DOI: 10.1111/prd.
12349
REVIEW ARTICLE
Probing periodontal microbial dark matter using
metataxonomics and metagenomics
Purnima S. Kumar1 | Shareef M. Dabdoub1 | Sukirth M. Ganesan2
1
Department of Periodontology, College of Dentistry, The Ohio State University, Columbus, Ohio, USA
2
Department of Periodontics, College of Dentistry and Dental Clinics, The University of Iowa, Iowa City, Iowa, USA
Correspondence
Purnima S. Kumar, Division of periodontology, College of Dentistry, 305 West 12th Avenue, Columbus, Ohio43210, USA.
Email: kumar.83@osu.edu
1 |  I NTRO D U C TI O N
To understand how bacteria cause disease, it is important to not
only understand how they colonize the host, what partners they re-
cruit to enable their host-associated lifestyle, how they evade the
immune system or create an environment of immune tolerance, but
also when, why, and how pathobionts are created. Our view of the
periodontal microbial community has been shaped by a century or
more of cultivation-based and microscopic investigations. While
these studies firmly established the infection-mediated etiology of
periodontal diseases, it was apparent from the very early days that
periodontal microbiology suffered from what Staley and Konopka
described as the “great plate count anomaly”, in that these cultur-
able bacteria were only a minor part of what was visible under the
1
microscope. For nearly a century, much effort has been devoted to
finding the right tools to investigate this uncultivated majority, also
known as “microbial dark matter”.
The discovery that DNA was an effective tool with which to “see”
microbial dark matter was a significant breakthrough in environmen-
tal microbiology, and oral microbiologists were among the earliest
to capitalize on these advances. By identifying the order in which
nucleotides are arranged in a stretch of DNA (DNA sequencing) and
creating a repository of these sequences, sequence databases were
created. Computational tools that used probability-driven analysis
of these sequences enabled the discovery of new and unsuspected
species and ascribed novel functions to these species. The discovery
that the evolutionary history of prokaryotes was recorded in cer-
tain molecules and that mapping this evolutionary distance was a
robust method to identify bacteria at the level of species led to a
new branch of study: metataxonomics. The philosophy that host-as-
sociated bacteria live in organized, cooperating communities led to
advances in studying the entire collection of microbial genes in an
© 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Periodontology 2000. 2020;00:1–16. 
ecosystem: the field of metagenomics. Here, we review the philo-
sophical, technological, and methodological advances that led to the
development of DNA sequencing as a tool to interrogate microbial
communities and how this contributed to advancing our understand-
ing of periodontal health and disease.
1.1 | Why sequence?
Our knowledge of the role played by bacteria in the etiology of
periodontal diseases has co-evolved with the development of tech-
niques and technologies for bacterial identification and characteri-
zation. The earliest evidence that dental plaque contains bacteria
came from the light microscopy-based observations of Anton von
Leewenhoek over 400 years ago. 2 He called them “animalcules” and
drew what we today recognize as fairly accurate representations
of cocci, bacilli, and fusiform bacteria, and even some mobile and
gliding bacteria. The work of Robert Koch, Fannie Hesse, and Julius
Petri enabled isolating pure cultures of bacteria.3 These methods,
when applied to characterizations of periodontal microbiota, pro-
pelled the discovery of cultivable species associated with gingivitis
and periodontitis, as well as periodontal health. Work by Noguchi,4
Mcintosh et al,5 Socransky et al,6 and Rosebury and Reynolds7 in
developing anaerobic cultivation methods quickly led to the discov-
ery of many anaerobic bacteria associated with periodontal disease.
Large-scale cultivation studies by Moore and Moore8 began the age
of “We know what we can grow”. Using 51 000 isolates obtained
from cases of periodontal health, adult gingivitis, and periodontitis
in circumpubertal and adult individuals, certain patterns of bacterial
colonization were identified.8 Actinomyces and streptococci played
a primary role in colonization of the tooth surface, and Fusobacterium
nucleatum emerged as a principal and frequent initiator of gingival
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2       KUMAR et al.
inflammation. These studies identified 28 nonspirochetal species,
five treponemal species, and at least one Mycoplasma species as im-
portant initiators of destructive periodontitis. These species would
later form the basis of the DNA-DNA checkerboard (see below).
However, when results from microscopy-based methods were cor-
related with cultivation-based approaches, it quickly became appar-
ent that a large fraction of organisms in the environment, including
the mouth, were uncultivated. Staley and Konopka1 called this the
“great plate count anomaly”.
As we moved away from the “single culprit” (eg, tuberculosis,
typhoid, cholera, malaria) to the “multiple culprit” model of disease
causation, “similarity” between organisms became an important
method of identifying potential pathogens. To do this, microbiolo-
gists of the late 1800s and early 1900s adopted principles that were
being used to classify eukaryotes.9 However, this was highly inef-
fective; unlike prokaryotes, eukaryotes such as plants and animals
are rich in morphologic and behavioral detail which can be used to
place them on the phylogenetic tree. One classic example is the mis-
10
classification of archaea as bacteria. These ancient organisms are
morphologically similar to bacteria and were known as archaebac-
teria based on cultivation and microscopic characterization. But it
is now established that these organisms are genetically more similar
to eukaryotes than to bacteria and have been placed in a domain of
their own.
Pioneering work by Zuckerkandl11 demonstrated that genetic
material (DNA, RNA, polypeptides) can be used to identify bacteria
with a much greater degree of accuracy than phenotypic character-
istics. According to the theory of “emergent evolution”, the first liv-
ing cell arose once a certain level of protein organization had been
9
achieved in the “primordial soup”. As the cell grew more organized,
and developed more specialized functions, the story of its evolution
was captured in certain molecules within its organization. Thus, the
modern cell is a record of its own evolutionary history. In this regard,
the DNA molecule is considered the first nucleic acid polymer to
evolve, as it carries both the memory of and the information needed
to create a protein. Evolution from the first DNA-containing cell to
the modern-day cell occurred through a series of mutations, each
of which had an incremental impact on cell physiology. The greater
the number of mutations in the DNA, the greater the changes to cell
organization and metabolism. Therefore, two organisms that share
the same evolutionary history will have more similarities in their
DNA, and therefore in their properties and behavioral attributes.
Zuckerkandl12called these molecules semantides and set forth the
following arguments to support the use of semantophoretic mole-
cules to assign phylogenetic relationships:
1. DNA is a linear permutation of one of four nucleotides (ad-
enine, cytosine, guanine, and thymine) and the amino acids
that they encode can exist in one of 20 states. This allows
for objective comparisons to be made between the sequences
of two organisms.
