Table 4. Effects of amplification method and reverse primer variants on observed microbial community alpha diversity.

Fecal gDNA was PCR amplified with 18-fold degenerate reverse primer pools (5 technical replicates), and with each unique reverse primer variant (RPV; 2 technical replicates). Data sets were rarefied to 1,800 sequences per sample, and Shannon indices (loge) were calculated. When using fully degenerate primer pools, average Shannon index was significantly higher for TAS methodology relative to DePCR methodology. When data from all reactions with individual RPVs were analyzed, average Shannon index was significantly lower for TAS methodology relative to DePCR methodology. Data from RPVs (1,800 sequences/sample) were pooled and re-rarefied to 1,800 sequences (five repetitions), and the resulting average Shannon index was significantly lower for the TAS methodology relative to DePCR methodology. Different approaches with the DePCR method did not generate significantly different Shannon indices (ANOVA P = 0.377), while the same approaches generated significantly different Shannon indices (ANOVA P < 0.001).

Comparison	# replicates analyzed	Average Shannon Index (SD), TAS	Average Shannon Index (SD), DePCR	ANOVA
Amplification with 18-fold degenerate primer pools	5	2.71 (0.03)	2.66 (0.04)	3.14E-05
Amplification with each RPV independently	33 (TAS) or 36 (DePCR)	2.4 (0.01)	2.58 (0.21)	5.95E-05
Summation of independent RPVs and re-rarefaction to 1800 sequences (5x)	5	2.48 (0.03)	2.69 (0.02)	7.40E-07
	ANOVA	3.69E-08	3.77E-01