Table 3.

Key method steps and categorization of the SOPs

Method group	SOP	Sample volume (mL)	Pre-treatment			Concentration step	Extraction	Molecular analysise	Concentration factor
			Pasteurization	Solids removal	Chemical addition
1	1.1	0.25	No	Nonea	None or RNA shield	None	Zymo Quick-RNA Fecal/Soil Microbe Microprep kit	Q	17
	1.2(H)	40	Half the samples				PureYield Plasmid Midiprep system	Q	500
	1.3	45	No				Qiagen RNeasy PowerSoil Total RNA kit	Q	450
1S	1S.1(H)	40	Half the samples	Yes (e.g., removal by centrifugation, filtration, or both)	None or salt addition (e.g., NaCl) prior to solids removal		PureYield Plasmid Midiprep system	Q	500
	1S.2H	40	All samples				Zymo III-P silica column	Q	200
	1S.3(H)	2	Half the samples				Qiagen QIAamp Viral RNA mini kit	D	5
2	2.1	30	No	Noneb	Beef extract or phosphate buffered saline (PBS)		Zymo Quick-DNA/RNA Viral kit	Q	60
	2.2	30	No				Zymo Quick-DNA/RNA Viral kit	Q	60
	2.3	225	No				Qiagen AllPrep PowerViral kit and Qiagen RNeasy PowerWater kit	D	1800
2S	2S.1	50	No	Yes (e.g., removal by centrifugation, filtration, or both)	None	Ultrafiltrationc	Qiagen RNeasy mini kit	Q	40–200
	2S.2	105	No				IDEXX Water DNA/RNA Magnetic Bead kit	Q	380–980
	2S.3	150	No				Invitrogen PureLink Viral RNA/DNA mini kit	Q	220–630
	2S.4(H)	50	Half the samples				Agilent Absolutely RNA Miniprep kit	Q	500
	2S.5	25	No				TRIzol	Q	63–280
	2S.6	30	No				Zymo Quick-RNA Miniprep kit	Q	16–18
3	3.1	50	No	None	Acid (HCl) to lower pH and (optionally) addition of salt (e.g., MgCl2)	HA filtrationd	NUCLISENS easyMAG	D	250
	3.2	100	No				Qiagen QIAamp Viral RNA kit	Q	880–2100
	3.3	50	No				Qiagen AllPrep PowerViral DNA/RNA kit	Q	280–470
	3.4	25	No				Qiagen RNeasy PowerMicrobiome kit using PowerBead tubes	D	420
	3.5	40	No				Qiagen RNeasy PowerMicrobiome kit using BashingBead tubes	Q	40–200
	3.6	30	No				Applied Biosystems MagMAX Viral/Pathogen Nucleic Acid Isolation kit	D	200–230
3S	3S.1	200	No	Yes (e.g., removal by centrifugation, filtration, or both)	Acid (HCl) to lower pH after solids removal		Qiagen AllPrep PowerViral DNA/RNA kit	Q	2000
	3S.2H	100	All samples				Phenol extraction	Q	380–1300
	3S.3H	50	All samples				Phenol extraction	Q	160–510
4	4.1	100	No	None	Salt (NaCl) and PEG		Qiagen QIAamp Viral RNA kit	D	60–96
	4.2	100	No				Qiagen QIAamp Viral RNA kit	D	53
	4.3	100	No				Qiagen QIAamp Viral RNA kit	D	55–83
	4.4	282	No				Qiagen QIAamp Viral RNA kit	Q	220
4S	4S.1(H)	40	Half the samples	Yes (e.g., removal by centrifugation, filtration, or both)	Salt (NaCl) and PEG after solids removal	PEG precipitation	TRIzol	Q	850–1300
	4S.2(H)	105	Half the samples				IDEXX Water DNA/RNA Magnetic Bead kit	Q	530
	4S.3	45	No				Qiagen RNeasy PowerMicrobiome kit using PowerBead tubes	D	130
	4S.4	36	No				Qiagen QIAamp Viral RNA kit	Q	590
	4S.5H	40	All samples				Qiagen AllPrep PowerViral DNA/RNA kit	Q	670
	4S.6(H)	200	Half the samples				NucleoMag Pathogen RNA Isolation kit	Q	170
	4S.7	40	No				Invitrogen PureLink Viral RNA/DNA mini kit	Q	34–170
	4S.8(H)	400	Half the samples				Qiagen QIAamp Viral RNA kit	D	470

^aSOP 1.3 centrifuged sample and analyzed solids.

^bSOP 2.3 separated solids and analyzed both solid and liquid fractions.

^cSOP 2S.5 used a concentrating pipette tip in the concentration step (similar principle to ultrafilter).

^dSOP 3S.1 filters the sample through an electropositive filter to remove solids and then elutes the viruses adsorbed to the filter with beef extract. The eluant is further concentrated with organic flocculation and ultrafiltration before extraction.

^e“Q” indicates reverse transcription quantitative PCR and “D” indicates reverse transcription digital PCR.