In silico evaluation and selection of the best 16S rRNA gene primers for use in next-generation sequencing to detect oral bacteria and archaea.
16S rRNA gene
Archaea
Bacteria
Coverage
Database
Mouth
Primer
Journal
Microbiome
ISSN: 2049-2618
Titre abrégé: Microbiome
Pays: England
ID NLM: 101615147
Informations de publication
Date de publication:
23 03 2023
23 03 2023
Historique:
received:
10
05
2021
accepted:
30
01
2023
entrez:
23
3
2023
pubmed:
24
3
2023
medline:
25
3
2023
Statut:
epublish
Résumé
Sequencing has been widely used to study the composition of the oral microbiome present in various health conditions. The extent of the coverage of the 16S rRNA gene primers employed for this purpose has not, however, been evaluated in silico using oral-specific databases. This paper analyses these primers using two databases containing 16S rRNA sequences from bacteria and archaea found in the human mouth and describes some of the best primers for each domain. A total of 369 distinct individual primers were identified from sequencing studies of the oral microbiome and other ecosystems. These were evaluated against a database reported in the literature of 16S rRNA sequences obtained from oral bacteria, which was modified by our group, and a self-created oral archaea database. Both databases contained the genomic variants detected for each included species. Primers were evaluated at the variant and species levels, and those with a species coverage (SC) ≥75.00% were selected for the pair analyses. All possible combinations of the forward and reverse primers were identified, with the resulting 4638 primer pairs also evaluated using the two databases. The best bacteria-specific pairs targeted the 3-4, 4-7, and 3-7 16S rRNA gene regions, with SC levels of 98.83-97.14%; meanwhile, the optimum archaea-specific primer pairs amplified regions 5-6, 3-6, and 3-6, with SC estimates of 95.88%. Finally, the best pairs for detecting both domains targeted regions 4-5, 3-5, and 5-9, and produced SC values of 95.71-94.54% and 99.48-96.91% for bacteria and archaea, respectively. Given the three amplicon length categories (100-300, 301-600, and >600 base pairs), the primer pairs with the best coverage values for detecting oral bacteria were as follows: KP_F048-OP_R043 (region 3-4; primer pair position for Escherichia coli J01859.1: 342-529), KP_F051-OP_R030 (4-7; 514-1079), and KP_F048-OP_R030 (3-7; 342-1079). For detecting oral archaea, these were as follows: OP_F066-KP_R013 (5-6; 784-undefined), KP_F020-KP_R013 (3-6; 518-undefined), and OP_F114-KP_R013 (3-6; 340-undefined). Lastly, for detecting both domains jointly they were KP_F020-KP_R032 (4-5; 518-801), OP_F114-KP_R031 (3-5; 340-801), and OP_F066-OP_R121 (5-9; 784-1405). The primer pairs with the best coverage identified herein are not among those described most widely in the oral microbiome literature. Video Abstract.
Sections du résumé
BACKGROUND
Sequencing has been widely used to study the composition of the oral microbiome present in various health conditions. The extent of the coverage of the 16S rRNA gene primers employed for this purpose has not, however, been evaluated in silico using oral-specific databases. This paper analyses these primers using two databases containing 16S rRNA sequences from bacteria and archaea found in the human mouth and describes some of the best primers for each domain.
RESULTS
A total of 369 distinct individual primers were identified from sequencing studies of the oral microbiome and other ecosystems. These were evaluated against a database reported in the literature of 16S rRNA sequences obtained from oral bacteria, which was modified by our group, and a self-created oral archaea database. Both databases contained the genomic variants detected for each included species. Primers were evaluated at the variant and species levels, and those with a species coverage (SC) ≥75.00% were selected for the pair analyses. All possible combinations of the forward and reverse primers were identified, with the resulting 4638 primer pairs also evaluated using the two databases. The best bacteria-specific pairs targeted the 3-4, 4-7, and 3-7 16S rRNA gene regions, with SC levels of 98.83-97.14%; meanwhile, the optimum archaea-specific primer pairs amplified regions 5-6, 3-6, and 3-6, with SC estimates of 95.88%. Finally, the best pairs for detecting both domains targeted regions 4-5, 3-5, and 5-9, and produced SC values of 95.71-94.54% and 99.48-96.91% for bacteria and archaea, respectively.