2. Evolutionary changes that impact the organization and function
of a cell typically occur through substitution of one amino acid in
50-300, or, based on the triplet code, one in 150-900 nucleotides.
Therefore, DNA is an excellent repository of not only evolution-
ary memory, but also the drivers of that evolutionary change.
And thus began an era of molecular typing methodologies. Early
techniques largely used restriction enzymes to digest DNA and
compared the resulting banding patterns as measures of genetic
relatedness.13-16 Plasmid DNA digests, restriction fragment length
polymorphism and terminal restriction fragment length polymor-
phism, pulsed-field gel electrophoresis, and ribotyping are all exam-
ples of restriction enzyme digestion.17 When these methods were
used to answer questions about periodontal pathogens, several im-
portant lessons were learnt:
1. Bacterial species demonstrate a variable amount of heteroge-
neity in their DNA content. This led to the identification of
“clonal types” based on differences in band patterns generated
on a gel following restriction enzyme digestion.18 For exam-
ple, Aggregatibacter actinomycetemcomitans demonstrated limited
genetic heterogeneity, with no more than two clonal types in
any one patient, while F. nucleatum demonstrated 5-18 clonal
types, with different clonal types emerging following successful
treatment of periodontal disease.
2. Clonal types of certain, but not all, bacterial species can be verti-
cally transmitted from mother to child, however, a similar pattern
is not observable with fathers. An organism that has been very
well studied in this regard is Streptococcus mutans.
3. Even although bacteria demonstrate prolific capabilities for hori-
zontal gene transmission, they exist as discrete taxonomic enti-
ties, and characterization of genetic diversity within species can
provide information regarding their role in pathogenesis.
Another popular method was DNA-DNA hybridization (pop-
ularly known as checkerboard), in which digoxigenin-labeled sin-
gle-stranded DNA from cultivated species were immobilized on a gel
and an environmental sample was hybridized to it.19 Because of the
need to obtain genomic DNA, this methodology was restricted to
cultivated species, thereby further perpetuating the “We know what
we can grow” paradigm. The 33 species (28 nonspirochetal species
and five treponemes, see above) that had been identified through
culturing were the ideal candidates for this method. Over 350 pub-
lications resulted from this approach, and we learnt much about the
abundances of these selected species in health and disease, as well
as shifts in their abundances following surgical and nonsurgical in-
terventions. However, these individual species are part of complex
communities and their role in disease causation or perpetuation can
only be fully understood when studied in an ecological context. This
was quickly borne out when their ability to act as diagnostic or prog-
nostic markers of periodontal diseases was tested and yielded no
consistent results.
It soon became apparent that the single-culprit or multiple-cul-
prit model of pathogenesis did not explain the variation in disease
incidence, severity, or progression and that cultivation, microscopy,
KUMAR et al. |
      3

F I G U R E 1   First-generation DNA sequencing technologies. Example DNA to be sequenced. (A) is illustrated undergoing either Sanger
(B) or Maxam-Gilbert (C) sequencing. (B) Sanger's “chain-termination” sequencing. Radio- or fluorescently labeled dideoxynucleotides of
a given type—which once incorporated, prevent further extension—are included in DNA polymerization reactions at low concentrations
(primed off a 5′ sequence; not shown). Therefore in each of the four reactions, sequence fragments are generated with 3′ truncations as a
dideoxynucleotide is randomly incorporated at a particular instance of that base (underlined 3′ terminal characters). (C) Maxam and Gilbert's
“chemical sequencing” method. DNA must first be labeled, typically by inclusion of radioactive P 32 in its 5′ phosphate moiety (shown
here by Ⓟ ). Different chemical treatments are then used to selectively remove the base from a small proportion of DNA sites. Hydrazine
removes bases from pyrimidines (cytosine and thymine), while hydrazine in the presence of high salt concentrations can only remove those
from cytosine. Acid can then be used to remove the bases from purines (adenine and guanine), with dimethyl sulfate being used to attack
guanines (although adenine will also be affected to a much lesser extent). Piperidine is then used to cleave the phophodiester backbone
at the abasic site, yielding fragments of variable length. (D) Fragments generated from either methodology can then be visualized via
electrophoresis on a high-resolution polyacrylamide gel: sequences are then inferred by reading “up” the gel, as the shorter DNA fragments
migrate fastest. In Sanger sequencing (left) the sequence is inferred by finding the lane in which the band is present for a given site, as the
3′ terminating labeled dideoxynucleotide corresponds to the base at that position. Maxam-Gilbert sequencing27 requires a small additional
logical step: Ts and As can be directly inferred from a band in the pyrimidine or purine lanes, respectively, while G and C are indicated by the
presence of dual bands in the G and A + G lanes, or C and C + T lanes, respectively. (Reused with permission from25)
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4       KUMAR et al.

F I G U R E 2   Second-generation DNA sequencing parallelized amplification. (A) DNA molecules being clonally amplified in an emulsion
PCR. Adapter ligation and PCR produces DNA libraries with appropriate 5′ and 3′ ends, which can then be made single-stranded and
immobilized onto individual suitably oligonucleotide-tagged microbeads. Bead-DNA conjugates can then be emulsified using aqueous
amplification reagents in oil, ideally producing emulsion droplets containing only one bead (illustrated in the two leftmost droplets, with
different molecules indicated in different colors). Clonal amplification then occurs during the emulsion PCR as each template DNA is
physically separate from all others, with daughter molecules remaining bound to the microbeads. This is the conceptual basis underlying
sequencing in 454, Ion Torrent, and polony sequencing protocols. (B) Bridge amplification to produce clusters of clonal DNA populations in a
planar solid-phase PCR reaction, as occurs in Solexa/Illumina sequencing. Single-stranded DNA with terminating sequences complementary
to the two lawn-oligos will anneal when washed over the flow-cell, and during isothermal PCR will replicate in a confined area, bending
over to prime at neighboring sites, producing a local cluster of identical molecules. (C) and (D) demonstrate how these two different forms
of clonally amplified sequences can then be read in a highly parallelized manner: emulsion PCR-produced microbeads can be washed over a
picotiter plate, containing wells large enough to fit only one bead (C). DNA polymerase can then be added to the wells, and each nucleotide
can be washed over in turn, and deoxynucleotide incorporation monitored (eg, via pyrophosphate or hydrogen ion release). Flow-cell–bound
clusters produced via bridge amplification (D) can be visualized by detecting fluorescent reversible-terminator nucleotides at the ends of a
proceeding extension reaction, requiring cycle-by-cycle measurements and removal of terminators. (Reused with permission from25)

or targeted DNA-based approaches such as PCR or checkerboard
were inadequate for capturing the richness and diversity of the oral
microbiome. One of the watershed moments in oral microbiologi-
cal research was the discovery that 60% of oral bacteria were as of
yet uncultivated, and that 33% of oral bacterial species had been
misclassified using phenotypic characterization methods. 20 Hence
began the era of sequencing in oral microbiology.
