INFECTION DIAGNOSTICS :: MICROBIOLOGY
Figure 2: Types of Next Generation Sequencing. The 3 types of Next Generation Sequencing are outlined above and within the text of the manuscript. Figure created from BioRender with Publication License
around time and no GC bias, yet has the disadvantage of nanopore technology is its high sequencing error rate.3 Bioinformatic Data Analysis: Bioinfor- matic sequencing data analysis involves a multistep well-established pipeline as a means to identify any pathogen sequences present in the sample. Generally speak- ing, the sequential major steps are quality filtering, human subtraction, alignment to a (pathogen) database, taxonomic char- acterization, and genome mapping. The confidence of the sample is proportional to the number of sequence reads identi- fied for the organism, normalized to the total number of reads present within the sample, and the overall genome coverage. Optional quantitative controls enable for the determination of the number of mol- ecules per milliliter of organism DNA in the original sample to be determined.5
The
direct clinical application of NGS to detect infectious agents is contingent on the avail- ability of a curated databases to provide a high level of confidence of matched reads against the organisms identified. For example, organism types may not be present in the database thus hampering their detection. Though nucleotide align-
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ment is the most commonly employed analysis strategy, amino acid /protein can be utilized to identify possible divergent organisms.5 NCBI possess a vast amount of curated and uncurated databases that are ever expanding. For example, more than 376,000 bacterial genomes are currently available.5,6 In order to offset the magnitude of data
achieved from NGS a number of software platforms have been developed, a sig- nificant investment cost that could likely hinder more universal acceptance within the clinical lab.11
A number of software
platforms, both commercially available (bioMérieux Episeq, Illumina, Bio-Rad’s SeqSense, Qiagen’s OmicSoft Suite) and open source software suites, are readily available. Episeq, designed developed by bioMérieux; as well as, several open source platforms provide cloud-based computing thus offering an attractive alternative to limited in-house analysis.11
Clinical utility and interpretation of the report analysis of NGS: Similar to all diagnostic assays, the clinical, real-world utility of NGS testing is depen- dent on a number of critical factors to con-
sider: (1) the patient presentation, symptom severity, and timing of sample collection; (2) the sample quality, location, and infection source type; and (3) the native operational characteristics of the assay, including but not limited to analytical sensitivity, specific- ity, and detection range.5 Post analyzation, a results report is gen- erated with clinically relevant information including the organism(s) identified with associated relevant sequencing metrics and comments for potentially clinically significant results. For example, detec- tion of contamination of endogenous or environmental flora or unusual or highly pathogenic organisms will likely be flagged with comments.5 The major clinical application within mi- crobiology laboratories are: whole genome sequencing, metagenomic NGS (mNGS), and targeted NGS (tNGS) (Fig 2).3,10
Whole-
genome sequencing involves sequencing and assembly of an entire template within a clinical sample, enabling simultaneous typing of any microorganism or virus genome; and in some cases, identifying resistance gene/mutations/prediction of antimicrobial susceptibility of a given strain.2
Generally speaking, a pure sample
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