Common Primers for Ribosomal Amplicon Sequencing
/Our customers commonly ask us what the best primers are to use for an amplicon sequencing project. This article is intended to be a resource to them based on the latest research and our experience performing analysis on numerous projects.
16S rRNA Amplicon Sequencing Primers [2,3,4]
The 16S rRNA small subunit (SSU) is a gene that is part of the 30S subunit of a prokaryotic ribosome. It is often used as a marker since every prokaryotic organism must have a ribosome but the sequence of the 16S component has several regions of variability which are useful for distinguishing between them.
| Targeted Region | Name | Sequence (5' -> 3') | Notes |
|---|---|---|---|
| V1 | 8F | TGGAGAGTTTGATCCTGGCTCAG | |
| V1 | 27F | AGAGTTTGATCMTGGCTCAG | Bacteria only. |
| V1 | F28 | GAGTTTGATCNTGGCTCAG | |
| V3 | R519 | GTNTTACNGCGGCKGCTG | |
| V3 | 341F | CCTACGGGAGGCAGCAG | |
| V3 | Pro341F | CCTACGGGNBGCASCAG | |
| V3 | F357 | CCTACGGGAGGCAGCAG | |
| V3 | 533R | TACCGCGGCTGCTGGCAC | |
| V4 | 515F | GTGCCAGCMGCCGCGGTAA | |
| V4 (alternate) | 515F | GTGYCAGCMGCCGCGGTAA | |
| V4 | 785R | GACTACHVGGGTATCTAATCC | |
| V4 | Pro805R | GACTACNVGGGTATCTAATCC | |
| V4 | 806R | GGACTACHVGGGTWTCTAAT | |
| V5 | 907R | CCGTCAATTCMTTTGAGTTT | Bacteria |
| V5 | R926 | CCGTCAATTCMTTTRAGT | |
| V5 (alternate) | 926R | CCGYCAATTYMTTTRAGTTT | |
| V5 | 799F | AACMGGATTAGATACCCKG | |
| V7 | 1193R | ACGTCATCCCCACCTTCC |
18S rRNA Amplicon Sequencing Primers [1,5]
Like the 16S rRNA, the 18S rRNA is an SSU which is part of the eukaryotic 40S ribsomal subunit.
| Position | Name | Sequence (5' -> 3') | Notes |
|---|---|---|---|
| 563 | 563F | GCCAGCAVCYGCGGTAAY | |
| 566 | 566F | CAGCAGCCGCGGTAATTCC | |
| 574 | 574F | GCCAGCAVCYGCGGTAAY | |
| 574 | 574*F | CGGTAAYTCCAGCTCYV | |
| 616 | 616F | TTAAAAVGYTCGTAGTYG | |
| 616 | 616*F | TTAAARVGYTCGTAGTYG | |
| 897 | 897f | AGAGGTGRAATTCTHRGA | |
| 952 | 952R | TTGGCAAATGCTTTCGC | |
| 957 | 1183F | AATTTGACTCAACACGGG | |
| 1132 | 1132R | CCGTCAATTHCTTYAART | |
| 1200 | 1200R | CCCGTGTTGAGTCAAATTAAGC | |
| 1246 | 1631R | TACAAAGGGCAGGGACGTAAT | |
| 1774 | EukbR | TGATCCTTCTGCAGGTTCACCTAC | |
| 1829 | 1391F | GTACACACCGCCCGTC |
Refer to this table for information about the coverage of various 18S primer sets on several taxonomic groups.