2 |  H I S TO RY O F S EQ U E N C I N G
Although Watson and Crick 21 deduced the double-turn helix struc-
ture of DNA using x-ray crystallography in 1953, the order in which
nucleotides were present in DNA could not be discerned until
1965, when Holley et al22 decoded the sequence of alanine trans-
fer RNA from Saccharomyces cereviciae. At the same time, Sanger
et al23 used two-dimensional fractionation to develop a collection
of ribosomal and transfer RNA sequences from various organisms.
Wu and Taylor's24 discovery that radioactive nucleotides could be
incorporated into DNA using DNA polymerase enabled the order
(or sequence) of nucleotides in a DNA fragment to be inferred (re-
viewed by Heather and Chain25). Sequencing was revolutionized in
1977 when Sanger et al26 developed the chain-termination (or dide-
oxy) technique. By incorporating radiolabeled chemical analogs of
deoxynucleotides into the reaction and identifying which one was
incorporated into the DNA strand, one could “read” the sequence of
a gene (Figure 1). This “first-generation” sequencing technique un-
derwent another innovation: a method to measure pyrophosphate
KUMAR et al. |
      5

during incorporation of deoxynucleotides was discovered, allow-
25
ing for multiple sequences to be analyzed in parallel (Figure 2).
This “second-generation” sequencing technique, also known as
“pyrosequencing”, was soon replaced by a method in which the de-
oxynucleotides are immobilized to a flow-cell surface, and barcoded
DNA is flowed over it (Figure 3). This method, also known as “next
generation” or “bridge sequencing”, allowed for massively parallel
sequencing of multiple samples (HiSeq and MiSeq are examples of
sequencing platforms that use this method). Sequence-based char-
acterizations of microbial communities can be achieved through
one of two techniques: amplicon sequencing or whole-genome
shotgun sequencing.
Amplicon sequencing refers to a method by which specific genes
(or regions within genes) are sequenced and compared with a data-
base for bacterial identification. Certain characteristics are central
to the success of amplicon sequencing methodology:
1. The gene is a viable “molecular clock”.9 A molecular clock is
any molecule whose sequence has undergone random changes
over certain periods of time. By comparing two or more species
carrying the same molecule, it becomes possible to measure
when their DNA began to diverge. Changes in sequence are
computed as a product of the rate at which the mutation
occurs in a population, and the time over which these changes
have occurred.28
2. The gene is ubiquitous. In order to make comparisons between
different species, it is important that all species possess this gene.
3. The gene carries out a sufficiently important cellular function that
its sequence is conserved.
Historically, several molecules have emerged as molecular clocks,
for example, the genes encoding elongation and initiation factors for
translation, RNA polymerase subunits, ATPase subunits, DNA gy-
rases, recA, heat-shock proteins, cytochrome c, and ribosomal RNA
genes. However, the 16S subunit of the ribosomal RNA gene, which
encodes the small subunit (30S) of the ribosome, 29 became the ideal
candidate because
• it is present in all organisms
• the ribosome is a central organelle in cellular function, ensuring
that the gene is conserved
• mutations occur in different parts of the gene at different rates,
enabling us to compute phylogenetic distances accurately
• they are large-sized genes (~ 1600 base pairs)
• there are fewer secondary structures (about 45 helical loops) in
the RNA, making it easy to sequence
• there are swathes of gene sequences that are conserved, making
it easy to design universal primers, while the nine hypervariable
regions (designated V1-V9) are documents of the phylogenetic
distances between species.
Comparison of 16S sequences led to the creation of the phylo-
genetic tree of life, 28 which demonstrated three distinct domains,
eukaryote, archaea, and bacteria. As sequencing technology im-
proved, it became possible to develop a collection of unambiguous,
full-length sequences, which improved the ability to identify spe-
cies using these genes. Curated sequence banks such as the Human
Oral Microbiome database, SILVA, greengenes, and the Ribosomal
Database Project are some of the classifiers that are in present use.30

F I G U R E 3   Third-generation DNA sequencing nucleotide detection. (A) Nucleotide detection in a zero-mode waveguide, as featured
in PacBio sequencers. DNA polymerase molecules are attached to the bottom of each zero-mode waveguide (*), and target DNA and
fluorescent nucleotides are added. As the diameter is narrower than the excitation light's wavelength, illumination rapidly decays traveling
up the zero-mode waveguide: nucleotides being incorporated during polymerization at the base of the zero-mode waveguide provide real-
time bursts of fluorescent signal, without undue interference from other labeled deoxynucleotides (dNTPs) in solution. (B) Nanopore DNA
sequencing as employed in Oxford Nanopore Technonologies's MinION sequencer. Double-stranded DNA gets denatured by a processive
enzyme (†), which ratchets one of the strands through a biological nanopore (‡) embedded in a synthetic membrane, across which a voltage
is applied. As the single-stranded DNA passes through the nanopore the different bases prevent ionic flow in a distinctive manner, allowing
the sequence of the molecule to be inferred by monitoring the current at each channel. (Reused with permission from25)
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6       KUMAR et al.
2.1 | What 16S sequencing taught us about the oral
microbiome in healthy individuals
Ten years after Kroes et al20 demonstrated that 48% of the oral
microbiome was uncharacterized, and that 37% of cultivated oral
species had been misidentified using phenotypic characterizations,
the Human Microbiome Project was undertaken.31,32 This effort ob-
tained 16S sequences from seven oral (buccal mucosa, hard palate,
keratinized gingiva, saliva, supra- and subgingival plaque, and tongue
dorsum) and two oropharyngeal sites (throat and palatine tonsils) of
182 individuals aged 18-40 years. Several studies were made pos-
sible as a result of technological advancements in high-throughput
deep sequencing as well as the development of bioinformatic pipe-
lines for sequence analysis. From all these studies, we learnt the
following:
1. The oral microbiome is second only to the gastrointestinal
tract in species richness and diversity. However, a “core” group
of organisms can be identified in the oral samples of 95% of
individuals.33,34 The number of “core species” varies from 22 in
saliva to 11 in supra- and subgingival plaque to seven on the
33
keratinized gingiva. However, the abundance of these “core
species” varies widely among individuals. Even although there
are five distinct colonization niches in the oral cavity, certain
species (belonging to the genera Fusobacterium, Streptococcus,
Veillonella, Granulicatella, and Gemella) are shared by all these
niches. This level of species uniformity is the greatest in the oral
cavity when compared with other body sites. On the other hand,
certain genera (notably Bacteroides, Prevotella, Corynebacterium,
Fusobacterium, Pasteurella, and Neisseria) contain species that
are highly related phylogenetically yet demonstrate distinct pre-
dilections for specific oral sites. For example, Corynebacterium
matruchotii was identified in supragingival plaque of most indi-
viduals, while a close relative, Corynebacterium argentoratense,
was most frequently detected in saliva and the hard palate.
