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First published online 8 August 2007
doi: 10.1242/dev.001073
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Review |
HHMI and Division of Biology, Caltech, 1200 E. California Blvd, Pasadena, CA 91125, USA.
* Author for correspondence (e-mail: pws{at}caltech.edu)
SUMMARY
In an era exploding with genome-scale data, a major challenge for developmental biologists is how to extract significant clues from these publicly available data to benefit our studies of individual genes, and how to use them to improve our understanding of development at a systems level. Several studies have successfully demonstrated new approaches to classic developmental questions by computationally integrating various genome-wide data sets. Such computational approaches have shown great potential for facilitating research: instead of testing 20,000 genes, researchers might test 200 to the same effect. We discuss the nature and state of this art as it applies to developmental research.
Introduction
The advent of high-throughput technologies (see glossary, Box 1) has greatly increased the amount of genetic information that is deposited in the public domain. In addition, the results of thousands of hard-won individual observations have been compiled in a consistent format (i.e. curated) in biological databases. Combined, these two sources have created genome-wide data sets that describe a wide range of biological processes (Fig. 1, see also Box 1).
As developmental biologists, we might be more interested in hypothesis-driven studies that focus on a small set of genes, rather than on generating genome-wide data. How can we best use all of these publicly available data to benefit our own research? Data integration has proven to be an effective strategy to extract biological meaning from heterogeneous data sets in both developmental research and other fields. It can be a powerful tool to identify candidate genes worthy of further study, and by automating the process it can allow the translation of genome-wide data into small-scale science.
It is highly likely that we have applied the principle of data integration
in our research all along. When seeking regulators of biological processes or
targets of gene functions, a common strategy is to compile a short list of
candidate genes and then experimentally test them. We are probably all
familiar with this type of simple `filtering'. In an example of this approach,
766 C. elegans genes were selected as germ-line enriched judging from
their microarray expression profiles, then the phenotypes of these genes were
analyzed by RNA interference (RNAi) to identify new genes necessary for
germ-line development (Piano et al.,
2002
). Along the same line, we might use RNAi to test for known or
predicted signaling proteins (Lehner et
al., 2006), transcription factors (e.g.
Parrish et al., 2006;
Fernandes and Sternberg,
2007), and so forth. Other `filters' include a protein or
transcript's potential phosphorylation sites, microRNA target sites or
subcellular localization. Sometimes, multiple filters are applied. If we are
looking for a transcriptional regulator active in a place and time of
interest, we might look up all the papers on genes expressed at that time and
place, look up all papers on transcription factors and determine if there is
any overlap. Of course, these searches are easier if there is a comprehensive
database that contains this information. The process of combining two or more
data sets to identify their intersection is the simplest form of data
integration.
Although data integration does not actually create new information, it can create new knowledge for the individual; as discussed above, it can limit the number of candidates a researcher should test, thereby allowing more time for an intensive analysis of each candidate. This strategy becomes even more desirable when a developmental process or the gene of interest does not have an easily screenable phenotype.
More-advanced techniques of data integration employ sophisticated
statistical models to improve the extraction of meaningful information. These
techniques may decrease false negatives, or attach a value of statistical
confidence to each data point. Most of these techniques were developed in
studies of Saccharomyces cerevisiae (e.g.
Marcotte et al., 1999;
Jansen et al., 2003), largely
owing to the wealth of genome-wide data that is available for this simple
organism. However, the impact of these techniques will be limited unless their
applications can be extended to other organisms or can reach the large
audience of small-scale research. Recently, several studies have successfully
applied genomics data integration strategies in metazoans, specifically to
explore mechanisms of development
(Gunsalus et al., 2005;
Zhong and Sternberg, 2006). In
this review, we highlight these examples to show how typical developmental
biology laboratories can use more-advanced data integration techniques to
benefit their own research. We first discuss the different types of genomic
data that are publicly available, and then describe the resources and methods
available to integrate these data. We also discuss the benefits and
limitations of such approaches in studying developmental biology.
