Design of 16S rRNA gene primers for 454 pyrosequencing of the human foregut microbiome

Table 1 Primers used in the study

Primer	# bases	Sequence (5’ to 3’)	Species with identical match (%)
Forward primers
8F	20	AGAGTTTGATCCTGGCTCAG	n/a1
343F	15	TACGGRAGGCAGCAG	99.1
517F	17	GCCAGCAGCCGCGGTAA	93.2
784F	15	AGGATTAGATACCCT	90.0
917F	16	GAATTGACGGGGRCCC	84.0
1099F	16	GYAACGAGCGCAACCC	73.1
Reverse primers
534R	18	ATTACCGCGGCTGCTGGC	91.8
798R	15	AGGGTATCTAATCCT	90.0
926R	20	CCGTCAATTYYTTTRAGTTT	83.0
1114R	16	GGGTTGCGCTCGTTRC	74.9
1407R	16	GACGGGCGGTGTGTRC	91.3
1541R	20	AAGGAGGTGATCCAGCCGCA	n/a1

¹Sequences near the ends of the 16S rRNA genes are often designed for primer binding and are not included in sequences deposited in GenBank. n/a: Not available. R = A, G; Y = C, T.

Table 2 Amplicons designed for analysis

Amplicon	Primers		Total length	HVR(s) included
	F	R
A	8F	534R	527	1,2,3
B	343F	798R	456	3,4
C	517F	926R	410	3,4,5
D	784F	1114R	331	5,6
E	917F	1407R	491	6,7
F	1099F	1541R	443	7,8,9

HVR: Hypervariable region; F: Forward primers; R: Reverse primers.

Table 3 Accuracy of taxonomic classification of 219 foregut species using 350-bp sequences

Amplicon	% accuracy compared to 900-bp amplicon/full length
	Phylum	Class	Order	Family	Genus
Forward reads
A	97.7/97.7	96.3/95.9	95.9/95.4	93.2/94.5	87.7/86.8
B	99.1/99.1	98.2/96.8	97.7/96.3	97.3/95.9	91.8/89.0
C	99.5/99.5	98.2/96.8	97.7/96.8	97.3/95.9	90.4/88.1
D	98.6/99.1	98.6/98.2	97.3/97.3	95.9/96.3	88.1/86.8
E	98.2/98.6	97.7/98.2	95.4/95.9	92.2/93.6	85.4/84.9
F	97.7/98.2	96.3/97.3	95.0/95.4	91.8/93.6	83.6/84.5
Reverse reads
A’	98.6/98.6	97.3/97.7	97.3/97.7	95.0/96.3	90.0/90.9
B’	99.5/99.5	98.2/96.8	97.7/96.8	97.7/96.3	93.6/91.8
C’	99.5/99.5	98.2/96.8	97.7/96.8	97.3/95.9	91.3/90.0
D’	99.5/100	98.6/99.5	96.8/98.2	94.5/96.3	87.2/89.5
E’	98.2/98.6	96.8/98.2	94.5/96.3	91.3/94.1	83.6/87.2
F’	98.2/98.6	96.8/98.2	95.0/96.4	92.2/95.0	82.6/85.8

Table 4 Foregut species misclassified using amplicon B’ compared with full length sequences

Species	Weight (of 9484)	Species identified using amplicon B
Atopobium AY959044	8	Atopobium parvulum
Bacteroides vulgatus	1	Uncultured bacterium
Bradyrhizobium japonicum	0	Blastobacter denitrificans
Bradyrhizobium liaoningense	1	Blastobacter denitrificans
Escherichia fergusonii	1	Shigella sonnei
Escherichia flexneri	4	Shigella sonnei
Haemophilus aegyptius	17	Haemophilus influenzae
Haemophilus haemolyticus	34	Haemophilus
Lactobacillus gasseri	2	Lactobacillus johnsonii
Leptotrichia wadeii	10	Leptotrichia shahii
Neisseria macaca	28	Uncultured bacterium
Prevotella melaninogenica	369	Uncultured bacterium
Pseudoramibacter	3	Uncultured bacterium
Streptococcus infantis	193	Streptococcus mitis
Total	671

Table 5 Esophageal species misclassified using amplicon B’ compared with Sanger sequences

Species	Weight (of 6800)	Species identified by Amplicon B’
Actinomyces naeslundii	2	Actinomyces viscosus
Atopobium AY959044	8	Atopobium parvulum
Bacteroides vulgatus	1	Uncultured bacterium
Bradyrhizobium japonicum	1	Blastobacter denitrificans
Campylobacter showae	4	Campylobacter
Escherichia flexneri	2	Shigella sonnei
Haemophilus aegyptius	17	Haemophilus influenzae
Haemophilus haemolyticus	33	Haemophilus
Lactobacillus gasseri	2	Lactobacillus johnsonii
Leptotrichia wadeii	7	Leptotrichia shahii turn
Neisseria macaca	25	Uncultured bacterium
Prevotella melaninogenica	288	Uncultured bacterium
Pseudoramibacter	1	Uncultured bacterium
Streptococcus infantis	77	Streptococcus mitis
Total	468

Table 6 Taxonomic coverage of domain bacteria by primers 347F and 803R

Primer	Optimization	Total species	Coverage at mismatches n (%)			Total sequence	Coverage at mismatches n (%)
			0	1	2		0	1	2
374F	Before	5165	3392 (65.7)	4835 (93.6)	5042 (97.6)	433 306	275 801 (63.7)	406 626 (93.8)	418 613 (96.6)
	After	5165	4703 (91.1)	4996 (96.7)	5114 (99.0)	433 306	391 695 (90.4)	418 832 (96.7)	424 756 (98.0)
803R	Before	5165	4584 (88.8)	5091 (98.6)	5159 (99.9)	433 306	352 827 (81.4)	417 612 (96.4)	430 967 (99.5)
	After	5165	4741 (91.8)	5131 (99.3)	5159 (99.9)	433 306	367 771 (84.9)	427 791 (98.7)	431 725 (99.6)