2. The oral microbiome plays an important role in vascular home-
ostasis, and the entero-salivary nitrate metabolic cycle is one
method by which it mediates its effects. Blood vessel diameter,
especially in the peripheral circulatory system, is controlled by
endothelial nitric oxide synthase, an enzyme whose activity is
catalyzed by nitric oxide. Humans do not have the capacity to pro-
duce nitric oxide from dietary nitrates and are entirely depend-
ent on their bacterial communities for this functionality. Certain
oral commensals are known to reduce salivary nitrates to nitrite.35
Moreover, using a daily antibacterial mouthwash results in a 90%
decrease in salivary nitrite levels, accompanied by a mean in-
crease of 3.5 mmHg in blood pressure.36 Together, these studies
attest to the significant role played by oral bacteria in controlling
peripheral blood flow.
3. Populations from different geographic locations demonstrate
distinct microbial profiles.37-40 Several rationalizations have
been proposed to explain these differences, including easier
person-to-person bacterial transmission in densely populated
areas, hunter-gatherer vs agrarian diet, and genotypic and ethnic
differences between cohorts. The geography of the oral cavity
itself appears to be a driver of microbial assemblages. Both tooth
location (molar vs incisor) as well as location on the tooth (buccal
vs lingual) are discriminants of community profiles, while mucosal
biofilms segregate along salivary flow gradients.41-44
4. Within the same geographic location, ethnicity is a determinant
of the oral microbiome, with the biggest differences between
African Americans and Caucasians.45 In fact, the difference be-
tween these two racial/ethnic groups living in the USA is greater
than that between either group and ethnicities from China or
Mexico. This pattern of microbial dissimilarity follows the differ-
ing prevalence of periodontal disease among these racial/ethnic
groups.46
5. There is persuasive evidence that the oral microbiome is a herit-
able feature, much like our genome. A recent study demonstrated
that the microbiome of predentate infants is a complete subset of
that of their mothers, and that 85% of this predentate microbiome
is retained throughout adult life.47 Newer studies are emerging
to show that children of parents with aggressive periodontitis ac-
quire a dysbiotic microbiome very early in life, and that this can-
not be altered through professional oral prophylaxis. However,
studies of twins reared together and apart demonstrate that both
genetics and environment can influence oral bacterial acquisition
and colonization.48,49
6. Anthropogenic behaviors play important roles in shaping the peri-
odontal microbiome. While the subgingival microbiome is stable
against small perturbations in the ecosystem such as food intake
(also known as pulses), sustained insults to the ecosystem (known
as pressed perturbations), for example, smoking,50-54 electronic
cigarette use,55 obesity,56 and alcohol use57 are all drivers of al-
tered community assemblages. The response of the subgingival
microbiome is specific to each type of exposure and has the ca-
pacity to customize the immuno-inflammatory machinery of the
host accordingly.
7. Systemic diseases and conditions play important roles in shifting
the subgingival microbial community structure.58 Among these,
there is strong evidence that pregnancy,59 rheumatoid arthritis,60
diabetes,61,62 chronic renal failure,63,64 pancreatic cancer,65 and
Fanconi's anemia66 all contribute to shifting species richness,
evenness, and diversity in unique ways. When anthropogenic in-
fluences are superimposed on these systemic diseases, the micro-
biome shifts in unpredictable ways.
8. Early studies using 16S sequencing reported that the periodontal
microbiome demonstrates a high degree of temporal stability over
a 2-year period in the absence of disease or other major perturba-
tions.67 However, several studies document temporal fluctuations
in the salivary68,69 and tongue70 communities over short or long
observation periods. Together, these data suggest that there is
significant interpersonal as well as location-dependent variability
in the stability of the oral microbiome.71
KUMAR et al.
2.2 | What 16S sequencing taught us about the
subgingival microbiome in periodontal disease
16S amplicon sequencing provided us with an unprecedented view
of the richness of the oral microbiome and enabled the discovery of
organisms that had never been cultivated or had been misidentified
during cultivation-dependent methods.72-74 It was initially estimated
that 60% of the subgingival microbiome74 was as of yet uncultivated,
however, this was later revised to 45%.73 The perspective provided
by Sanger sequencing, and later by next-generation sequencing,
taught us the following:
1. Oral dysbiosis differs from that of the gut, where disease is
accompanied by a reduction in the number of indigenous com-
mensals and an increase in density of one or more species.75 By
contrast, the subgingival microbiome of a periodontally healthy
individual contains 45-90 species,72,76 whose abundances do not
vary significantly following initial colonization. About one-third
of these species are influenced by the universal characteristics
of the subgingival environment and do not exhibit significant
interpersonal variability.33 By contrast, the defining elements
of established periodontitis are a microbiome that exhibits high
alpha and beta diversity (a greater number of species that
demonstrate a wide range of abundances)77 and significant
heterogeneity between individuals78 and within disease sites
79
in the same mouth. This microbial heterogeneity poses a
challenge to identifying microbial markers of disease onset or
progression.
2. A highly diverse and species-rich microbiome is associated with
80
aggressive periodontitis, especially localized aggressive peri-
odontitis. The microbiome not only includes bacterial species, but
also members of the domain Archaea (Methanobrevibacter oralis,
Methanobacterium curvum/congolense, and Methanosarcina mazeii)
81,82
and viruses.
3. The peri-implant sulcus provides a unique colonization niche for
oral bacteria, and the peri-implant microbiome is distinct from the
adjoining periodontal ecosystem.83-87 Peri-implantitis and peri-
53
implant mucositis are microbiologically similar entities, giving
rise to the paradigm that peri-implant mucositis is a pivotal event
in the progression to irreversible destruction of peri-implant
tissues.
3 | E VO LU TI O N O F M E TAG E N O M I C S
From an ecological perspective, 16S amplicon sequencing answers
the question: Who is out there? However, ribosomal genes contrib-
ute to <0.1% of the total genome, and hence, short-read amplicon
sequencing techniques do not resolve the question: What are these
communities capable of doing? This question is best answered by
determining the proteins encoded by the microbial community and
differentiating samples based on microbial functions rather than
type of species. The process of collecting genes from members of
|
      7
a microbial community from a specific environment was defined
“metagenomics” by Handelsman et al.88
The foundation for metagenomic exploration of human microbial
ecosystems was laid by pioneering studies on soil and ocean ecosys-
tems. 25,89 Venter et al90 carried out the first whole-genome shotgun
sequencing in the ocean ecosystem and identified 1.2 million un-
known genes and 148 previously unidentified bacterial phylotypes
(bacteria whose presence is only known by their DNA sequence).
