Available online at www.sciencedirect.

com

Next generation sequencing and bioinformatic bottlenecks:

the current state of metagenomic data analysis
Matthew B Scholz1,2, Chien-Chi Lo1,2 and Patrick SG Chain1,2
The recent technological advances in next generation
sequencing have brought the field closer to the goal of
reconstructing all genomes within a community by presenting
high throughput sequencing at much lower costs. While these
next-generation sequencing technologies have allowed a
massive increase in available raw sequence data, there are a
number of new informatics challenges and difficulties that must
be addressed to improve the current state, and fulfill the
promise of, metagenomics.
Addresses
1
Genome Science Group, Los Alamos National Laboratory, Los Alamos,
NM 87545, United States
2
Microbial and Metagenome Program, Joint Genome Institute, Walnut
Creek, CA 94598, United States
Corresponding author: Chain, Patrick SG (pchain@lanl.gov)

however this particular goal has been largely unattainable

owing to technological limitations in bacterial isolation/
DNA recovery as well as in sequencing capacity (cost and
throughput).

Community profiling
Early efforts to describe whos there have relied upon
cataloging species as designated by conserved changes in
their rDNA sequence, either via targeted sequencing of
amplicons, microarray technologies (Phylochip [1]), or
electrophoretic sizing techniques that are primarily
restricted to differentiating between communities (DGGE
and T-RFLP [24]). Each methodology has its own set of
limitations, and most share a reliance on the known set of
target 16S rDNA sequences, as well as the assumption
that 16S sequence can serve as a sufficient marker for
species level identification.

Current Opinion in Biotechnology 2012, 23:915

This review comes from a themed issue on
Analytical biotechnology
Edited by Wei E. Huang and Jizhong Zhou
Available online 9th December 2011
0958-1669/$ see front matter
# 2011 Elsevier Ltd. All rights reserved.
DOI 10.1016/j.copbio.2011.11.013

The discipline of metagenomics

Until recently, analysis of any bacterial community was
severely limited by available technology and a shortage of
reference genomes. The advent of next generation
sequencing (NGS; Table 1) has allowed an explosion
in sequencing of individual genomes, and started a revolution in metagenomic sequencing and analysis. The
increased throughput and decrease in costs of sequencing,
coupled with additional technological advances have
transformed the landscape of metagenomics.
The goal for any metagenome sequencing project is the
full characterization of a community (essentially whos
there?, what are they doing?), and thus usually includes
efforts to understand: 1) community composition/structure, including the taxonomic breakdown and relative
abundance of the various species, 2) genic contribution
of each member of the community, including number and
functional capacity, and 3) intra-species or intra-population
heterogeneity of the genes. Ideally, one would be able to
completely reconstruct all genomes within a given sample;
www.sciencedirect.com

Functional gene metagenomic studies are of interest to

those specifically investigating what are they doing in
detail. A methodologically similar approach to 16S community profiling can also be applied using gene families of
particular enzymatic function, which again can be
explored using either microarray (e.g. Geochip [5]) or
direct sequencing techniques [6]. While not mutually
exclusive, there is little, if any, data to correlate the
presence of a particular species as indicated by 16S rDNA
sequence to the enzymes identified by functional gene
assays. These profiling approaches are now becoming less
cost effective and more time consuming than direct
sequencing of a community.

Shotgun sequencing of metagenomes

Limited sequencing throughput constrained most early
metagenomic shotgun sequencing efforts to the characterization of simple microbial communities: enriched
bioreactor communities or those found in extreme
environments [7]. One high-cost deep metagenomic survey of the ocean was performed using this technology
(GOS), although in-depth analysis could still only be
performed with the most dominant organisms [8].
NGS technologies have allowed exploration of complex
communities by sequencing at lower costs and higher
throughput than Sanger based sequencing (Table 1). This
new sequencing capacity can be utilized for more comprehensive characterization of more diverse and complex
microbial communities such as animal-associated microflora (e.g. termite hindgut [9], human intestinal tract [10],
human saliva [11,12], and cow rumen [2,13]), and has
Current Opinion in Biotechnology 2012, 23:915

10 Analytical biotechnology

Table 1
List of NGS sequencing platforms and their expected throughputs, error types and error rates. Each platform has distinct advantages
owing to cost, error rate, read length, and so on
Platform

