Identification and Quantification of Abundant Species from Pyrosequences of 16S rRNA by Consensus Alignment.
ABSTRACT 16S rRNA gene profiling has recently been boosted by the development of pyrosequencing methods. A common analysis is to group pyrosequences into Operational Taxonomic Units (OTUs), such that reads in an OTU are likely sampled from the same species. However, species diversity estimated from error-prone 16S rRNA pyrosequences may be inflated because the reads sampled from the same 16S rRNA gene may appear different, and current OTU inference approaches typically involve time-consuming pairwise/multiple distance calculation and clustering. I propose a novel approach AbundantOTU based on a Consensus Alignment (CA) algorithm, which infers consensus sequences, each representing an OTU, taking advantage of the sequence redundancy for abundant species. Pyrosequencing reads can then be recruited to the consensus sequences to give quantitative information for the corresponding species. As tested on 16S rRNA pyrosequence datasets from mock communities with known species, AbundantOTU rapidly reported identified sequences of the source 16S rRNAs and the abundances of the corresponding species. AbundantOTU was also applied to 16S rRNA pyrosequence datasets derived from real microbial communities and the results are in general agreement with previous studies.
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Identification and Quantification of Abundant Species from Pyrosequences of 16S
rRNA by Consensus Alignment
School of Informatics and Computing, Bloomington, IN 47408, U.S.A
Abstract—16S rRNA gene profiling has recently been boosted
by the development of pyrosequencing methods. A common
analysis is to group pyrosequences into Operational Taxonomic
Units (OTUs), such that reads in an OTU are likely sampled
from the same species. However, species diversity estimated
from error-prone 16S rRNA pyrosequences may be inflated
because the reads sampled from the same 16S rRNA gene
may appear different, and current OTU inference approaches
typically involve time-consuming pairwise/multiple distance
calculation and clustering. I propose a novel approach Abun-
dantOTU based on a Consensus Alignment (CA) algorithm,
which infers consensus sequences, each representing an OTU,
taking advantage of the sequence redundancy for abundant
species. Pyrosequencing reads can then be recruited to the
consensus sequences to give quantitative information for the
corresponding species. As tested on 16S rRNA pyrosequence
datasets from mock communities with known species, Abun-
dantOTU rapidly reported identified sequences of the source
16S rRNAs and the abundances of the corresponding species.
AbundantOTU was also applied to 16S rRNA pyrosequence
datasets derived from real microbial communities and the
results are in general agreement with previous studies.
keywords—16S rRNA gene; pyrosequencing; Operational Tax-
onomic Unit (OTU); abundant species
16S rRNA gene profiling has been applied to the anal-
ysis of complex microbial populations since the middle
1990s , but has recently been boosted by advances in
sequencing techniques that can produce large 16S rRNA
datasets containing hundreds thousands of 16S RNAs frag-
ments spanning the hypervariable regions of 16S rRNA
genes, enabling deep views into hundreds of microbial
communities . 16S rRNA pyrosequencing studies have
revealed much greater species diversity in many environ-
ments (e.g., soils, ocean water, and human bodies) than
previously anticipated—although it was shown in one study
 that some of the projects may overestimate the species
diversity—and have great potential in a variety of applica-
16S rRNA pyrosequences can be mapped onto the phy-
logenetic tree of known 16S rRNA sequences to provide
a view of the taxonomic distribution of the species they
represent. In this type of approach, however, 16S rRNA
pyrosequences can only be mapped to known species or
branches. Alternatively, taxonomy independent analysis can
be applied when sequences are classified into Operational
Taxonomic Units (OTUs) of specified sequence variations,
although it is still arguable which similarity cutoff should be
used to define species (or genus) . Typically, sequences
with < 3% dissimilarity are assigned to the same species,
while those with < 5% dissimilarity are assigned to the same
Typical methods of OTU inference cluster sequences into
groups—either through pairwise comparison or multiple
alignment, followed by sequence clustering, as in mothur
 and ESPRIT , or derived by heuristic clustering of se-
quencing reads as in FastGroupII  and CD-HIT —
and then a consensus sequence may be derived for each
group of sequences. The pitfalls of these approaches are: 1)
without knowing the consensus sequence (central sequence)
of a group, sequences may be mistakenly classified into
the group; 2) deriving consensus sequence from a group of
sequences is nontrivial, often requiring a multiple sequence
alignment; and 3) clustering algorithms often involve all-
against-all pairwise comparison of the reads, which requires
large memory and long computational time .