| Box 1. Glossary Bayesian network. A way of using Bayes theorem to calculate the probability of an outcome based on a network of information. The information consists of a set of observations correlated with the outcome, which allows the assignment of a probability of the outcome given the observation. Controlled vocabulary. Defined as a set of terms that ensures their consistent use among many people. Controlled vocabulary terms can include synonyms. For example, FlyBase developed a vocabulary of body parts that covers many aspects of Drosophila anatomy. WormBase has a set of life-stages. Genome-wide. Covering a large proportion of the genome; for example, 80% of 20,000 genes. High-throughput. Rapid data collection, usually using automation such as robotics and image processing. Likelihood ratio. The frequency in a positive training set divided by the frequency in the negative training set. The likelihood ratio is 1 if there is no difference between the training sets. A likelihood ratio of >1 indicates a positive predictor; a likelihood ratio of <1 indicates a negative predictor. Ontology. A defined set of concepts with defined relationships among the concepts. TILLING. The efficient identification of mutations in an organism by mutagenesis and the resequencing of genes of interest. Training set. A set of data that can be used to teach (`train') a computer program. Such sets could range, for example, from a set of human faces and a set of other animal faces, to a set of genes that are known to interact and a set that are known not to interact. By using a training set, computer scientists can train computer programs to discriminate which of two sets of data an unclassified object (or gene) is likely to belong to.
|
We focus on data integration rather than on approaches to mine individual
genomic data sets because it provides the best use of the publicly available
data. It is thus especially beneficial to developmental biologists, who might
not be able to generate these genomic data themselves. We focus on the
applications of these strategies in developmental biology investigations,
rather than on the technical advances in data integration methods (for a
review, see Joyce and Palsson,
2006). Therefore, rather than discuss in detail the bioinformatic
techniques that are available in the data integration field, we introduce a
few typical methods and key concepts that are of particular use to studies of
developmental biology. This is also not a comprehensive review of the genomic
data available for each organism (see
Antoshechkin and Sternberg,
2007; Lee, 2005;
Eppig et al., 2007;
Crosby et al., 2007;
Rhee et al., 2003).
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In this section, we select five types of commonly used genome-wide data
sets and briefly discuss the current state, strengths and weaknesses of these
data. Although only experimental data are described here, it should be noted
that non-experimental data can also be used in data integration. For example,
the co-occurrence of gene names in the literature has been used, in addition
to experimental data, in data integration to predict functional interactions
(Lee et al., 2004).
Numerous databases have been developed to accommodate the growing amount of genomic data. Often, the data that we are interested in are covered by multiple databases. Which databases shall we use? Table 1 lists some popular, publicly accessible resources for the genomic data mentioned above. This is by no means a comprehensive list.
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Expression data
There are two major types of gene expression data: (1) annotation-based
data obtained from experiments such as reporter gene assays and in situ
hybridization and (2) data from microarray experiments.
Annotation-based data indicate the developmental stage and the tissue of
gene expression, often using consistent nomenclature. High-throughput studies
in this category mostly employ RNA in situ hybridization or promoter-reporter
gene fusion assays. Large-scale in situ hybridization data are available for:
gene expression patterns during D. melanogaster embryogenesis
(Tomancak et al., 2002); all
stages in C. elegans development (NEXTDB,
http://nematode.lab.nig.ac.jp/index.html);
Xenopus embryogenesis (Pollet et
al., 2005); mouse embryogenesis
(Visel et al., 2004;
Christiansen et al., 2006);
and expression patterns in the adult mouse nervous system
(Gray et al., 2004;
Magdaleno et al., 2006;
Lein et al., 2007).
Large-scale reporter gene assays require transgenic strain construction; thus,
it can be difficult to reach high-throughput efficiency in certain organisms.
The technique was first successfully applied on a genome scale to study
protein subcellular localization in S. cerevisiae
(Kumar et al., 2002;
Huh et al., 2003).
Subsequently, several high-throughput projects have emerged to study
developmental profiles in C. elegans
(McKay et al., 2004;
Dupuy et al., 2004;
Dolphin and Hope, 2006) and
gene expression in the mouse central nervous system
(Gong et al., 2003).
Microarray studies measure mRNA levels on a genome scale and have become a
powerful tool with which to study development. Soon after the invention of the
technique (Schena et al.,
1995), developmental gene expression profiles were studied in
several model organisms, including C. elegans
(Hill et al., 2000;
Kim et al., 2001) and D.
melanogaster (White et al.,
1999; Furlong et al.,
2001). Now, microarray techniques have been extensively applied to
the study of development in various mutant backgrounds and under different
conditions. Accordingly, strategies for analyzing microarray data have also
advanced tremendously. An interesting example is a cross-species microarray
data integration study conducted by Stuart et al.
(Stuart et al., 2003). Stuart
et al. first identified conserved genes (meta genes) that have orthologs in
humans, fruitflies, C. elegans and yeast. By examining expression
data from these species, 
22,000 pairs of meta genes were found to be
co-expressed across species, revealing conserved functional modules in core
biological processes, such as the cell cycle, during transcription, and
signaling. Their discoveries demonstrated the power of cross-species data
integration.
