Table 1.

List of modified Illumina V3-V4 primers and resulting read counts from the second feeding study 16S rRNA gene survey.

	Sample ID	Forward primer	Reverse primer	PCR amplification	Demultiplexed read count
Pooled sample 1	Wood1	F1	R1	−	−
	Wood2	F2	R1	−	−
	XTWa	F3	R1	+	58,488
	WTW	F1	R2	+	81,646
	2WF	F2	R2	−	−
	2WM	F3	R2	+	44,379
	2WH	F1	R3	+	40,820
	3WF	F2	R3	−	−
	3WM	F3	R3	+	44,457
	3WH	F1	R4	+	25,011
Pooled sample 2	2XF	F1	R1	+	7,298
	2XM	F2	R1	+	12,536
	2XH	F3	R1	+	22,560
	3XF	F1	R2	+	5,149
	3XM	F2	R2	+	14,524
	3XH	F3	R2	+	28,446
	4XF	F1	R3	−	−
	4XM	F2	R3	+	12,715
	4XH	F3	R3	+	10,219
	XTWb	F1	R4	+	43,047
Forward primer structure		5′-Overhang_adapter – Trimer_Index – Forward_16S_primer-3′
	F1	5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-GTT-CCTACGGGNGGCWGCAG
	F2	5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-CAT-CCTACGGGNGGCWGCAG
	F3	5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-TCC-CCTACGGGNGGCWGCAG
Reverse primer structure		5′-Overhang_adapter - Trimer_Index – Reverse_16S_primer-3′
	R1	5′-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-CCA-GACTACHVGGGTATCTAATCC
	R2	5′-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-TGA-GACTACHVGGGTATCTAATCC
	R3	5′-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-GTA-GACTACHVGGGTATCTAATCC
	R4	5′-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-TAC-GACTACHVGGGTATCTAATCC

The primers were modified to include a unique trinucleotide sequence between the 16S rRNA primer and overhang adapter. Successful amplification from the sample is indicated by (+).