Run time (h)

Roche
454 FLX+
454 FLX Titanium
454 GS

1820
10
10

Illumina
GAIIx
HiSeq 2000
HiSeq 2000 V3
MiSeq

14
8
10
1

Life technologies
SOLiD 4
SOLiD 5500xl

12
8

Ion torrent
PGM 314 Chip
PGM 316 Chip
PGM 318 Chip

3
3
3

Pacific biosciences
RS

14/8 Smart Cells

Read length (bp)

700
400
400

Throughput per run (Mb)

Error type

Error rate (%)

900
500
50

Indel
Indel
Indel

1
1
1

2  150
2  100
2  150
2  150

96,000
400,000
<600,000
1000

Substitution
Substitution
Substitution
Substitution

>0.1
>0.1
>0.1
>0.1

50  35
75  35 PE
60  60 MP

71,000
155,000

A-T Bias
A-T Bias

>0.06
>0.01

100
100+
200

10
100
1000

Indel
Indel
Indel

1500

45/SC

Insertions

1
1
1
15

even made it feasible to begin investigating soil and

pelagic microbial communities of extreme complexity.

necessitate planning for at least several hundred gigabytes of data storage per sample.

New technologies for metagenomic

sequencing: advantages and drawbacks

Analysis methods for metagenomics

As new NGS technologies continue to emerge, the field of

metagenomics has adapted to the new types of sequencing data. However, each NGS technology has advantages
and disadvantages that must be addressed to enable
appropriate use of metagenome sequencing data. The
volume of NGS data coupled with their relatively short
reads raises the question of how to analyze these data so as
to maximize their scientific value. While 454 pyrosequencing has been used for metagenome analyses, the number
of reads obtained from the Illumina (and SOLiD) platform (Table 1) makes this short read technology the
most well suited to deep-coverage sequencing and
analysis of a shotgun metagenome. However, these reads
are very short (currently 36150 bp), making many read
based analyses difficult, incomplete, or impossible.
Therefore, new methods are required to analyze Illumina-sequenced metagenomes.
Novel algorithms able to process millions to billions of
very short reads are being developed worldwide, for
sequence alignment, assembly, as well as read annotation
[14] among other applications (Table 2). Although this
provides many useful resources, it has delayed or prevented the selection of standard best practice tools for
analysis. Any NGS research in metagenomics will require
significant computational resources, and a core of bioinformaticians with skills to install, update, and run the
latest tools. Furthermore, using NGS platforms can
Current Opinion in Biotechnology 2012, 23:915

While the ultimate goal is to reconstruct all the genomes

within an environment, this is not feasible owing to the
computational complexity involved. Instead there are two
general types of analyses performed as a proxy for complete genome reconstruction (Figure 1), both with
inherent limitations: 1) assembling the reads into contigs,
and performing taxonomic classification and functional
assignments, or 2) read-based reconstruction of the functional and taxonomic components of the metagenome.
Several problems particular to the assembly of metagenomes exist. The algorithms for short read assembly require
crippling amounts of memory to reconstruct the many
genomes found within a metagenome. Additionally, the
wide range of abundances of the genomes within a sample
further complicates the issue, and low abundance genomes
may not assemble at all. Furthermore, population heterogeneity within lineages can fragment otherwise contiguous
sequences, and the similarity of closely related lineages can
result in chimeric assembly. Read-based methods to
achieve both functional and taxonomic classification of
NGS data suffer from the sheer number of reads (long
analysis time) and from short read length (high error rates).
Assembly

The currently accepted methods most capable of assembling NGS data utilize Kmer DeBruijn graph traversalbased methods, including programs such as Velvet, SOAPdenovo, ALLPATHS, ABySS, the CLC Bio commercial
www.sciencedirect.com

Current state of metagenomic data analysis Scholz, Lo and Chain 11

Table 2
Currently available software tools for analysis and assembly of metagenomes
References

Software/algorithm
Annotation and analysis

MG-RAST
IMG-M
Eragatis
DIYA
CloVR
RATT
VMGAP
CAMERA
METAREP

[38]
[39]
[27]
[40]
http://clovr.org/
[41]
[31]
[42]
[43]

