TABLE 2.

Effect of specimen preprocessing on the analytical sensitivity of pathogen detection by NGS

Organism	Samples	PCR result (CT)		NGS result (No. of reads mapped to pathogen genome)			NGS result (% genome coverage)
		Unprocessed	Processed	Unprocessed	Processeda	Enrichment factor (processed/unprocessed)	Unprocessed	Processedb
Spiked pathogens
S. pneumoniae	NPW1	29.5	30.8	397	3,417	8.6	2.20	13.22
	CSF1	31.9	33.2	362	1,059	2.9	1.57	3.18
	NPW2	35.9	36.4	6c	2,086	347.7	0.08	9.76
E. coli	CSF1	29.2	29.9	57,204	275,178	4.8	65.14	93.19
	CSF2	34.6	34.3	7,024	46,010	6.6	6.89	37.37
	CSF3	35.0	35.4	10,834	37,064	3.4	10.18	30.47
H. influenzae	CSF1	27.7	27.8	163,923	767,109	4.7	92.44	93.78
	NPW2	35	35	41	5,712	139.3	0.41	26.60
	CSF2	Undetermined	Undetermined	8,963	41,250	4.6	2.55	5.26
	CSF3	Undetermined	Undetermined	10,450	29,216	2.8	2.78	4.16
N. meningitidis	CSF2	23.7	24.2	840,062	4,895,057	5.8	95.63	97.60
	CSF3	24.3	25.1	1,283,102	3,692,081	2.9	96.54	97.36
S. agalactiae	CSF2	30.2	32.6	22,809	39,962	1.8	65.39	78.98
	CSF3	31.3	33.9	26,498	14,454	0.5	62.82	45.31
B. pertussis	NPW2	23.7	24.5	31,862	4,248,717	133.3	67.17	100.00
Human adenovirus	NPW1	17.3	17.6	31,927	782,835	24.5	99.96	100.00
	CSF1	21.0	21	626,057	3,055,828	4.9	99.57	99.69
	NPW2	22.2	23.7	8,243	729,057	88.4	98.83	99.52
Herpes simplex virus 2	CSF2	22.3	23.3	260,048	991,123	3.8	98.55	99.18
	CSF3	23.2	24.3	343,310	798,024	2.3	98.76	99.18
	NPW1	23.3	24.8	4,493	68,752	15.3	82.88	97.32
	CSF1	24.6	26.6	51,468	118,020	2.3	95.26	97.60
Unspiked pathogens/organisms detected in NPA specimens
Human adenovirus	NPW3	21.9	22.5	11,330	242,141	21.4	99.98	99.98
M. catarrhalis	NPW1			599	904	1.5	3.47	4.32
H. influenzae	NPW1			118	1025	8.7	0.91	5.59
S. mitis	NPW1			76	220	2.9	0.51	1.37
S. sanguinis	NPW1			10	271	27.1	0.08	1.21
S. constellatus	NPW2			7	796	113.7	0.1	4.87
A. xylosoxidans	NPW2			56,046	1,679,351	30.0	13.5	31.08
Streptococcus intermedius	NPW2			18	3,134	174.1	0.18	16.77
S. epidermidis	NPW2			43	1,278	29.7	0.49	5.82
C. aurimucosum	NPW2			258	46,713	181.1	1.68	11.44
C. diphtheriae	NPW2			117	37,642	321.7	0.02	6.16
F. nucleatum	NPW2			4	1,030	257.5	1.48	8.84

^aSignificantly different from unprocessed specimens (P < 0.01).

^bSignificantly different from unprocessed specimens (P < 0.001).

^cBold indicates spiked pathogen undetectable in unprocessed samples but detected in processed samples.