methods can be utilized, but concentrations of the remaining pathogens are often small, and it is challenging to obtain adequate sensitivity levels. Because it is crucial to obtain adequate sequencing coverage of the pathogens to ensure the best sensitivity and specificity, molecular biologists trained in NGS should be consulted with respect to sample collection, sample extraction, library preparation methods, and sequencing parameters.

Another issue: while clinical laboratories are often comfortable with the wet lab techniques of extraction, library prep, and/or whole genome sequencing, it is just as important to recognize the critical role of data analysis of the complex data sets generated by NGS. Data analysis for pathogen detection is a highly complex process, as dozens or even hundreds of microorganisms must be analyzed in a single test. Further, pathogenic versus non-pathogenic determinations must be made, as all clinical samples will contain many non-pathogenic microorganisms. A few data analysis tools for pathogen detection have become available, but they have not yet been validated for clinical use. Further challenges for pathogen NGS data analysis involve the generation, safety, storage, transport, computation, and archiving of these massive data files (up to 2 terabytes).

Looking ahead

While these challenges may seem intimidating, engaging strong strategic partners with expertise in NGS and complex data analysis can make adoption of NGS pathogen diagnostics achievable today. The best strategic partners will be able to provide recommendations for sample collection, extraction, and preparation, as well as be able to handle the complex data analysis phase of pathogen identification. Balancing the complexities, along with engaging experts, will facilitate a complete transformation of pathogen diagnostics through NGS. Today is an exciting time for clinical laboratories as this important technology is being embraced. Imagine a world where a single clinical sample is able to yield all of the data needed for pathogen identification, pathogen drug response, and host-pathogen immune response…this world is quickly approaching!


  1. Lyme Disease Association, Inc. National Institutes of Health study on Lyme disease reveals significant chronic symptoms and common misdiagnosis. 2005.
  2. Tomas MY, Getman D, Donskey CJ, Hecker MT. Over-diagnosis of urinary tract infection and under-diagnosis of sexually transmitted infection in adult women presenting to an emergency department. J Clin Microbiol. 2015.
  3. Centers for Disease Control and Prevention. Antibiotic resistance threats in the United States, 2013. Executive summary.

Crystal Icenhour, PhD, is the founding Chief Executive Officer of Aperiomics, a Northern Virginia company currently offering the Absolute*NGSSM  Pathogen Detection suite of products.