ITS Amplicon Sequencing Primers for Fungal Studies [6]
A diagram of the eukaryotic ribosomal region indicating the positions of the 18S, ITS, 5.8S, and 28S sequences. Source: Wikipedia
The most commonly used marker used for fungal studies is the internal transcribed spacer (ITS) regions (see image above). The ITS sequences site between the 18S and 5.8S sequences and the 5.8S and 28S sequences. The average length of this region is approximately 550bp[9].
| Amplicon | Name | Sequence (5' -> 3') | Notes |
|---|---|---|---|
| ITS1 | ITS1F | CTTGGTCATTTAGAGGAAGTAA | |
| ITS1 | ITS2 | GCTGCGTTCTTCATCGATGC | |
| ITS2 | 3271-ITS2F | CARCAAYGGATCTCTTGG | |
| ITS2 | 3271-ITS2R | GATATGCTTAAGTTCAGCGGGT | |
| ITS2 | ITS4 | TCCTCCGCTTATTGATATGC |
There are many other regions that can be useful for species-specific or strain-specific identification. For example, recA, gyrB, rpoB, and others are often used [7]. For the case of Propionibacterium acnes, researchers found a single locus sequencing typing (SLST) that could identify specific P. acnes sequence types [8].
Do you have questions about the best locus for identifying your targets of interest? Please tell us about your project!
References
Kounosu, A., Murase, K., Yoshida, A. et al. Improved 18S and 28S rDNA primer sets for NGS-based parasite detection. Sci Rep 9, 15789 (2019). https://doi.org/10.1038/s41598-019-52422-z
Apprill, A., McNally, S., Parsons, R., & Weber, L. (2015). Minor revision to V4 region SSU rRNA 806R gene primer greatly increases detection of SAR11 bacterioplankton. Aquatic Microbial Ecology, 75(2), 129–137.
Caporaso, J. G., Lauber, C. L., Walters, W. A., Berg-Lyons, D., Lozupone, C. A., Turnbaugh, P. J., Noah Fierer, N., & Knight, R. (2011). Global patterns of 16S rRNA diversity at a depth of millions of sequences per sample. Proc Natl Acad Sci USA 108, 4516–4522.
Caporaso, J. G., Lauber, C. L., Walters, W. A., Berg-Lyons, D., Huntley, J., Fierer, N., Owens, S. M., Betley, J., Fraser, L., Bauer, M., Gormley, N., Gilbert, J. A., Smith, G., & Knight, R. (2012). Ultra-high-throughput microbial community analysis on the Illumina HiSeq and MiSeq platforms. ISME J 6, 1621–1624.
Hugerth LW, Muller EE, Hu YO, Lebrun LA, Roume H, Lundin D, Wilmes P, Andersson AF. Systematic design of 18S rRNA gene primers for determining eukaryotic diversity in microbial consortia. PLoS One. 2014 Apr 22;9(4):e95567. doi: 10.1371/journal.pone.0095567. Erratum in: PLoS One. 2015;10(1):e0117636. PMID: 24755918; PMCID: PMC3995771.
Cui, L., Morris, A. & Ghedin, E. The human mycobiome in health and disease. Genome Med 5, 63 (2013). https://doi.org/10.1186/gm467
Holmes DE, Nevin KP, Lovley DR. Comparison of 16S rRNA, nifD, recA, gyrB, rpoB and fusA genes within the family Geobacteraceae fam. nov. Int J Syst Evol Microbiol. 2004 Sep;54(Pt 5):1591-1599. doi: 10.1099/ijs.0.02958-0. PMID: 15388715.
Scholz CF, Jensen A, Lomholt HB, Brüggemann H, Kilian M. A novel high-resolution single locus sequence typing scheme for mixed populations of Propionibacterium acnes in vivo. PLoS One. 2014 Aug 11;9(8):e104199. doi: 10.1371/journal.pone.0104199. PMID: 25111794; PMCID: PMC4128656.
Nilsson RH, Tedersoo L, Ryberg M, Kristiansson E, Hartmann M, Unterseher M, Porter TM, Bengtsson-Palme J, Walker DM, de Sousa F, Gamper HA, Larsson E, Larsson KH, Kõljalg U, Edgar RC, Abarenkov K. A Comprehensive, Automatically Updated Fungal ITS Sequence Dataset for Reference-Based Chimera Control in Environmental Sequencing Efforts. Microbes Environ. 2015;30(2):145-50. doi: 10.1264/jsme2.ME14121. Epub 2015 Mar 19. PMID: 25786896; PMCID: PMC4462924.
