Table 2.

Overview of the three PCR batches, detailing the samples and experimental settings

Assay number	Sample	Template amount	Cycles	Primer F	Primer R	Inhibitor
1	Negative control	up to VF	25	341F (0.5 μM)	805R (0.5 μM)	none
1	Faeces DNA (positive control)	12.5 ng	25	341F (0.5 μM)	805R (0.5 μM)	none
1	Human DNA	12.5 ng	25	341F (0.5 μM)	805R (0.5 μM)	none
2	Negative	up to VF	35	341F (0.5 μM)	805R (0.5 μM)	none
2	Faeces DNA (positive control)	12.5 ng	35	341F (0.5 μM)	805R (0.5 μM)	none
2	Faeces DNA	12.5 ng	35	5 cons (0.5 μM)	805R (0.5 μM)	none
2	Human DNA	50 ng	35	5 cons (0.5 μM)	805R (0.5 μM)	none
2	Human DNA	50 ng	35	341F (0.5 μM)	805R (0.5 μM)	none
2	Human DNA	50 ng	35	341F (0.5 μM)	805R (0.5 μM)	none
2	Human DNA	50 ng	35	341F (0.5 μM)	805R (0.5 μM)	none
2	Human DNA	50 ng	35	341F (0.5 μM)	805R (0.5 μM)	0.25 μM
2	Human DNA	50 ng	35	341F (0.5 μM)	805R (0.5 μM)	0.5 μM
3	Human DNA	50 ng	35	341F (0.5 μM)	805R (0.5 μM)	0.25 μM
3	Human DNA	50 ng	35	341F (0.5 μM)	805R (0.5 μM)	0.5 μM
3	Human DNA	50 ng	35	341F (0.5 μM)	805R (0.5 μM)	1 μM

Note: Concentrations are reported in molarity (M). The term ‘5’ cons’ refers to the oligonucleotide designed based on the off-target 5′ consensus and used as a forward primer, while ‘inhibitor’ refers to the same oligonucleotide modified with a 3’ C3-spacer.