Thanks to the establishment of data standards in the field
(Quackenbush, 2004),
microarray data have the advantage of being made available in a single format
(per platform). However, recent studies suggest that more standards are needed
to enable the results from different experiments, laboratories and platforms,
to be more comprehensively and accurately compared
(Irizarry et al., 2005;
Larkin et al., 2005;
Members of the Toxicogenomics Research
Consortium, 2005). One disadvantage of microarray experiments is
that the data often contain a high background. Data from in situ hybridization
gives good spatial and temporal resolution, but there is still noise present
resulting from sample preparation and processing. Reporter gene assays, using
lacZ or GFP variants, give superb spatial and temporal
resolution but might not reflect the endogenous gene owing to the inclusion of
incomplete sequences in the transgene, or artifacts resulting from factors
such as the stability of the reporter. In addition, reporter gene constructs
often do not include the gene's 3' UTR, a major source of
post-transcriptional regulation - for example, by microRNAs
(Ambros and Chen, 2007).
Interactome data
Interactome data provide an invaluable source to study molecular mechanisms
that underlie development. Interactome data include both physical and genetic
interactions. High-throughput studies on protein-protein physical interactions
have become a rapidly developing field. In S. cerevisiae, there have
been multiple genome-wide studies from different research groups, who have
made use of different techniques, such as yeast two-hybrid assays
(Ito et al., 2001;
Uetz et al., 2000) and
affinity purifications coupled with mass spectrometry
(Gavin et al., 2006;
Gavin et al., 2002;
Ho et al., 2002). Genome-wide
yeast two-hybrid studies have also been conducted in flies
(Giot et al., 2003), worms
(Li et al., 2004) and humans
(Rual et al., 2005;
Stelzl et al., 2005).
In comparison to physical interaction studies, there have been relatively
few high-throughput genetic interaction studies. The first genome-wide genetic
interaction study was a synthetic lethality screen conducted in S.
cerevisiae using a library of deletion mutants
(Tong et al., 2001;
Tong et al., 2004). These
studies indicated the topology of the genetic interaction network
(Boone et al., 2007), as well
as specific discoveries such as genes involved in DNA replication in response
to DNA damage (Budd et al.,
2005). In multicellular organisms, RNAi technology
(Fire et al., 1998) enables
researchers to overcome the problem of using only a small number of available
mutants and to design high-throughput (or at least genome-wide) genetic
interaction screens (Baugh et al.,
2005; Lehner et al.,
2006; van Haaften et al.,
2004; Dietzl et al.,
2007). The scope of these studies has extended from studying a
specific process, such as the DNA-damage response
(van Haaften et al., 2004), to
investigating a broad spectrum of multiple signaling pathways
(Lehner et al., 2006). We have
also seen the advent of sophisticated analyses of quantitative data to detect
genetic interactions (Baugh et al.,
2005; Schuldiner et al.,
2005; Collins et al.,
2007). Quantitative interaction data typically describe the
fraction of organisms that have a wild-type phenotype (e.g. survival); thus,
if wild type is 100% viable, mutant A is 80% viable and mutant B is 60%
viable, a synergistic effect of A and B would be inferred if the A-B double
mutant were significantly less viable than 48% (the product of 0.60 and 0.80),
the expected value if A and B affect independent processes.
In addition to such high-throughput data sets, the extent of interaction
data compiled from small-scale studies has also grown dramatically. In some
model organisms, such as S. cerevisiae and C. elegans, the
number of these interactions has reached the same scale as the number of
interactions discovered by high-throughput methods
(Reguly et al., 2006;
Bieri et al., 2007).
However, interactome data are far from perfect. Only a small portion of
physical interactions has been confirmed by more than one data set or by
genetic interactions (von Mering et al.,
2002; Reguly et al.,
2006), suggesting that these data are far from complete. In
addition, the yeast two-hybrid method can have high false-positive rates, up
to 50% in some cases (Fields,
2005), but false positives are likely to vary between experiments
and among individual results in a given screen. Several factors can contribute
to false positives. For example, two proteins might interact in yeast nuclei
but might never be expressed in the same cell in vivo. The particular
construct used can also affect the results, so one domain might interact, but
the full-length protein might not. However, large-scale analyses include
estimates of error rates, and users of these data can have more confidence in
them if they understand the methods used in each study. In addition, it is
desirable to develop computational methods to explore potential interactions
and to improve data reliability.