CONCLUSIONS
Given the three amplicon length categories (100-300, 301-600, and >600 base pairs), the primer pairs with the best coverage values for detecting oral bacteria were as follows: KP_F048-OP_R043 (region 3-4; primer pair position for Escherichia coli J01859.1: 342-529), KP_F051-OP_R030 (4-7; 514-1079), and KP_F048-OP_R030 (3-7; 342-1079). For detecting oral archaea, these were as follows: OP_F066-KP_R013 (5-6; 784-undefined), KP_F020-KP_R013 (3-6; 518-undefined), and OP_F114-KP_R013 (3-6; 340-undefined). Lastly, for detecting both domains jointly they were KP_F020-KP_R032 (4-5; 518-801), OP_F114-KP_R031 (3-5; 340-801), and OP_F066-OP_R121 (5-9; 784-1405). The primer pairs with the best coverage identified herein are not among those described most widely in the oral microbiome literature. Video Abstract.
Identifiants
pubmed: 36949474
doi: 10.1186/s40168-023-01481-6
pii: 10.1186/s40168-023-01481-6
pmc: PMC10035280
doi:
Substances chimiques
RNA, Ribosomal, 16S
0
DNA Primers
0
Types de publication
Video-Audio Media
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
58Informations de copyright
© 2023. The Author(s).
Références
Nucleic Acids Res. 2013 Jan 7;41(1):e1
pubmed: 22933715
Cent Eur J Immunol. 2015;40(2):194-200
pubmed: 26557034
Nucleic Acids Res. 2014 Jan;42(Database issue):D633-42
pubmed: 24288368
J Mol Biol. 2019 Jul 26;431(16):2957-2969
pubmed: 31103772
Int J Med Microbiol. 2015 Oct;305(7):682-8
pubmed: 26324012
Front Cell Infect Microbiol. 2019 Oct 11;9:347
pubmed: 31681625
J Periodontol. 2020 Oct;91 Suppl 1:S56-S67
pubmed: 32533776
Anaerobe. 2012 Jun;18(3):373-7
pubmed: 22561061
Sci Rep. 2014 Oct 13;4:6602
pubmed: 25308100
Pharmacol Res. 2013 Mar;69(1):137-43
pubmed: 23201354
J Clin Microbiol. 2011 Apr;49(4):1464-74
pubmed: 21289150
J Oral Microbiol. 2017 Jan 23;9(1):1270614
pubmed: 28326153
Sci Rep. 2017 Jun 16;7(1):3703
pubmed: 28623321
J Oral Maxillofac Pathol. 2019 Jan-Apr;23(1):122-128
pubmed: 31110428
J Endod. 2017 Aug;43(8):1309-1316
pubmed: 28648489
Bioinformatics. 2009 Jun 1;25(11):1422-3
pubmed: 19304878
Bioinformatics. 2008 Aug 15;24(16):1757-64
pubmed: 18567917
Mol Ecol Resour. 2020 Nov;20(6):1558-1571
pubmed: 32599660
Mol Syst Biol. 2011 Oct 11;7:539
pubmed: 21988835
Database (Oxford). 2020 Jan 1;2020:
pubmed: 32761142
Clin Oral Investig. 2021 May;25(5):3033-3042
pubmed: 33057825
Genome Res. 2009 Jul;19(7):1141-52
pubmed: 19383763
Front Nutr. 2016 Aug 08;3:26
pubmed: 27551678
J Clin Periodontol. 1998 Feb;25(2):134-44
pubmed: 9495612
PLoS Biol. 2007 Mar;5(3):e16
pubmed: 17355171
J Clin Periodontol. 2017 Mar;44 Suppl 18:S135-S144
pubmed: 28266112
J Oral Microbiol. 2019 Aug 9;11(1):1653123
pubmed: 31489129
Nat Methods. 2016 Jul;13(7):581-3
pubmed: 27214047
PLoS Biol. 2007 Mar;5(3):e77
pubmed: 17355176