A fast approach, AbundantOTU, is proposed that first
finds consensus sequences, without any clustering, followed
by recruitment of reads to the consensus sequences. A
consensus sequence may represent an OTU, and thus the
reads recruited to the same consensus sequence can contain
sequences of different strains of the same species, or error-
prone reads from the same strain. This approach was inspired
by the fact that if one knew the species composition of a
microbial community, it would be easy to assign error-prone
reads to their source 16S rRNAs. AbundantOTU utilizes the
redundant sequence information (even though the reads may
contain sequencing errors), so that consensus sequences can
be derived by a Consensus Alignment (CA) algorithm. For
low abundant sequences, it is difficult to determine if they
are sampled from rare species (they represent rare species),
or if they cannot be recruited due to high sequencing errors.
Although characterizing rare species is not the focus of
the paper, AbundantOTU can be applied first to group
the abundant sequences, and then the remaining sequences
(typically a small proportion of the original sequences) can
be further analyzed by using other tools, such as mothur 
and PyroNoise .
using consensus alignment. (a) Consensus alignment by using a dynamic
programming algorithm, adding one nucleotide at a time. (b) Abundant
OTU inference by deriving consensus sequence and recruiting reads to the
consensus sequence iteratively.
A schematic demonstration of AbundantOTU algorithm by
AbundantOTU starts by finding the consensus sequence of
a group of sequences by a consensus alignment algorithm,
followed by assigning sequences to the consensus sequence
(Fig. 1). It may also be used as a generic tool for con-
sensus sequence inference for a predefined group of DNA
sequences. The consensus alignment algorithm is similar
to the algorithm that was proposed by Li and colleagues
for finding similar regions in many strings  and an
algorithm that was developed for repeat detection in genomic
A. Consensus alignment algorithm
Given a collection of m pyrosequences S = s1,s2,...,sm,
the consensus alignment problem is to find the optimal
consensus sequence that is similar to the most input se-
quences. Denote a consensus sequence as sc. The consensus
alignment evaluates the similarity between the consensus
sequence and all of the input sequences by an objective
similarity function as,
where Sim(sc,si) is the sequence similarity between the
consensus sequence sc, and an input sequence si.
To obtain a consensus sequence with the optimal sim-
ilarity function, a frequent l-mer (l = 40 by default) is
first identified in the input sequences, using the hashing
technique. The frequent l-mer, which serves as a seed,
can then be extended forward and backward to obtain the
entire consensus sequence. A greedy strategy is adopted that
adds one nucleotide at a time with the highest similarity
between the growing consensus sequence with the input
sequences that share the same seed (Fig. 1a). The forward
and backward extension can be achieved by using the same
algorithm. Here I use the forward extension as an example
to illustrate the algorithm.
Assume k − 1 nucleotides has been extended to the
consensus sequence, denoted as sc
niis the nucleotide at position i; note here the position index
is relative to the end of the seed). The optimal nucleotide
(either A, T, C or G) to be added at position k in the
consensus sequence, nk, can be computed as,
Here ? is the alignment bandwidth that is used to speed up
the alignment between the consensus sequence sc
pyrosequence si(i.e., position k of the consensus sequence
will be allowed to align to a limited number of positions
of sequence i). We used ? = 5 by default, considering that
sequences grouped in the same species-level OTU can not
differ too much from each other1. Also, only the sequences
that have the seed are compared to the consensus sequence.
The best alignment score between the consensus sequence
and sequence i with aligned positions of k (in the consensus
sequence) and j (in sequence i) can be computed by a
dynamic programming algorithm as follows.
j) = max
j−1) + Score(n,si
j) − g
j−1) − g
nucleotides n and si
consider a simple scoring function: Score(n,si
n = si
The initialization of the alignment is as follows.
j) is the similarity score between two
j, and g is the gap penalty. Here we
j) = 1 if
j, and 0 otherwise.
j) = −g ∗ j ∀ 0 ≤ j ≤ ?
k−?−1) = −∞ ∀ k > 1
B. OTU inference
OTU inference can be achieved by applying iteratively
the consensus alignment algorithm until no more consensus
sequences can be found that recruit a minimum number of
reads (here 5 is used, considering that fewer reads will be
insufficient for the inference of their consensus sequence),
1Note that it takes exponential time to explore all possible sequences if
the seeds are extended rigorously.
as shown in Fig. 1b. Once a new consensus sequence is
found, all the reads that are nearly identical (e.g., with
sequence difference ≤ 3%) to the consensus sequence are
recruited to the consensus sequence, and assigned to the
same species-level OTU. Note a read that does not share the
same seed as the consensus sequence (so was not used for
defining the consensus sequence) can still be recruited to the
same consensus sequence as long as their overall sequence
difference is ≤ 3%. Finally, the consensus sequences that
recruit < 5 reads each are discarded.
