16S rRNA gene-based profiling of the human infant gut microbiota is strongly influenced by sample processing and PCR primer choice

Table 2 Mean percentage of bacteria in specific families detected following 454 sequence analysis using the different primer sets across all samples

	Primer sets
Forward primer	27f-YM^a	27f-Mix^b	27f-Bif^c	Bif164^d
Reverse primer	534r	534r	534r	Bif662-r
Family	Percentage of bacteria detected per family
Bifidobacteriaceae	17.19 ± 2.37	47.30 ± 4.26	95.53 ± 1.25	100 ± 0.000001
Lactobacillaceae	16.03 ± 4.61	8.59 ± 2.58	0.07 ± 0.05	0 ± 0.00
Enterococcaceae	2.01 ± 1.71	1.31 ± 1.13	0.02 ± 0.02	0 ± 0.00
Streptococcaceae	9.41 ± 2.43	4.83 ± 1.08	0.11 ± 0.07	0 ± 0.00
Staphylococcaceae	1.07 ± 0.56	0.44 ± 0.22	0.04 ± 0.04	0 ± 0.00
Clostridiaceae	16.02 ± 5.27	10.65 ± 4.04	0.74 ± 0.48	0 ± 0.00
Lachnospiraceae	9.54 ± 2.40	8.60 ± 3.06	1.02 ± 0.43	0 ± 0.00
Veillonellaceae	5.38 ± 3.04	3.92 ± 0.91	0.13 ± 0.07	0 ± 0.00
Bacteroidaceae	10.15 ± 4.62	8.48 ± 3.74	0.24 ± 0.15	0 ± 0.00
Enterobacteriaceae	10.62 ± 1.90	3.80 ± 0.66	0.04 ± 0.03	0 ± 0.00
Other	2.56 ± 0.95	2.07 ± 0.56	2.05 ± 0.97	0 ± 0.00

Values given are the mean of all data, plus or minus SE of the mean. Data results from analysis of samples from two babies ages 2–14 weeks, at 17 time points, extracted using method 1 (FastDNA SPIN Kit for Soil) and amplified with four primer sets giving 68 sequence datasets. Average number of sequences per sample = 1645
^aThe degenerate primer 27f-YM sequence has two mismatches with the Bifidobacterium genus
^b27f-Mix—4:1:1:1:1 molar mix of forward primers (27f-YM, 27f-Chl, 27f-Bor, 27f-Bif, 27f-Ato, respectively)
^cSpecific for bifidobacteria but designed to have similar amplification efficiency as the other primers
^dStandard specific primers for detection of bifidobacteria

ISSN: 2049-2618