Transcriptional regulation data
There are many rich sources of data about transcriptional regulation from
which developmental biologists would like to infer genetic regulatory
networks. We will not review in detail the methods involved in data
integration because this active area of bioinformatics deserves its own
review, but instead will provide two examples of where automated data
integration is useful. Much data integration in this area deals with the
association of transcriptional regulators with the sites to which they bind
and with gene expression. Individual types of analyses are often successful
but have limits, which can be overcome partially by data integration.
An increasingly important technique, chromatin immunoprecipitation (ChIP),
detects protein-DNA interactions in vivo and thus indicates where proteins
bind to DNA. Genome-wide ChIP analysis has been achieved by detecting DNA
fragments precipitated with a particular protein using whole-genome tiling
microarrays, the conventional sequencing of fragment ends or, more recently,
by sequencing the DNA fragments themselves
(Johnson et al., 2007).
However, proteins can bind to DNA without having a known functional
consequence. These data can be integrated with sequence motifs, conservation
of sequence alignment, and with gene expression data in order to make
predictions about the genes regulated by a particular transcription
factor.
Sandmann et al. (Sandmann et al., 2007) found 2000 regions that bind
the DNA-binding protein/transcriptional activator Twist in Drosophila
using ChIP and a whole-genome tiling array. They narrowed down these results
to 500 candidate gene regions by integrating information about the
co-expression of an associated gene and Twist, and genetic trans-regulation
data. Six TWIST-bound regions were tested and all were sufficient to drive
reporter gene expression in vivo. In a similar approach, Zeitlinger et al.
(Zeitlinger et al., 2007)
found hundreds of regions that bind three factors (Dorsal, Twist and Snail).
Up to 80% of these regions had motifs for a given factor, as compared with
35% of randomly chosen sequences. Conservation across 12
Drosophila species was used to gain further confidence in these
enhancer regions, of which seven were shown to function as transcriptional
enhancers in vivo. In both studies, ChIP data were combined with other data to
accurately predict enhancer function.
Genomic DNA sequence comparisons of orthologous genes can identify
conserved sequence alignments, as well as over-represented sequence motifs. Of
course, a single important DNA site can be indistinguishable from a background
of sequence if, for example, every gene on average has one copy of a sequence.
However, with other information, a single site can be identified. For example,
in C. elegans the RFX-family regulator DAF-19 binds a 14-mer site
(called the Xbox) (Blacque et al.,
2005). Blacque et al. compared mRNA from ciliated neurons with all
neurons to identify transcripts enriched in ciliated neurons. They then
selected those that had an Xbox near the start codon, and successfully
predicted expression in ciliated neurons. However, only 42% of genes with
correctly positioned Xboxes were expressed in ciliated neurons.
Phenotypic data
Phenotypic data are accumulating at an exponential rate, boosted by
technologies such as RNAi and TILLING
(Wienholds et al., 2003;
Henikoff et al., 2004) (see
glossary in Box 1). Genome-wide
high-throughput phenotype characterizations have been conducted in yeast using
a deletion library (Giaever et al.,
2002), in worms by RNAi
(Kamath et al., 2003;
Simmer et al., 2003) and in
Drosophila by RNAi on cell lines
(Boutros et al., 2004) or by
expression of inverted repeats in transgenic flies
(Dietzl et al., 2007).
Although there is no doubt that high-throughput in vivo studies have
identified gene functions during various developmental events, studies in
Drosophila and mammalian cell lines have also contributed to our
knowledge of development by identifying new pathway components and by
characterizing drug responses and cellular events (e.g.
Berns et al., 2004;
Eggert et al., 2004). In
addition to high-throughput results, data from small-scale studies of
individual genes are also being compiled through the literature curation that
is provided by model organism databases such as FlyBase and Mouse Genome
Informatics (MGI). These data provide more-detailed information than do
high-throughput data, whereas the high-throughput data have better
consistency. Some high-throughput studies focus on a subset of genes instead
of the entire genome, which allows results to be analysed in more detail. For
example, the study by Piano et al. (Piano
et al., 2002) described in detail 47 different early embryonic
phenotypes created by the inactivation of 766 genes in C.
elegans.
|
)]. RNAi experiments may not effectively knockdown the
function of many genes. For example, in genome-wide RNAi experiments in C.
elegans, only 10% of tested genes displayed visible phenotypes on the
standard wild-type laboratory strain
(Kamath et al., 2003). When a
more sensitive strain, rrf-3, was used, the number of genes
associated with phenotypes increased to 23%
(Simmer et al., 2003);
however, this still leaves most genes without functional information.
