INFECTION DIAGNOSTICS :: MICROBIOLOGY


The clinical value of next-generation sequencing integration within medical laboratories


By Stephen Vella Ph.D. I


nfectious diseases (ID) remain at the forefront as the leading cause of morbid- ity and mortality globally. The intrinsic ability of ID to spread quickly, stealthily to surmount the immune system, and rapidly evolve through beneficial mutations con- ferred through natural selection is made no more evident through the global dis- ruption caused by COVID19.1,2


Fred Sanger developed the first platform of DNA sequencing that was rapidly and significantly utilized for decades in research and clinical genetics.3


Later in 1983, Kary


Mullis invented the polymerase chain reaction (PCR). These two technologies served as the fundamental foundation of modern day microbial/molecular diagnos- tics, commonly referred to as nucleic acid amplification tests (NAATs).2


In 1977,


of NGS has been exemplified, not only at the individual patient level, but as well as, NGS has been utilized to help govern and direct public health and (hospital) infection control strategies. For example, NGS contributed (and still contributes) to the discovery and tracking of SARS-COV2 variants including alpha, delta, omicron, and possible future variants throughout the course of the ongoing global pandem- ic and currently, Public Health England routinely employs whole-genome NGS to track spread of antimicrobial resistance of M. tuberculosis.7,8 Given these developments, the U.S.


Even with the


advent of NAATs, traditional methodolo- gies such as culture, strain identification, antigen, and antibody detection remain a key component of laboratory diagnostics.2,4,5 Sanger sequencing commonly utilizes


clonal amplification of adaptor-ligated DNA fragments across the surface of a glass flow cell, yet it is limited in terms of low throughput and complexity.6


Major


improvements and advancements in molecular biology that were transitively incorporated into sequencing technolo- gies led to the development of second and third generation sequencing methodolo- gies, commonly termed next-generation sequencing (NGS). Such innovations led to the milestone achievement of comple- tion of the human genome project.1,3,6 Nowadays, sequencing turnaround time and cost have dramatically reduced, as well as have become more automated and compact since the early 2000s, thus enabling easier adoption and more prac- tical widespread utilization within the clinician setting and beyond.2,3 An immense amount of curated clinical,


genetic, and genomic data has emerged through NGS, helping foster the develop- ment of more precision based medicine, laboratory diagnostics, and clinical treat- ment.2,3


In addition to microorganism


identification, NGS has been utilized for detection of antibiotic resistance, single nucleotide polymorphism (SNPs), and the host immune response.3


The clinical value 18 JUNE 2022 MLO-ONLINE.COM


Food and Drug Administration (FDA) has outlined the guidelines for designing, developing, and validation of approved NGS tests.3


Generally speaking, both


second and third generation sequenc- ing technologies share nearly identical three step workflows: (1) preparation and extraction of nucleic acid template; (2) preparation of library including clonal amplification; and (3) sequencing and alignment of short reads.3


Science and methodology of NGS within the laboratory: Generally speaking, NGS can be divided into the Sequencing and Data analysis phase (Fig 1A). With regards to the clinical lab, NGS possesses several steps and variables that must be taken under considerations if a clinician or labora- tory manager desires to implement NGS within its clinician pipeline, the details of which are outlined in Fig 1B. Sample Collection and Preprocessing: As with any diagnostic assay, optimal specimen collection with sufficient volume is fundamental to obtain meaningful se- quencing results. The DNA of the intended target needs to be at a sufficient threshold for detection. Thus, the timing of specimen collection serves as an important factor and needs to be taken under significant consideration. For example, if samples are obtained from a patient that is undergoing or about to receive antibiotics, this treat- ment may adversely impact the levels of DNA needed for quality results.5,9 Nucleic Acid Extraction: Similar to nearly all NAAT based assays, the first


step is nucleic acid extraction from the specimen. Due to the enhanced sensi- tivity and capability of detecting DNA or RNA from any organism, precaution needs to be taken to limit the risk of contamination of the extraction reagents. For example, the commensal flora of laboratory personnel can contaminate laboratory reagents and risk leading to inappropriate patient diagnoses.5 Library Preparation and Clonal Ampli-


fication: Post extraction of sample nucleic acid (whether it be DNA or RNA), the specimen is further processed in order to ensure compatibility and optimiza- tion for high-throughput sequence analysis. Library preparation is a delicate process comprising several steps that seek to preserve or enrich the pathogen sequences present within the sample, while maintaining the complex, native diversity, that is intrinsic to the sample. Depending on the type and target of the NGS assay (targeted, whole genome, or metagenomic, discussed later), pathogen genetic material can be selectively en- riched using differential lysis, DNase or RNAse, mitochondrial and/or ribosomal RNA depletion, or whole genome hybrid- ization. However, most clinical labora- tories will likely employ an unbiased strategy utilizing total nucleic acid to more broadly identify for the presence of pathogen DNA. If a more targeted or refined approach is desirable, com- monly, spiked targeted primers specific to conserved regions of either bacteria (16S rRNA), fungal (internal transcribed spacer region) or to different clades of viral targets will be added.3,5,10 The final step required for creation of the library is the addition of sample bar- codes and sequencing adaptors, using standard, common techniques. Sample barcodes are short DNA sequences ligated to the ends of each sample library that allows for the pooling of multiple samples for sequencing analysis and sample iden- tity using bioinformatics. Sequencing adaptors are specialized and specific oli- gonucleotide adaptors tailored to a given sequencing platform and are commonly added through either adapter ligation or transposase-mediated addition.5


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