Assembly

RAY
Velvet
SOAPdenovo
Newbler
ABySS
ALLPATHs
Genovo
CLCbio
Meta-IDBA
MetaVelvet

[44]
[21]
[45]
[20]
[46]
[16]
[24]
http://clcbio.com
[47]
http://metavelvet.dna.bio.keio.ac.j

Mapping/alignment

BWA
Bowtie
Novoalign
SOAP
MrFAST
CloudBurst
BFAST
MUMer
MOSAIK
BLAST
MAQ

[48]
[49]
[50]
[51]
[52]
[53]
[54]
[26]
http://bioinformatics.bc.edu/marthlab/Mosaik
[26]

product as well as number of newer assemblers under

development [15,1622] (Table 2). The Kmer approach
was developed in an attempt to overcome the time limitations of traditional overlap-based assembly strategies. The
primary limitation of overlap-based assemblers is that they
require a number of calculations proportional to N2 (the
square of the number of reads in the assembly). As NGS
output surpassed the multi-million read mark, this process
became prohibitively slow. While there are several excellent reviews of Kmer-based assembly [15], it is important
to note two things: first, this method reduces the time of
assembly, but at the cost of requiring significant RAM
which is proportional to the size of the genome(s) being
assembled and/or the amount of data, which de facto limits
the total size of the metagenome being assembled; second,
this method is non-deterministic. Because reads are broken
down into smaller pieces of defined length (Kmers), reads
themselves are no longer the target of assembly, leading to
the potential introduction of assembly errors.
Metagenomes have presented a number of additional
assembly challenges. Multiple genomes are represented
disproportionately owing to uneven community composition resulting in poor or no coverage of many parts of
many genomes. Assembly of reads into contigs is often
www.sciencedirect.com

limited owing to complex population composition [23],

thus more data are required to cover the diverse genomes,
at the cost of computational memory and time requirements. Species level (and population) heterogeneity can
further confuse assembly by presenting forks or bubbles
in the DeBruijn graph. While there are a number of
effective assemblers for single genomes, only three
(Genovo, MetaVelvet and Meta-IDBA [24]) currently
attempt to address these metagenome issues, and one
(Genovo) is limited to a few million reads. MetaVelvet
and Meta-IDBA attempt to bin genomes by abundance
in the population by separating short-read data by Kmer
frequency and/or collapsing the graph. Alternative
approaches include finding ways to partition or bin reads
into discreet groups that are more amenable to assembly.
Read-based analyses

Read-based approaches can also allow exploration of gene

functions and taxonomic classification. However, only a
subset of the data can be assigned to either function or
species, and of those that can be classified, the degree of
confidence can vary as a result of both the reference
databases, and the shortness of reads. When coupled with
the sheer volume of reads, which threatens to overwhelm
current available online annotation and local analysis
Current Opinion in Biotechnology 2012, 23:915

12 Analytical biotechnology

Figure 1

NGS-Based Metagenome Analysis Methods

Your Sequencing Method of Choice
Amplicon Sequencing
16s RiboTags

Character- or
HomologyBased
Approaches

Whole Sample Sequencing

Gene-Targeted
Metagenomics

OTU Based
&
HypothesisTesting
Approaches

Shotgun Metagenomics

Read
Binning
Gene
Calling

Taxonomy/Function-subfamily
Community Profiling

FunctionSubfamily
Profiling

Assembly

Reads

Contig
Binning

Contig
Annotation

Read
Annotation

Contig-based
Taxonomy
and
Functional
Profiling

Read-based
Taxonomy
and
Functional
Profiling

Final Analytical Data Set for Analysis and Community (Metagenome) Comparisons
Current Opinion in Biotechnology

Analytical stages and steps for analysis of metagenomic data from either amplicon sequencing or whole sample shotgun metagenome sequencing.