C. Computational time of AbundantOTU
For a single step of consensus alignment, the computa-
tional complexity is O(?Lmseed), where ? is the bandwidth,
L is the average length of a consensus sequence (which is
approximately the average length of the input sequences),
and mseed is the total number of input sequences that
contain the seed to be extended (mseed ≤ m; m is the
total number of input sequences). As ? is a small constant
(5 by default), the computational complexity of a single
step of consensus alignment is equivalent to O(Lmseed).
Since AbundantOTU involves iterative consensus alignment
until no more abundant OTU can be inferred, the total
computational complexity of AbundantOTU is O(kLmseed),
where k is the total number of consensus sequences that can
be inferred, a number that is typically much smaller than m,
the total number of input sequences.
D. Taxonomic analysis of the consensus sequences
Once the consensus sequences are derived, they can be
passed to other tools for further taxonomic analysis. One
of the advantages of using consensus sequences is that the
number of consensus sequences is significantly smaller than
the original pyrosequences, and thus dramatically decreases
the computational time of downstream analysis, such as
phylogenetic mapping using megablast or other rigorous
phylogentic mapping methods (; ). Here we used
the RDP online classifier (http://rdp.cme.msu.edu/) , and
BLAST search against Greengene 16S rRNA gene database
 downloaded from http://greengenes.lbl.gov/cgi-bin/nph-
index.cgi (as of Jan 20, 2010) for taxonomic assignments.
E. Benchmarks and tests
We tested AbundantOTU on two mock datasets, for
which the microbial composition and reference sequences
are known, and three metagenomic datasets derived from
real communities (see Table I for the summary of the
datasets). The first mock dataset (designated as Priest09)
is the ‘divergent sequence’ dataset from  that contains
amplified and pyrosequenced sequences from 23 divergent
16S rRNA fragments spanning V5 (the pyrosequences
dataset and reference sequences were downloaded from
The second mock dataset (designated as Mock07) contains
short sequences generated by pyrosequencing PCR amplicon
libraries of 43 known 16S rRNA gene fragments spanning
V6 using the Roche GS20 system, generated in a study
of sequencing errors . This dataset was downloaded
from http://genomebiology.com/2007/8/7/R143. The three
real metagneomic datasets contain reads from oral 
and skin  samples, respectively, downloaded from the
NCBI Short Read Archive (SRA) with accession numbers
SRR002260 (oral/plaque), SRR002259 (oral/saliva), and
The sources codes of AbundantOTU are available at
The AbundantOTU results are summarized in Table I.
The results show that AbundantOTU can generate consensus
sequences that are identical or very similar to the known
reference sequences in the mock communities. For the
16S rRNA datasets derived from environmental samples,
the results are in general agreement with previous studies.
Note that we focused on comparisons with methods that
do not require computationally expensive pairwise/multiple
alignments and/or clustering algorithms. For both CD-HIT
and FastGroupII, we used 97% similarity (i.e., 3% dissim-
ilarity) threshold. And our results show that AbundantOTU
tolerates sequencing errors and gives reliable estimations of
abundance of the species represented in the dataset. Due
to the page limit, we only show detailed analysis of two
A. Evaluation on mock community Priest09
Priest09 dataset contains reads sampled from the V5
regions of 23 divergent 16S rRNA genes. Since the reference
sequences are known, we mapped each of the reads to
the reference sequence with lowest distance to get the
expected abundance level for each of the reference se-
quences, using the crossmatch program from the phrap
package (http://www.phrap.org/). AbundantOTU reported 24
abundant OTUs (the least abundant OTU contains only 14
reads), which recruited most of the sequences included in
the dataset (99.9%). We also compared the representative
sequences of these abundant OTUs (their consensus se-
quences) to the known reference sequences. Overall, the
consensus sequences inferred by AbundantOTU are identical
or very similar to the known reference sequences: 5 are
identical to the reference sequences and 13 differ from the
reference sequences by one indel or mismatch (Fig. 2). By
contrast, the representative sequences derived by CD-HIT
are less similar to the known reference sequences. Further,
the abundance-rank curves of this database derived by three
different methods and the expected abundance-curve (Fig.
3) show that AbundantOTU produced the abundance rank
SUMMARY OF THE ABUNDANTOTU RESULTS OF FIVE DATASETS.
Number of abundant OTUs DatasetsNumber of reads Reads recruited toRunning time
The most abundant OTU
All abundant OTUs
OTUs that recruit at least 5 reads are considered as abundant; all the calculations were carried out on a linux computer (Intel Xeon 2.93GHz)
Number of reference sequences
reference sequences. The differences are measured as the total number of
mismatchs and indels involved in aligning a reference sequence with the
inferred sequence. The difference of 0 means that the inferred sequence is
identical to the corresponding reference sequence.