Once community DNA has been sheared and the fragments
sequenced, one of two approaches is possible: genome-centric or
gene-centric. In the first approach, the fragments are arranged on a
scaffold to recreate complete bacterial genomes, and thereby esti-
mate the microbial content of the community. This is known as as-
sembly-driven metagenomics and it investigates the metabolic and
lifestyle capabilities of an ecosystem in the context of the organ-
isms that inhabit them. However, this approach requires substantial
sequencing depth to allow for meaningful genome assembly. In the
second approach, the information enclosed in the fragmented se-
quences can be used to deduce the metabolic and other functional
capabilities of a community, and two samples can be compared ei-
ther at the level of gene families, operons, or cellular processes. This
approach, known as comparative metagenomics, allows for compar-
ison of not only the functional capabilities of the two communities
but also the taxonomic composition of these communities, as taxon-
omy can be deduced from the 16S sequences in the metagenome or
from other marker genes.
In 2006, Gill et al91 characterized the functionalities encoded in
the gut metagenome of healthy humans for the first time. Metabolic
reconstruction of the gut microbiomes of two unrelated healthy
adults showed significant enrichment for genes involved in several
metabolic pathways that had not been previously reported: the me-
tabolism of xenobiotics, glycans, and amino acids, the production of
methane, and the biosynthesis of vitamins and isoprenoids through
the 2-methyl-D-erythritol 4-phosphate pathway. In another land-
mark study by Turnbaugh et al,92 the host phenotype (obesity) cor-
related with microbial genes and specific metabolic pathways. The
obesity-associated gut microbiome showed an increased capacity to
promote fat deposition. This study not only highlighted the merit
of comparative metagenomic studies but also indicated a way in
which in silico observations could be used to design translatable ex-
perimental models and in turn determine the mechanism of human
diseases.
3.1 | What comparative metagenomics taught us
about periodontal disease
It was not until 2012 that comparative metagenomic approaches en-
tered periodontal literature. A pilot study of 15 subgingival plaque
samples from two patients with periodontitis and three healthy
controls by Liu et al,93 using the Illumina sequencing platform
(2  × 76 base pairs paired end), reported taxonomic and functional
changes associated with periodontitis for the first time. Despite
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8       KUMAR et al.
limitations such as high levels (~90%) of contamination with human
DNA, and lower sequencing coverage, the authors were able to con-
struct several oral microbes, and preliminarily characterize some
system-level differences between periodontal health and disease.
Of note, they reported that the subgingival microbiome in periodon-
titis was characterized by the presence of previously uncultivated
TM7s and enrichment in several degradation pathways and virulence
factors.
In 2013, Wang et al94 used a deeper sequencing approach
(Illumina HiSeq 2 × 100 base pairs) for their comparative analysis
of 16 subgingival plaque samples from six healthy and five subjects
with periodontitis. They established a core-disease microbiota, iden-
tified almost 30 novel and previously unidentified species in the oral
microbiome, and reported the contribution of phages to disease in
this ecosystem. Additionally, they reported an overrepresentation
of several functional categories traditionally attributed to virulence
and pathogenicity such as bacterial chemotaxis, lipopolysaccharide,
flagellar assembly, and glycan biosynthesis.
Several microbial genes associated with virulence factors and
functional components related to amino acid metabolism, glycos-
aminoglycan, and pyrimidine degradation were enriched in peri-
odontitis in a targeted functional gene array approach with ~135
000 probes by Li et al.95 In a longitudinal study, Shi et al96 established
shifts in the functional potential of the subgingival microbiome in
response to treatment. Distinct shifts in taxonomic composition and
an increase in co-occurrence between bacterial members occurred
in the disease sites, but not in the resolved sites. Along with the
levels of anaerobes such as Campylobacter, Treponema, Tannerella,
and Porphyromonas, there were associated shifts in functions such
as chemotaxis and flagellar motility, which were associated with
disease sites (n = 12). Also, specific functions, for example, lysine
degradation, were overrepresented in disease sites, while lysine bio-
synthesis was overrepresented in shallow sites. When three patients
were followed up for a third visit (baseline with disease, after scaling
and root planing, maintenance visit) after therapy, the authors were
able to use the state of the microbiome at the second visit to predict
the clinical trajectory at the third visit with an accuracy of 81.1%.
The first comprehensive site-specific functional characterization
of the oral microbiome encompassing all the members of the eco-
system, including viral, fungal, and archaeal species in periodontal
health and disease, in a larger sample size with robust statistical com-
79
parisons was published by our group in 2016. This study was the
first undertaking in the field of oral microbiome research to: (a) es-
tablish a core metabolic network associated with periodontal health
and disease; (b) identify the discriminatory functional markers; and
(c) determine contributing members of the functional networks. Our
findings of upregulated virulence functions such as lipopolysaccha-
ride synthesis, iron metabolism, and bacterial chemotaxis in disease,
upheld the definition of the “disease microbiome” as described by
other groups using whole-genome shotgun sequencing approaches.
The disease state was also associated with an increase in abundance
of archaeal members that correlated with a corresponding increase
in anaerobic/fermentation pathways and increased functional
inter-kingdom cooperativity. Interestingly, we also identified that the
taxonomic and functional profiles of sites that were clinically healthy
in patients diagnosed with periodontitis were similar to active dis-
ease sites. This finding indicated a global dysbiosis in the ecosystem
and not a site-specific dysbiosis, as previously believed, thus shift-
ing the paradigm in periodontal microbial ecology. With significant
microbial heterogeneity, a predominance of specialist species, and
the presence of novel functions in a disease-associated microbiome,
we were able to identify 30 genes encoding 14 distinct functions as
discriminators of periodontitis.
3.2 | Strain-resolved metagenomics and what it can
teach us about periodontal disease
Metagenomics has traditionally been used to provide a global view of
microbial communities in the context of their environments. Recent
efforts, however, have been aimed at using this approach to exam-
ine gene content and the functional potential of bacterial strains.97
Microbial strains are low-level taxonomic units that maintain the
same phenotype under different exposures/conditions; however,
they show evident genotypic heterogeneity. In other words, the
same microbial species can have substantially different strains. The
argument made in favor of this approach is that bacterial strains are
the building blocks of any microbiome, and that accessory genes
present in only specific strains can encode important functions that
allow the organism to survive and thrive in a particular environment.