systems, these issues strengthen the argument to use both

assembly and read-based approaches together to examine
metagenomes.
Read mapping methods based on the BurrowsWheeler
aligners, such as BWA, Bowtie MAQ, and others (Table 2)
can perform fast alignments of reads to a given set of
reference sequences [14]. This method has specific limitations with regards to the reference sequences, but is
extremely rapid. MUMmer [25] is less limited by database
size, but is also much slower. BLAST [26] is more sensitive
and can be used to find distant homologous sequences for
taxonomy and functional attributions. However this comes
at too high a cost in terms of CPU time. Depending on
computational resources and method of alignment, results
for tens to hundreds of millions of reads can generally be
obtained within hours (Burrows Wheeler methods), days
(Nucmer) or weeks (BLAST). Other fast approaches, such
as using pre-developed Hidden Markov Models (HMMs),
can also be used to find conserved domains, however these
do not function very well for short reads, and typically have
a limited range (e.g. a single gene of interest).
Current Opinion in Biotechnology 2012, 23:915

Metagenome analysis and comparative metagenomics

The annotation of metagenomic contigs is often viewed

as simply an extension of bacterial genome annotation.
However, for most metagenomes, there will also be viral
and eukaryotic components. There is currently no centralized method to annotate such diverse sequences at the
same time. Bacterial and archaeal annotations can be
performed in-house by use of available gene calling
and functional annotation workflows, including those in
the Ergatis web-based workflow management system
[27], the command-line cg-pipeline [28]. Online metagenome annotation services such as IMG-M [29] and
MG-RAST [30], are also available. While a virus specific
annotation pipeline, VMGAP [31] also exists, there is
currently no available system that combines all necessary
pipelines for complex metagenome samples, thus a strong
need for such development exists.
CAMERA provides a framework to design and implement data analysis pipelines. Other installable workflow
managements systems like Galaxy [32], Ergatis, and
others, already have hundreds of tools for analyzing
www.sciencedirect.com

Current state of metagenomic data analysis Scholz, Lo and Chain 13

genomic and metagenomic data [33]. Customized features can be added by users, and analysis pipelines can be
built and shared easily among scientists within the
research community.

are still drastic improvements that can be made to these

tools, and novel algorithms that are sorely needed to
process NGS data, however it is clear that metagenomic
sequencing/analysis is finally hitting its stride.

IMG-M, CAMERA and MG-RAST allow some analyses

beyond annotation, including taxonomic and functional
assignments, and pathway reconstruction, making these
resources popular portals that can provide a single platform
for depositing, locating, analyzing, visualizing and sharing
data. These resources are incredibly useful for those individual laboratories without access to high performance
computers to perform analysis, and have grown to accommodate browsing functional and taxonomic comparisons of
metagenomes in detail and as a whole. METAREP is
another tool developed for high-performance comparative
metagenomics. Users can analyze and compare annotated
metagenomics datasets by graphical summaries for taxonomic and functional classifications and perform statistical
tests. If metadata is available, these sites/tools can provide
multivariate statistical analysis, principal component
analysis (PCA) and nonmetric multidimensional scaling
to cluster samples and show the major factors which
contribute most to the observed functional or taxonomic
compositions of the metagenomic populations [11].

It is expected that sequencing will soon cease to be the

limiting factor in metagenomic studies. The computational
resources required for assembly, annotation and analysis
have already become the main bottlenecks for metagenomics projects. It is highly probable that sequencing
centers will begin to serve principally as bioinformatics
resources that lend computational resources and expertise
to the community. However, without novel algorithms for
assembly and analysis, it is clear that the sheer volume of
sequencing data will overwhelm available resources.

Finally, the comparison of multiple metagenomes can

reveal relationships between different environments or
different time points within the same environment, based
on taxonomic and functional profiling. Dinsdale et al. conducted a broad metagenomic comparison, contrasting
15 million sequences from 45 distinct microbiomes
and 42 distinct viromes [34]. More recently, a number of
other metagenomic comparisons have been conducted,
such as comparisons between deep-sea hydrothermal
microbial communities [35], biogas fermenters [36], oceanic viral communities [37], and saliva microbiomes [12].
Metagenome comparison platforms will become more and
more important to gain scientific understanding of how
community structure and functional profiles relate to
environmental perturbations.

Acknowledgements
This study was supported partly by Laboratory-Directed Research and
Development of Los Alamos National Laboratory under grant number
20100034DR, by the U.S. Department of Energy Joint Genome Institute
through the Office of Science of the U.S. Department of Energy under
Contract No. DE-AC02-05CH11231, and by grants from the U.S. Defense
Threat Reduction Agency under contract numbers B104153I and B084531I.

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Current Opinion in Biotechnology 2012, 23:915