Design of 16S rRNA gene primers for 454 pyrosequencing of the human foregut microbiome.
OBJECTIVE
To design and validate broad-range 16S rRNA primers for use in high throughput sequencing to classify bacteria isolated from the human foregut microbiome.


METHODS
A foregut microbiome dataset was constructed using 16S rRNA gene sequences obtained from oral, esophageal, and gastric microbiomes produced by Sanger sequencing in previous studies represented by 219 bacterial species. Candidate primers evaluated were from the European rRNA database. To assess the effect of sequence length on accuracy of classification, 16S rRNA genes of various lengths were created by trimming the full length sequences. Sequences spanning various hypervariable regions were selected to simulate the amplicons that would be obtained using possible primer pairs. The sequences were compared with full length 16S rRNA genes for accuracy in taxonomic classification using online software at the Ribosomal Database Project (RDP). The universality of the primer set was evaluated using the RDP 16S rRNA database which is comprised of 433 306 16S rRNA genes, represented by 36 phyla.


RESULTS
Truncation to 100 nucleotides (nt) downstream from the position corresponding to base 28 in the Escherichia coli 16S rRNA gene caused misclassification of 87 (39.7%) of the 219 sequences, compared with misclassification of only 29 (13.2%) sequences with truncation to 350 nt. Among 350-nt sequence reads within various regions of the 16S rRNA gene, the reverse read of an amplicon generated using the 343F/798R primers had the least (8.2%) effect on classification. In comparison, truncation to 900 nt mimicking single pass Sanger reads misclassified 5.0% of the 219 sequences. The 343F/798R amplicon accurately assigned 91.8% of the 219 sequences at the species level. Weighted by abundance of the species in the esophageal dataset, the 343F/798R amplicon yielded similar classification accuracy without a significant loss in species coverage (92%). Modification of the 343F/798R primers to 347F/803R increased their universality among foregut species. Assuming that a typical polymerase chain reaction can tolerate 2 mismatches between a primer and a template, the modified 347F and 803R primers should be able to anneal 98% and 99.6% of all 16S rRNA genes in the RDP database.


CONCLUSIONS
347F/803R is the most suitable pair of primers for classification of foregut 16S rRNA genes but also possess universality suitable for analyses of other complex microbiomes.
Nossa
Carlos W
CW
Department of Veterans Affairs New York Harbor Health System, 423 E 23rd Street, New York, NY 10010, USA.
Oberdorf
William E
WE
Yang
Liying
L
Aas
Jørn A
JA
Paster
Bruce J
BJ
Desantis
Todd Z
TZ
Brodie
Eoin L
EL
Malamud
Daniel
D
Poles
Michael A
MA
Pei
Zhiheng
Z
D000230:Adenocarcinoma / Q000382:microbiology / Q000503:physiopathology; D018450:Disease Progression; D004926:Escherichia coli / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology; D004938:Esophageal Neoplasms / Q000382:microbiology / Q000503:physiopathology; D005764:Gastroesophageal Reflux / Q000382:microbiology / Q000503:physiopathology; D020459:Genes, rRNA / Q000235:genetics*; D016480:Helicobacter pylori / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology; D006801:Humans; D054892:Metagenome / Q000235:genetics*; D012313:RNA / Q000235:genetics*; D012336:RNA, Ribosomal, 16S / Q000235:genetics*; D017423:Sequence Analysis, RNA / Q000379:methods*; D013296:Streptococcus pneumoniae / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology; D041742:Upper Gastrointestinal Tract / Q000382:microbiology*
C034477:RNA primers; D012336:RNA, Ribosomal, 16S; D012313:RNA
Nucleic Acids Res. 1981 Mar 25;9(6):1451-61
6785727
J Bacteriol. 1991 Jan;173(2):697-703
1987160
Proc Natl Acad Sci U S A. 1990 Jun;87(12):4576-9
2112744
PLoS One. 2009 Oct 09;4(10):e7401
19816594
Nucleic Acids Res. 2004 Jan 1;32(Database issue):D101-3
14681368
Mol Biol Evol. 2007 Aug;24(8):1596-9
17488738
Appl Environ Microbiol. 1998 Oct;64(10):3854-9
9758810
ISME J. 2007 Aug;1(4):283-90
18043639
Nucleic Acids Res. 1996 Sep 1;24(17):3381-91
8811093
Appl Environ Microbiol. 1996 Feb;62(2):625-30
8593063
J Infect Dis. 2008 Oct 15;198(8):1131-40
18717638
World J Gastroenterol. 2005 Dec 14;11(46):7277-83
16437628
J Mol Biol. 1975 May 25;94(3):441-8
1100841
Genome Res. 2009 Apr;19(4):521-32
19339662
Appl Environ Microbiol. 2007 Aug;73(16):5261-7
17586664
J Clin Microbiol. 2005 Nov;43(11):5721-32
16272510
Proc Natl Acad Sci U S A. 1998 Jun 9;95(12):6854-9
9618502
Proc Natl Acad Sci U S A. 2006 Jan 17;103(3):732-7
16407106
J Clin Microbiol. 2004 Oct;42(10):4686-96
15472328
PLoS One. 2008 Jul 30;3(7):e2836
18665274
Appl Environ Microbiol. 2008 Mar;74(5):1453-63
18192407
Gastroenterology. 2009 Aug;137(2):588-97
19394334
Proc Natl Acad Sci U S A. 1985 Apr;82(8):2437-41
3921961
BMC Microbiol. 2008 Mar 06;8:43
18325110
J Microbiol Methods. 2007 May;69(2):330-9
17391789
Microbiol Rev. 1987 Jun;51(2):221-71
2439888
Nucleic Acids Res. 2007;35(18):e120
17881377
Nature. 2005 Sep 15;437(7057):376-80
16056220
Nucleic Acids Res. 2006 Jul 1;34(Web Server issue):W394-9
16845035
Genome Res. 2009 Dec;19(12):2317-23
19819907
Proc Natl Acad Sci U S A. 2004 Mar 23;101(12):4250-5
15016918
Nucleic Acids Res. 2007 Jan;35(Database issue):D169-72
17090583