Ontologies and functional annotation
The most common format of these genomic data is a list of genes that have
been annotated with their expression patterns or associated with phenotypes in
a controlled vocabulary. A more resourceful annotation method involves the use
of ontologies, a more structured and controlled vocabulary (see glossary in
Box 1 and
Fig. 2). Ontology defines each
annotation term and the relationship among terms, enabling us to associate
genes with related, but not identical, functions
(Fig. 2). For example, if one
gene is annotated as causing embryonic lethality and another as defective
gastrulation, they will not be associated unless a phenotype ontology is used
that defines defective gastrulation as a specific case of embryonic lethality.
Currently, the Mouse Phenome Database (MPD) and WormBase (see
Table 1) have constructed mouse
and worm phenotype ontologies, respectively, and provide annotations using
these ontology terms. Anatomy ontologies have also been developed by the
Zebrafish Information Network (ZFIN)
(Sprague et al., 2006) and
WormBase (see Table 1).
The Gene Ontology (GO) Consortium (GO
Consortium, 2006) provides one of the most widely used platforms
of gene function annotations. The ontology contains three sets of controlled
vocabularies to describe gene functions: biological process (e.g. embryonic
development), cellular component (e.g. nucleus) and molecular function (e.g.
protein kinase activity). To ensure consistency, the ontology (see
Fig. 2C, for example) was
defined by professional curators from multiple model organisms. Each gene is
associated with these annotation terms and an evidence code (e.g. IEP,
Inferred from Expression Pattern).
GO data are considered to be of high quality by bioinformaticians because
they are manually inspected to ensure accuracy, and because they provide
detailed information for a large number of genes. Although a developmental
biologist might prefer more detail concerning gene function, there is a
balance between granularity and coverage when conducting computational
studies. In the context of genome-wide coverage, GO provides impressive data
quality. Because of this, the GO data set has been used not only as a type of
data to be integrated (Jansen et al.,
2003), but also as a benchmark to test the performances of various
data integration methods (Lee et al.,
2004).
When using GO data, it is important to remember that data with different evidence codes should be processed differently. For example, data with the IEA (Inferred from Electronic Annotation) code may be less reliable than manually curated data. Some GO data may overlap with other biological data sets. For example, in C. elegans the RNAi phenotypic data have been converted into GO biological process annotations (e.g. the phenotype `embryonic lethal' has been converted to the GO process `embryonic development'). The redundancy of these data sets should be noted if a computational method requires each data set to be independent.
Data integration: why?
Data integration is a useful tool for biologists to navigate through this seemingly overwhelming amount of sometimes contradictory genomic data. By combining various types and sources of information, we can achieve a better assessment of what is known about a protein or process, which will then facilitate our own research.
Take our own experience, for example. We integrated phenotypic, expression,
interactome and GO data from yeast, flies and worms to predict genetic
interactions in worms (Zhong and
Sternberg, 2006). Among the results was a set of novel predicted
interactors for let-60, which encodes a member of the RAS family and
is a crucial regulator of the extent of vulval induction
(Beitel et al., 1990;
Han et al., 1990;
Han and Sternberg, 1990).
These interactions were tested by RNAi in a let-60(gf) background for
suppression or enhancement of the multivulva phenotype. Twelve of 49 of the
predictions yielded significant effects. As a striking confirmation of these
results, the functional interaction between let-60 and
tax-6, one of our verified predicted interactions, was also
identified in an independent classical genetic-interaction analysis (W.
Johnson and M. Han, personal communication). In our study of let-60
modifiers in vulva development, data integration facilitated our research by
prioritizing which candidates to test.
To further illustrate the benefit of applying data integration to one's own
research, we compare two scenarios in which the same study would be conducted
with and without data integration. Without computational data integration,
genetic screens would be undertaken by a conventional method to study genetic
interactions. Lehner et al. (Lehner et
al., 2006) conducted an RNAi screen for modifiers of 30
genes. They applied RNAi to mutants and searched for synthetic phenotypes,
such as lethality and growth defects. They found 345 genetic interactions from
a total of 65,000 pairwise tests, making their efficiency (measured as
the ratio of discovery versus effort) 345/65,000=0.53%. In the second
hypothetical scenario, the same study would be conducted but now with the data
integration results. