Table 3.

Thresholds for identification pathogen by mNGS in the diagnosis of lower respiratory tract infections.

Study	Disease	Sample types	Platform	Thresholds for identification pathogen by mNGS
Zhang Y. et al. [81]	Pneumocystis pneumonia	Sputum, blood, lung tissue and BALF	NA	•Pneumocystis jirovecii ’s specific reads ranking among top15 or its relative reads proportion in fungi higher than 85%
Wang J. et al. [7]	Mixed pulmonary infection	BALF and lung tissue	NA	The infectious pathogen was determined if it met any of the following thresholds:
				•culture and/or histopathological examination positive of bacteria, virus or fungi, IPA was defined using galactomannan antigen and PCR;
				•at least 50 unique reads from a single species of bacteria, virus or fungi; for pathogen with unique reads less than 50, it still can be diagnosed as infectious pathogen with the consistent clinical situation;
				•at least one unique read from MTBC.
Li H. et al.[5]	Pulmonary infection	Lung tissues	BGISEQ-500	The infectious pathogens were determined if it met any of the following thresholds:
				•30% relative abundance at the genus level in bacteria or fungi
				•culture and/or histopathological examination positive and at least 50 unique reads from a single species of bacteria or fungi
				•at least one unique read from MTBC
Huang J. et al. [54]	Peripheral pulmonary infection	Lung tissue, BALF, and protected-specimen brush.	BGISEQ-100	The criteria for a positive mNGS test result included:
				•the relative abundance of bacteria (Mycobacterium tuberculosis excluded) and fungi was >30% at the genus level;
				•Mycobacterium tuberculosis was considered to be positively detected if at least one read was aligned to the reference genome at species or genus level;
				•positive virus detection was considered when the SMRN was no less than 3;
				•when the pathogen was detected by traditional pathogen detection methods and the mNGS reads number was >50, this pathogen can also be considered as positively detected by mNGS.
Langelier C. et al. [17]	LRTIs	BALF	illumina	Microbes identified were classified as confirmed pathogens if
				•both clinical testing and mNGS identified the microbe;
				•there existed literature evidence of pathogenicity in the lungs;
				•a developed Z-score was as least twofold greater than that of any other microbe of the same type (virus, bacteria, or fungus) identified in the patient.
				•Microbes were considered new potential pathogens if mNGS alone identified the microbe and criteria 2 and 3 described here were met;
				•all other microbes were considered unlikely or uncertain pathogens.
Charalampous, T. et al. [3]	Bacterial lower respiratory infection	Sputum, BALF and ETAs	MinION	•≥1% of classified reads, with a WIMP assignment q-score ≥20 (within .csv files).
Wang, H. et al. [55]	Severe nonresponding pneumonia	BALF	BGISEQ-100	•Bacterial/mycoplasma/chlamydia: SDSMRNG ≥ 3, if SDSMRN ≥ 3, species was reported; otherwise, the genus was reported
				•DNA Virus/fungus: SDSMRN ≥ 3
				•RNA Virus: SDSMRN ≥ 1
				•Parasite: SDSMRN ≥ 100
				•MTC: SDSMRNG ≥ 1

Abbreviations: BALF, broncho-alveolar lavage fluid; ETA, endotracheal tube aspirate; IPA, invasive pulmonary aspergillosis; MTBC, Mycobacterium tuberculosis complex; MTC, Mycobacterium tuberculosis complex; NA, no accessible; SDSMRN, the number of reads stringently mapped to pathogen species; SMRN, stringent map read number; WIMP, ‘What’s In My Pot?’ pipeline.