BMC Microbiol. 2012 May 03;12:66
pubmed: 22554309
Nucleic Acids Res. 2016 Jan 4;44(D1):D7-19
pubmed: 26615191
Microbes Infect. 2015 Jul;17(7):505-16
pubmed: 25862077
Appl Environ Microbiol. 2006 Jul;72(7):5069-72
pubmed: 16820507
J Microbiol Biotechnol. 2014 Jun 28;24(6):812-22
pubmed: 24722376
Front Microbiol. 2017 Mar 28;8:494
pubmed: 28400755
Curr Protoc Mol Biol. 2018 Apr;122(1):e59
pubmed: 29851291
Sci Total Environ. 2018 Mar 15;618:1254-1267
pubmed: 29089134
J Oral Microbiol. 2020 Sep 13;12(1):1814674
pubmed: 33062199
Proc Natl Acad Sci U S A. 2011 Mar 15;108 Suppl 1:4516-22
pubmed: 20534432
Oral Microbiol Immunol. 2009 Feb;24(1):32-7
pubmed: 19121067
Nucleic Acids Res. 2013 Jan;41(Database issue):D590-6
pubmed: 23193283
PCR Methods Appl. 1993 Dec;3(3):S30-7
pubmed: 8118394
J Dent Res. 2015 Oct;94(10):1425-31
pubmed: 26198391
mSystems. 2018 Dec 4;3(6):
pubmed: 30534599
PLoS One. 2017 Aug 11;12(8):e0182992
pubmed: 28800622
Int J Mol Sci. 2016 Nov 25;17(12):
pubmed: 27898021
Sci Rep. 2015 Nov 13;5:16498
pubmed: 26563586
BMC Bioinformatics. 2018 Sep 29;19(1):343
pubmed: 30268091
Nat Biotechnol. 2020 Sep;38(9):1079-1086
pubmed: 32341564
Front Microbiol. 2019 Dec 05;10:2796
pubmed: 31866971
Sci Rep. 2016 May 11;6:25775
pubmed: 27166431
Front Cell Infect Microbiol. 2020 Dec 11;10:596490
pubmed: 33425781
J Clin Periodontol. 2015 Nov;42(11):1015-23
pubmed: 26412568
J Pharm Bioallied Sci. 2017 Jul-Sep;9(3):155-163
pubmed: 28979069
Int J Oral Sci. 2022 Mar 2;14(1):14
pubmed: 35236828
Int J Syst Evol Microbiol. 2009 Mar;59(Pt 3):509-16
pubmed: 19244431
Sci Rep. 2015 Mar 12;5:9053
pubmed: 25761675
Microorganisms. 2020 Feb 23;8(2):
pubmed: 32102216
Int J Syst Bacteriol. 1996 Jul;46(3):745-52
pubmed: 8782684
J Oral Microbiol. 2020 Oct 11;12(1):1830623
pubmed: 33149844
J Med Libr Assoc. 2010 Apr;98(2):171-5
pubmed: 20428285
Environ Microbiol Rep. 2019 Aug;11(4):487-494
pubmed: 30058291
World J Gastroenterol. 2010 Sep 7;16(33):4135-44
pubmed: 20806429
Microbiome. 2020 May 15;8(1):65
pubmed: 32414415
J Dent Res. 2019 May;98(5):500-509
pubmed: 30894042
Mol Oral Microbiol. 2016 Jun;31(3):234-42
pubmed: 26194817
Nucleic Acids Res. 2016 Jan 4;44(D1):D67-72
pubmed: 26590407
J Microbiol Methods. 2003 Dec;55(3):541-55
pubmed: 14607398
Microb Genom. 2020 Aug;6(8):
pubmed: 32706331
Caries Res. 2009;43(4):308-13
pubmed: 19494490
Nucleic Acids Res. 2016 Jan 4;44(D1):D733-45
pubmed: 26553804
Oral Dis. 2017 Apr;23(3):276-286
pubmed: 27219464
Arch Microbiol. 2018 May;200(4):525-540
pubmed: 29572583
Ann Epidemiol. 2016 May;26(5):311-21
pubmed: 27180112
PeerJ. 2018 Jun 12;6:e5030
pubmed: 29910992
Database (Oxford). 2010 Jul 06;2010:baq013
pubmed: 20624719
Nucleic Acids Res. 2015 Sep 18;43(16):7762-8
pubmed: 26250111
ISME J. 2012 Jul;6(7):1440-4
pubmed: 22237546