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16S rRNA indexed primers amplify phylogenic markers for microbiome sequencing analysis

The microbiome and its impact on human and environmental health

The 16S rRNA gene is frequently used in microbiome studies to identify the subset of microbes present in biological samples. Researchers amplify short hypervariable regions from this gene, tag the amplified products with unique barcodes, perform highly multiplexed sequencing runs, and compare the sequences to the known bacterial genome database. However, primer design for such analyses can be challenging given the massive sequence variability in sampled lifeforms. Read about the development and design of these primers, and how you can obtain your own custom, high fidelity versions of these sequences.

Microorganisms nourish the plants on this planet and work in unison to keep our environment healthy. A microbiome represents the subset of those microorganisms that reside in a defined environment. The ecological community of microorganisms within and on the human body is composed of an enormous number of bacterial, archaeal, and eukaryotic microbes, as well as viruses [1]. Even though individual microbial cells can be much smaller than a typical human cell, it is estimated that the microbiome found in a healthy human body harbors several million distinct genes, outnumbering the unique genes in our own genomes by a factor of at least 100 [2]. These commensal or pathogenic microorganisms interact with human bodies to shape our immune systems and influence metabolism, which in turn directly or indirectly affect most, if not all, of our physiological functions [3].

Technological advances and challenges in microbiome analysis

Although microbes have been known to exist throughout the earth’s environment and within the human body, microbial diversity and the functional significance of the microbiome have long been underappreciated. However, this is changing, primarily driven by the technological advancements of culture-independent profiling and the collaborative efforts of researchers aiming to characterize microbial ecology on our planet [4]. In 2010, the Earth Microbiome Project (EMP) was established with the ambitious goal of constructing a catalog of microbial diversity from habitats across the world [5]. The consortium also proposed protocols and reagents to standardize sample processing and data analysis that will facilitate subsequent global comparison [6].

Of the characterized prokaryotic genes, the 16S ribosomal RNA (rRNA) gene serves as an excellent marker for investigation of bacterial phylogeny [7,8]. The prokaryotic 16S rRNA gene is approximately 1500 bp in length and contains 9 variable regions interspaced by conserved regions. The variable regions of the 16S rRNA gene are frequently interrogated for phylogenetic classification. Typically, to understand the constituents of a bacterial population, researchers amplify short hypervariable regions from this gene, tag the amplified products with unique barcodes, perform highly multiplexed sequencing runs, and compare the sequences to the known bacterial genome database [9]. However, primer design for hypervariable region amplification can be challenging given the massive sequence variability in sampled lifeforms. Ideally, the degenerate primer pair should be deliberately designed to meet the following parameters:

  • Recognize a vast number of known 16S rRNA genes from previously identified microbes and amplify them with approximately equal efficiency
  • Include sufficient degeneracy to amplify sequences from unknown but related species
  • Generate amplicons of suitable length for high-throughput analysis on available NGS platforms

16S rRNA primer designs and amplification strategies

Originally described in 2011, the PCR primers, 515F/806R, which amplify the V4 hypervariable region of 16S rRNA, was selected by the EMP to amplify prokaryote genomes (bacteria and archaea), followed by sequencing on an Illumina platform [9,10]. Since then, this primer pair has been modified to accommodate new discoveries in microbial ecology. For instance, as proposed by Parada et al. and Apprill et al., degenerate bases were added to both forward and reverse primers to avoid unwanted biases towards certain marine taxa [11,12]. It is also noteworthy that the sample-specific 12 nt Golay barcode, which used to be located on the reverse primer, has been moved to the forward primer (515F). This enables pairing of a universal forward primer with various reverse primers, when longer amplicons are desired [13]. Similar primer sets have been developed for the study of microorganisms in other kingdoms. Euk1391F-EukBr amplifies a portion of the 18S rRNA gene in eukaryotic microbial lineages, and ITS1F-ITS2 targets fungal rRNA [14,15].

