allows to characterize a lot of infections, for example infections due to staphylococcus aureus, lyme disease, candidiasis, tuberculosis and influenza. also, this approach can be used for cancer classification. rnaseq analysis have a lot of other purposes and applications such as to identify novel or under appreciated host–microbial interactions directly from clinical samples, to make indirect diagnosis on the basis of a pathogen specific human host response and to discriminate infectious versus noninfectious causes of acute illness. challenges clinical utility even though, most of the metagenomics outcomes data generated consist of case reports which belie the increasing interest on diagnostic metagenomics. accordingly, there is an overall lack of penetration of this approach into the clinical microbiology laboratory, as making a diagnosis with metagenomics is still basically only useful in the context of case report but not for a true daily diagnostic purpose. as of 2018, cost-effectiveness modelling of metagenomics in the diagnosis of fever of unknown origin concluded that, even after limiting the cost of diagnostic metagenomics to $100 – 1000 per test, it would require 2.5-4 times the diagnostic yield of computed tomography of the abdomen and pelvis in order to be cost neutral and cautioned against ‘widespread rush’ to deploy metagenomic testing. furthermore, in the case of the discovery of potential novel infectious agents, usually only the positive results are published even though the vast majority of sequenced cases are negative, thus resulting in very biased information. besides, most of the discovery work based in metagenomic that precedes the diagnostic-based work even mentioned the known agents detected while screening unsolved cases for completely novel causes. laboratory validity to date, most published testing has been run in an unvalidated, unreportable manner. the ‘standard microbiological testing’ that samples are subjected to prior to metagenomics is variable and has not included reverse transcription-polymerase chain reaction (rt-pcr) testing for common respiratory viruses or, routinely 16s/its pcr testing. given the relative costs of validating and performing metagenomic versus 16s/its pcr testing, the second one is considered an easier and more efficient option. a potential exception to the 16s/its testing is blood, given the huge amount of 16s sequence available, making clean cutoffs for diagnostic purposes problematic. furthermore, almost all of the organisms detected by metagenomics for which there is an associated treatment and thus would be truly actionable are also detectable by 16s/its testing (or 16s/its-ngs). this makes questionable the utility of metagenomics in many diagnostic cases. one of the main points to accomplish laboratory validity is the presence of reference standards and controls when performing mngs assays. they are needed to ensure the quality and stability of this technique over time. sense and sensitivity in clinical microbiology labs, the quantitation of microbial burden is considered a routine function as it is associated with the severity and progression of the disease. to achieve a good quantitation a high sensitivity of the technique is needed. whereas interfering substances represent a common problem to clinical chemistry or to pcr diagnostics, the degree of interference from host (for example, in tissue biopsies) or nonpathogen nucleic acids (for example, in stool) in metagenomics is a new twist. in addition, due to the relative size of the human genome in comparison with microbial genomes the interference can occur at low levels of contaminating material. another challenge for clinical metagenomics in regards to sensitivity is the diagnosis of coinfections where there are present high-titer pathogens that can generate biased results as they may disproportionately soak up reads and make difficult to distinguish the less predominant pathogens. in addition to issues with interfering substances, specially in the diagnosis area, accurate quantitation and sensitivities are essential as a confusion in the results can affect to a third person, the patient. for these reason, practitioners currently have to be keenly aware of the index-swapping issues associated with illumina sequencing which can lead to trace incorrectly barcoded samples. since metagenomics has typically been used on patients for whom every other test to date has been negative, questions surrounding analytical sensitivity haven been less germane. but, for ruling out infections causes being one of the more important roles for clinical metagenomics it is essential to be capable to perform a deep enough sequencing to achieve adequate sensitivities. one way could be developing novel library preparation techniques. cost considerations as of 2018, the illumina monopoly on high-quality next-generation sequencing reagents has meant that the sequencing reagents alone cost more than fda-approved syndromic testing panels. also additional direct costs of metagenomics such as extraction, library preparation, and computational analysis have to be considered. in general, metagenomic sequencing is most useful and cost efficient for pathogen discovery when at least one of the following criteria are met: the identification of the organism is not sufficient (one desires to go beyond discovery to produce data for genomic characterization), a coinfection is suspected, other simpler assays are ineffective or will take an inordinate amount of time, screening of environmental samples for previously undescribed or divergent pathogens.