This strain-level heterogeneity can explain the differences in the
virulence of pathogens.98 Identifying strains from metagenomic data
can be performed by mapping metagenomic sequence reads against
reference genomes and obtaining a catalog of single-nucleotide vari-
ant patterns or marker genes across different samples or time points.
Several computational tools are available to facilitate this analysis.
Meta-multi locus sequence typing99 identifies a group of hypervari-
able loci in a species and creates a sequence type for each strain. By
comparing the metagenome under consideration with a database of
multi locus sequence types, not only can the data be resolved at the
strain level, but new sequence types which diverge from those in
the database can reveal the presence of novel strains. StrainPhlAn100
extends this approach to species for which at least one reference
genome is available. Pangenome-based phylogenomic analysis,101
on the other hand, uses the presence/absence of genes to create
a strain-specific gene repertoire. Lineage102 and ConStrains103 har-
ness the potential of single nucleotide polymorphisms within univer-
sal genes to identify strain-level differences. In another approach,
assembled contiguous sequences can be grouped into “bins”, rep-
resenting distinct organisms, known as metagenome-assembled ge-
nomes. De novo Extraction of Strains from MetAgeNomes104 uses
bins derived from de novo co-assembly of multiple samples to re-
solve sequence variants within core genes and designate them as
haplotypes and strains.
One of the earliest studies in the human microbiome
to document strain-level genomic variations along with
KUMAR et al.
genotypic-phenotypic interplay was published by Morowitz
105
et al in 2011. However, this study only investigated selected
taxa (Citrobacter spp.). A comprehensive single-nucleotide vari-
ant analysis of 252 samples from 207 individuals from Europe
and North America identified individual specific strains and es-
tablished temporal stability in the genomic variation patterns in
longitudinal samples.106 Consequently, in 2015 and 2016, sev-
eral groups utilized a marker-species or pan-gene approach to
track the strain-level variations in the metagenomes across dif-
ferent samples. However, attaining the accuracy and resolution
necessary to isolate genomes and reconstruct variants was still
challenging. In 2017, Truong et al107 utilized a novel strain identi-
fication approach in more than 1500 metagenomes from subjects
in five different continents and demonstrated: (a) subject-specific
strain variation; (b) stability of the identified strains in species; and
(c) geographic/host factor-specific variation in the microbial strain
structure. Strain variation in Bacteroides was the most consistent
while Prevotella copri was the most plastic colonizer.
While the literature related to strain-resolved metagenomics is
principally in the environmental and gut microbiomes, subject-spe-
cific, site-specific, and strain-specific colonization of the most prev-
alent oral commensal Neisseria has been demonstrated across the
different niches in the oropharynx. This work, published by Donati
et al108 in 2016, laid the foundations for future pan-genome stud-
ies in the oral cavity. In a study of oral, vaginal, and gut microbiome
109
in pregnant women, Goltsman et al were able to associate strain
variations in the oral microbiome with pregnancy complications such
as preeclampsia. Currently, few investigations explore strain reso-
lution in the oral metagenome, and no evidence of metagenomic
strain-level variations associated with periodontal health or disease
is available.
4 | E VO LU TI O N O F Q UA NTITATI V E
B I O LO G Y
One of the most consequential realizations in the field of molecular
biology was that genetic information (whether DNA, RNA, or protein)
could be recorded digitally as strings of characters, each represent-
ing a single base or amino acid. Once Frederick Sanger determined
the sequence of insulin,110 it quickly became clear that computers
would be essential in this arena, as multiple sequence comparisons
by hand would be impractical and, sure enough, developments in
sequencing were necessarily accompanied by advances in software
for making use of the resulting data.
The assembly of the human genome took a “shotgun” approach
where large insert clones were fragmented and sequenced, with each
sequence read assigned per-base quality scores and reassembled by
overlap consensus. The most common approach for achieving this
111
were the software packages Phred (whose scoring approach is
still used today), Phrap,112 Consed,113 and PolyPhred.114,115 Adjacent
clones were then aligned and assembled employing a guided ap-
proach using a Tiling Path map, with Gigassembler being the primary
|
      9
tool for implementing this approach to assemble original draft
human genome for the Human Genome Project.116
With improved efficiency and automation, and decreased costs
of sequencing, large-scale analysis of microbial communities became
a reality. The recognition that the information content of ribosomal
RNA (particularly the nearly universal 16S subunit), with its mix of
highly conserved and hypervariable regions, would provide a means
to identify organisms with unprecedented accuracy, provided “pow-
erful new tools to the microbial ecologist”.117 Now two approaches
could be pursued. First, databases of known sequences correspond-
ing to organisms could be compiled and searched. Indeed, in 1980
the Approved Lists recorded 1791 valid species. By 2007 that number
had increased to 8168,118 and the current release of the SILVA small
subunit non-redundant 99 database contains 510 984 sequences
separated by a 99% identity cutoff.119 Second, novel species could
be determined by a process of elimination against such databases
and by clustering with self-similar sequences from environmental
samples. Both approaches required new bioinformatic tools for com-
putational search and alignment techniques.
One of the first bioinformatic approaches developed for se-
quence similarity was the Needleman-Wunsch global alignment
technique.120 It is an optimal matching algorithm that assigns a score
(taking into consideration matches, mismatches, and gaps) to every
possible alignment between two sequences. While, practically, this
means that the algorithm is quite slow, it is still used today when
exact global alignment is a primary concern. In 1981, the Smith-
Waterman algorithm was proposed to perform local sequence align-
ment.121 As opposed to the global alignment approach, this algorithm
considers alignments of all lengths between two sequences. While
this approach was still quite slow, it allowed for much wider applica-
tion given the underlying biology and sequencing approaches. When
comparing two sequences, depending on the source and where the
sequencing happened to start, a global alignment is much less likely
than a partial, or local, alignment. Returning to targeted sequencing
of 16S ribosomal RNA, we can see that the combination of sequence
starting point and biological variation make local alignment a more
reasonable approach.
However, while local alignment provided expanded applicabil-
ity, the Smith-Waterman algorithm was impractical for the kind
of large-scale investigations enabled by expanding databases and
improved sequencing technologies. Consequently, it was gener-
ally replaced by heuristic-enabled algorithms that were less exact-
ing, but substantially more computationally efficient. The FASTA
algorithm122,123 published in 1985 was one of the more popular
of this type. In addition to DNA-DNA alignment, it allowed for
translated alignments for comparing DNA sequences with protein
databases. Soon after, in 1990, a breakthrough algorithm was pub-
lished: the basic local alignment tool, known as BLAST,124 which
has become one of the most widely used tools in bioinformatics.
