Next-generation sequencing (NGS) technology has become an effective instrument for the discovery, recognition, and study of human pathogens. There are several benefits over traditional approaches, as sequences generated can be used to diagnose and characterize pathogens more reliably, test for the occurrence of resistant mutations/genes, forms of vaccine escape, recombination or reassortment, and variables of virulence and pathogenicity. This has enabled NGS and bioinformatics a more practical and increasingly acceptable aspect of scientific and public health laboratories around the world, combined with increases in sequencing error rates and simplified laboratory methods, and the declining costs of NGS and computational requirements. Furthermore, the introduction of NGS and bioinformatics techniques as routine testing and tracking methods include advanced information infrastructure and quality control programs that are capable of meeting public health laboratory objectives.
There are a number of applications within NGS to address, in addition to the range of sequencing platforms on the market. Pathogen enrichment or host depletion protocols should be addressed in cases of alleged low pathogen concentration or presence of pathogens in samples with a high host nucleic acid content.
Through the study of sequenced genomes and/or transcripts from an environmental sample, Metagenomics is the analysis of an entire population of species and usually results in the identification of organisms from all areas of existence. This technique is typically undertaken in surveillance and diagnostics where other more-directed assays such as polymerase chain reaction (PCR) fail. Due to the advent of a novel pathogen, genetic evolution of an existing pathogen, or poor design of the assay, these assays could fail. Blood, stool, cerebrospinal fluid (CSF), semen, or nasopharyngeal swabs have become the most widely used materials for metagenomic sequencing in pathogen exploration, where researchers have sought to classify the etiological agent responsible for an outbreak or other clinical syndrome. Generally speaking, where at least 1 of the following requirements is met, metagenomic sequencing is most beneficial and cost-effective for pathogen discovery: (1) the detection of the organism is not adequate (one needs to go beyond discovery to generate genomic characterization data), (2) coinfection is assumed, (3) other simplified tests are unsuccessful or take an unnecessary amount of time, and/or (4) the purpose is to scan environmental samples for recently undiscovered or divergent pathogens.
Numerous target enrichment techniques have been developed to improve the chance of collecting pathogen-derived transcripts and/or genomes because of elevated levels of background noise in metagenomic sequencing. To pick an effective enrichment strategy and amplify the sequence of interest, previous knowledge of the pathogenic genomic history can be used. In general, there are two major methods that can be used to maximize the volume of a sample's pathogen signal: negative sorting and positive enrichment.
Negative selection (background depletion or subtraction) targets and removes the genetic history of the host and microbiota while striving to retain the nucleic acid extracted from the pathogens of concern.
Positive enrichment, which is used to improve the pathogen signal rather than suppress background noise, is a simplified strategy that results in less loss of target. This is usually achieved through hybridization-based target capture by probes, which are used for downstream amplification and sequencing to take out nucleic acid of interest.