Comparison of the differences between the inferred and known
that is closest to the expected curve, whereas FastGroupII
tends to produce more but smaller OTUs.
B. Evaluation on a skin-associated microbial community
AbundantOTU analysis of the skin dataset revealed sev-
eral very abundant species (see Table II). The top species
identified by AbundantOTU are in general agreement with
the abundant species in the original report () (the top three
species are the same), with some exceptions. Consensus
sequence 8 and 9 were classified as from the same genus
but represent different species—these two sequences only
share 91% sequence identify. BLAST search shows that
consensus sequence 8 is identical to Streptococcus salivar-
ius, and consensus sequence 9 is identical to Streptococcus
sanguinis strain GumJ19. Interestingly, consensus sequence
6 is identical to a fragment in the Arachis hypogaea (peanut)
chloroplast rRNA gene, but there is no discussion about
whether reads sampled from chroloplast rRNA are present
or if these large number of sequences (more than 7000
sequences are recruited to this consensus sequence by Abun-
dantOTU) were filtered out in .
Note that the dataset we downloaded from NCBI SRA
contains 496,499 sequences, while the analysis presented in
Figure 3. The abundance-rank curves of the Priest09 dataset using different
methods. OTUs/clusters are plotted from most to least abundant along the
x-axis, with their abundances displayed on the y-axis. The curves only
show the high abundant OTUs/clusters. The reference curve shows the best
result that any method can achieve, in that the reference sequences are
known so that sequencing reads can be mapped to the references directly.
The AbundantOTU curve overlaps nicely with the reference curve.
SUMMARY OF THE TOP 10 MOST ABUNDANT OTUS IDENTIFIED BY
ABUNDANTOTU FROM THE SKIN DATASET
Note the consensus sequences were taxonomically assigned using the
online RDP classifier (http://rdp.cme.msu.edu/).
 was based on a filtered dataset that contains only 351,630
sequences, i.e., 71% of the original sequences (others did
not pass the quality control ). AbundantOTU revealed
144 abundant OTUs, which recruited 434,617 (87.5%) of
the pyrosequences. This suggests that AbundantOTU is
sequencing error tolerant, and can utilize sequences that
otherwise will be discarded due to sequencing errors.
AbundantOTU takes advantage of the redundancy of the
pyrosequences of 16S rRNA to infer the OTUs and their
representative sequences, using a consensus alignment algo-
rithm, assuming that there should be more reads that have
the correct nucleotide than reads with sequencing errors at
a particular position. As such, AbundantOTU is robust, and
less affected by sequencing errors with sequencing errors.
Since it relies on sequence redundancy, it cannot be used to
identify rare species, which are only represented by one or
very few sequences. It is challenging to infer rare species
correctly, since these species are barely represented by se-
quencing reads and sequencing errors may be difficult to spot
(if not impossible). AbundantOTU can report the sequences
that are not recruited to the abundant OTUs, so that further
analysis can be carried out on these ‘singleton” sequences.
But we believe that these rare sequences (sampled from rare
species or sequences from abundant species but with high
sequencing errors) should be treated cautiously; otherwise,
they may cause inflated species diversity estimations (). In
this paper, the algorithm has been tested on pyrosequences
of 16S rRNA genes. But it can also be applied to sequences
of 16S rRNA genes derived from any sequencing machine,
as long as redundant sequences exist.
AbundantOTU can be combined with other taxonomy-
based analysis of pyrosequences of 16S rRNAs. For ex-
ample, as we demonstrated in the analysis of skin dataset,
taxonomic assignment of the representative sequences of the
abundant OTUs can be achieved using the RDP classifier.
Using AbundantOTU can reduce the computational time,
since often the pyrosequence datasets are dominated by a
few abundant species, and deriving these sequences with
AbundantOTU is fast in comparison to taxonomic assign-
ment on the entire dataset. Rigorous but time-consuming
taxonomic assignment may then be pursued on the few
consensus sequences derived from AbundantOTU. Another
potential application of AbundantOTU is to combine it
with more time-consuming OTU inference methods that rely
on clustering of reads, based on their pairwise distances.
Abundant OTUs can be inferred by AbundantOTU (taking
advantage of the fact that it achieves fast and accurate
inference of abundant OTUs), and the remaining sequences
can then be analyzed by those methods.
The author would like to thank Drs. Haixu Tang and
Thomas G. Doak for helpful discussions and reading the
manuscript. This work was supported by National Institutes
of Health grants (1R01HG004908-02 and 1U01HL098960-
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