Its emphasis on speed through its novel heuristic algorithm was
vital to enable searches of large databases such as GenBank.125,126
Part of its successful approach was the introduction of “seeding”,
where it quickly finds significant short matches (high-scoring
 |
10       KUMAR et al.
segment pairs) between two sequences aided by evolutionarily
127
guided scoring matrices (eg, BLOcks SUbstitution Matrix)
fore continuing to a more exact matching process by extending the
seeds to longer ungapped alignments, followed by gapped align-
ments.128 This screening process allowed it to rapidly discard large
numbers of sequences that could not provide a significant match.
Furthermore, a significant contributor to its widespread use
was making a web-accessible interface available on the National
Center for Biotechnology Information website.125,126,129
Following its initial release, the core algorithm has been im-
proved (basic local alignment search tool +),130 extended to enable
large simultaneous query sets (mega basic local alignment search
tool),131,132 to examine distantly related proteins (position-specific
133
iterative basic local alignment search tool), and has been central
processing unit-, graphics processing unit-, and field-programmable
gate-accelerated.134-136 In addition, it has inspired a subsequent ex-
plosion of heuristic local alignment algorithms, including Sequence
137
Search and Alignment by Hashing Algorithm, miBLAST,
BLAT,139 Bowtie,140 USEARCH,141 Bowtie 2,142 High Speed Basic
Local Alignment Search Tool Nucleotide,128 double index alignment
of next-generation sequencing data,143 and Many-against-Many se-
144,145
quence searching.
The wide applicability, speed, and accuracy of these tools has
seen them being commonly used for read-mapping analysis of
whole-genome shotgun sequencing (whole genome sequencing or
whole metagenome sequencing) data, where sequenced reads are
mapped to general or specialized sequence and protein databases
125,126
such as National Genetic Sequence Data Base, the pepti-
dase database,146 Kyoto encyclopedia of genes and genomes,147
1458,149 150,151
Pfam, Clusters of Orthologous Groups, Swiss-Prot,
SEED,153 Reference Sequence Database, 154
UniRef,155 UniProt,156
Virulence Factors Data Base,157 evolutionary genealogy of genes:
Non-supervised Orthologous Groups,158 Carbohydrate-Active
159
Enzyme database, database of carbohydrate-active enzyme
(CAZyme) sequence and annotation,160 and Integrated Microbial
Genome/Virus.161
While local alignment algorithms have achieved widespread
use across molecular biology, targeted sequencing and phyloge-
netic analysis require algorithms implementing multiple sequence
alignment, where three or more sequences (DNA, RNA, protein)
are compared simultaneously to determine sequence homology
and evolutionary relationships. The original approach is the pro-
gressive multiple sequence alignment method first introduced by
Feng and Doolittle.162 This method started with pairwise alignment,
using the Needleman-Wunsch algorithm mentioned earlier, to align
closely related sequences first then continuing to add more distantly
related sequences as the alignment grows. The CLUSTAL W algo-
rithm followed including additional heuristics and distance-based
clustering to improve alignments and speed, and its family of algo-
rithms are still the most popular of this type (eg, multiple alignment
163
using fast Fourier transform ). The software Multiple Sequence
164
Comparison by Log-Expectation represents further improvement
of the current generation by iteratively reassessing the alignment
of previously added sequences to avoid the problem of poor ini-
be- tial alignment choice in progressive alignment. Another variation in
these algorithms involves the use of hidden Markov models to con-
struct probabilistic inference models to assign likelihood scores to
sets of multiple sequence alignments. Biosequence analysis using
profile hidden Markov models165 is the most popular of this variety.
One issue with using local alignment algorithms for amplicon se-
quencing is that they attempt to find all homologous sequences at a
detailed level. While the basic local alignment search tool and simi-
lar algorithms were significantly faster than the previous generation
of optimal matching algorithms, they were not designed to identify
redundancy, precisely the result of targeted sequencing employing
PCR amplification. In moderate complexity environments such as the
oral cavity, or even high complexity environments such as oceans or
soils, the number of unique organisms is orders of magnitude smaller
than the number of sequences which can be derived from those en-
vironments, especially with the current generation of sequencing
138
machines. Thus, sequence clustering algorithms were employed or
developed to quickly reduce the number of sequences considered
for matching to databases with local alignment algorithms. One of
the first approaches was CD-HIT,166 which was created to reduce
the size of protein databases by eliminating highly homologous se-
quences. It selected the longest sequence and proceeded through
the rest of the data in decreasing order classifying sequences as re-
dundant or representative (new cluster), employing word indexing,
and counting tables to rapidly accomplish this task. A parallelized
version was released in 2012166 to handle the substantial increase
in sequencing depth provided by high-throughput sequencing tech-
nologies. UCLUST141 represented an improvement in speed and sen-
152
sitivity over CD-HIT by exploiting the fact that “similar sequences
tend to have short words in common”. By sorting sequences in de-
creasing order of common words, substantial improvements could
be achieved.
These tools were highly popular in 16S sequencing analysis, as
evidenced by their inclusion in the software pipeline for microbial
ecology, Quantitative Insights Into Microbial Ecology.167 The other
most widely used pipeline mothur168 employed the PHYLogeny
Inference Package169 to create a pairwise distance matrix between
sequences, followed by the unweighted-pair group method using
average linkage clustering to group the sequences, resulting in a
similar end product to CD-HIT and UCLUST. More recently, these
relatively general clustering algorithms have been replaced by highly
targeted algorithms focused on the characteristics of the 16S ribo-
somal RNA (or another amplicon sequencing target) gene as well as
the sequencing platforms themselves and their error distributions.
These algorithms belong to a new class that produce amplicon se-
quence variants170 as a successor to the operational taxonomic units
produced by the previous generation of clustering algorithms. The
tools taking this approach include UPARSE,171 Minimum Entropy
Decomposition,172 Divisive Amplicon Denoising Algorithm,173 and
deblur.174 By focusing on controlling sequencing error, these algo-
rithms can resolve sequences down to single nucleotide variants
without relying on arbitrary similar cutoffs (eg, 97%) and provide
KUMAR et al.
a means for comparing labels across studies without requiring
reclustering.
In the realm of 16S sequencing, there are four primary gen-
eral purpose (nonhabitat-specific) databases used for analysis175:
SILVA,176 the Ribosomal Database Project,177 the National Center
for Biotechnology Information,178 and greengenes.179 Each varies
in their data sources, number of included sequences, methods for
assigning and resolving taxonomic assignment, covered domains of
life, included ribosomal targets, and frequency of updates. SILVA
focuses on classifications for bacteria, archaea, and eukarya, cu-
rating 16S and 18S sequence data primarily from International
Nucleotide Sequence Database Collaboration members. Taxonomic
labeling is based on Bergey's Manual of Systematic Bacteriology,180
181
List of Prokaryotic names with Standing in Nomenclature, and
the International Society of Protistologists, but is ultimately man-
ually curated.175,176 The Ribosomal Database Project covers bacte-
ria, archaea, and fungi, differing from SILVA by inclusion of the 28S
marker. It sources sequence data from GenBank and the European
Bioinformatics Institute as well as from direct submissions. As
with SILVA, taxonomy in the Ribosomal Database Project is based
on Bergey's Manual of Systematic Bacteriology and LPSN, but fun-
175,177
gal assignment is conducted through their own efforts. The
National Center for Biotechnology Information database is solely
focused on submitted 16S sequence data. Bacteria and archaeal
sequence data are available as separate BioProjects for download
(PRJNA33175 and PRJNA33317, respectively). For identification
purposes, a pre-built BLAST database is also provided.175,178 It uti-
lizes manual curation of naming based on over 150 sources, both
general and organism-specific. Its efforts are an ongoing process
in conjunction with data submissions, and daily updates are issued.
Finally, greengenes is similarly focused on 16S sequences for bac-
teria and archaea. Taxonomic classification is drawn primarily from
National Center for Biotechnology Information in conjunction
with de novo phylogenetic tree construction utilizing alignments
175
based on sequence and secondary structure.
Although there are many general similarities between the da-
tabases in their sources for data and naming, the exact sequences
included, filtering and chimera-checking, tree creation, and simple
human error, all combine to create differences in assignment accu-
racy (outside of tool choice). Even from a simple naming perspective
there are substantial differences. In their recent article, Balvočiūtė
175
and Huson attempted mapping each of these databases to each
other by simple phylogeny. They found that 63%-90% of taxa at
various levels of the taxonomic ranks were unique to one of the
four databases. National Center for Biotechnology Information and
SILVA shared the most with each other: 60% at the phylum, class,
and order ranks, and 10% at the genus and species ranks. However,
National Center for Biotechnology Information appeared to be the
most inclusive of all the databases. Another recent study published
182
by Park and Won utilized a mock community analysis to compare
assignment accuracy among SILVA, greengenes, and EzBioCloud183
(no longer publicly available). Among each of the databases they
reported up to 20% false-positive assignments at the genus level.
|
      11
They attributed these differences to database size, sequence source
(amplicon vs genome assemblies), and sequences with missing taxo-
nomic information.
4.1 | How sequencing analysis
pipelines and bioinformatics shaped our
understanding of the oral microbiome
The oral microbiome is known to harbor between 600 and 700 spe-
cies of bacteria, with some estimates as high as 1200.76 Archaeal and
viral species are also present, but less well characterized; Gannoum
et al184 reported 101 fungal species. Furthermore, of the known bac-
terial constituents, ~ 30% have not yet been cultivated.73 In order to
provide comprehensive and reliable references for studies involving
the oral cavity, given the issues discussed, efforts have been taken
to develop oral-specific databases. Two habitat-specific databases
have been created to fill this role: the Human Oral Microbiome
Database185 and CORE, a “phylogenetically curated 16S rDNA data-
base of the core oral microbiome”.186
The Human Oral Microbiome Database aims to provide both a
resource for taxonomic identification as well as functional charac-
terization of oral samples by providing both 16S and whole genome
reference databases. It was originally created to provide a “stable tax-
onomic structure for the unnamed oral taxa” by introducing a human
oral taxon ID, such that each named species and phylotype cluster is
assigned an ID and therefore independent of naming. The original da-
tabase was constructed from 600 16S ribosomal RNA gene libraries
and over 35 000 clone sequences. As per the latest release, version
15.2 (2019/09/02), the Human Oral Microbiome Database contains
1015 full-length 16S sequences, 1570 complete and draft genomic
sequences, and has been expanded to represent the aero-diges-
tive tract; it is now called the expanded Human Oral Microbiome
Database.187 The CORE database was created with a similar objec-
tive, and with a focus on correcting classification and naming incon-
sistencies in oral species found in existing 16S databases. The initial
data source was a combination of known named and cultured oral
species from GenBank and 20 000 Sanger-generated 16S sequences
collected from 200 human subjects, ultimately resulting in 1043
sequences representing 636 species.186 In both cases, the authors
reported substantial improvements in oral sample characterization
over the general purpose databases discussed above.185-187 As with
any database, however, an unavoidable issue is its finite nature.
Known oral species mined from the literature are necessarily re-
stricted by the patients and study subjects they were isolated from.
With known differences in microbiome composition by ethnicity and
geography,45,188 certain study designs may benefit by incorporating
multiple databases in an analysis. Indeed, with any single database, it
can be difficult to know whether an unidentified sequence is simply
missing, represents a novel organism, or is the result of sequenc-
ing error. Lourenço et al189 studied the oral and gut microbiomes in
individuals with and without periodontal disease. Utilizing both the
greengenes and Human Oral Microbiome databases, they reported
 |
12       KUMAR et al.
the presence of pathogenic species associated with inflammation
and periodontal tissue destruction in the gut microbiomes of their
subjects regardless of periodontal health status. Shi et al96 extracted
16S sequences from whole-genome shotgun sequencing data and
utilized both the Human Oral Microbiome and CORE databases, as
well as SILVA. To verify these taxonomic results, they constructed
a customized database of genomic sequences and mapped their
whole-genome shotgun sequencing reads against it. Ultimately,
they found the results of each of the methods to be consistent with
each other. Another recent study utilized a similar approach with the
stated goal of increasing the resolution in an understudied group:
individuals of Arab heritage. Al-Hebshi et al190 combined modified
versions of the Human Oral Microbiome database, the expanded
Human Oral Microbiome database, and greengenes based on their
previous experiences with low resolution results using a single da-
tabase. As sequencing becomes less and less expensive and more
globally available, databases will certainly improve, but the issues we
have discussed should be kept in mind when designing and executing
future studies.
5 |  CO N C LU S I O N
In summary, DNA sequencing methodologies have expanded our
understanding of the oral and periodontal microbial ecosystem ex-
ponentially. While these methods are not without bias or error, they
provide a means to identify previously unknown or unsuspected
species in these niches, explore their contributions to health and
their role in disease etiology, and pave the way for targeted, preci-
sion therapeutics.
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How to cite this article: Kumar PS, Dabdoub SM, Ganesan
SM. Probing periodontal microbial dark matter using
metataxonomics and metagenomics. Periodontol 2000.
2020;00:1–16. https://doi.org/10.1111/prd.12349