The widely-adopted 515F/806R primers provide an example of the typical components that often comprise a 16S rRNA indexed primer recommended by the EMP:

  • An Illumina adapter sequence that binds to the flow cell
  • A Golay barcode that enables a high level of multiplexity (on forward primer only)
  • A 10 nt primer pad to prevent hairpin formation
  • A 2 nt primer linker that shares no homology to any 16S rRNA sequence at the corresponding positions [9]
  • A loci-specific sequence that binds to evolutionarily conserved regions

These components are depicted in Figure 1A, with a schematic of their use in single-indexed amplification shown in Figure 1B.

art190-MH-16S primers_Fig1A
Figure 1A. Sequence composition of 515F and 806R primers, color-coded by different functional components. Both primers are shown in the 5′ to 3′ orientation, which is the orientation that should be used for online ordering through IDT. Recently modified positions are highlighted in purple (“Y” on 515F and “N” on 806R). The nucleotide sequences of the 2 primers are listed on the EMP website (

art190-MH-16S primers_Fig1B
Figure 1B. 16S rRNA primer composition and use in single-indexed amplification and sequencing. Using a one-step, single-indexed amplification approach, the V4 region of the 16S rRNA gene is amplified with the primers 515F and 806R tagged with Illumina P5 and P7 adapter sequences.

Alternative strategies, such as dual-indexing amplification and sequencing approaches, have also been used to characterize the composition of microbial communities [16,17]. Unlike the EMP primers discussed above, sample-specific barcodes (Index 1 and Index 2) are included on forward and reverse primers, and are used to amplify the V3–V4 hypervariable regions of the 16S rRNA gene to generate an amplicon of approximately 460 bp [16,18,19]. As described by Fadrosh and others, in this case, a two-step amplification reaction is performed. The first round of amplification appends the overhang adapter sequences for compatibility with Illumina sequencing, while a subsequent, limited-cycle amplification adds multiplexing indices and sequencing adapters (Figure 2) [16]. With fewer barcodes needed to reach the same level of multiplexity, dual-indexing approaches likely reduce the cost of oligo synthesis and improve experimental flexibility.

art190-MH-16S primers_Fig2
Figure 2. Two-step, dual-indexing amplification and sequencing approach.

IDT primers recommended for microbiome analysis

IDT is recommended as the oligo supplier by the EMP [20]. With our industry-leading synthesis technologies, we provide the highest quality primers, and help ensure that microbiome analysis is reproducible and accurate. Given the length of these indexed primers, we often recommend our customers order them as standard desalted Ultramer® Oligonucleotides. Our proprietary Ultramer synthesis platform provides unprecedented coupling efficiency, which results in a high percentage of full-length products without the need for extra purification. For even greater convenience, standard desalted Ultramer Oligos are formulated and delivered at guaranteed yields.

Contact us at with any questions about oligo ordering or to discuss your experimental design with our scientific applications experts.


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  13. Walters W, Hyde ER, et al. (2016) Improved bacterial 16S rRNA gene (V4 and V4-5) and fungal internal transcribed spacer marker gene primers for microbial community surveys. mSystems, 1(1):e00009–15.
  14. Amaral-Zettler LA, McCliment EA, et al. (2009) A method for studying protistan diversity using massively parallel sequencing of V9 hypervariable regions of small-subunit ribosomal RNA genes. PLoS One, 4(7):e6372.
  15. Gardes M, Bruns TD. (1993) ITS primers with enhanced specificity for basidiomycetes--application to the identification of mycorrhizae and rusts. Mol Ecol, 2(2):113–118.
  16. Fadrosh DW, Ma B, et al. (2014) An improved dual-indexing approach for multiplexed 16S rRNA gene sequencing on the Illumina MiSeq platform. Microbiome, 2(1):6.
  17. Kozich JJ, Westcott SL, et al. (2013) Development of a dual-index sequencing strategy and curation pipeline for analyzing amplicon sequence data on the MiSeq Illumina sequencing platform. Appl Environ Microbiol, 79(17):5112–5120.
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Published Jul 17, 2017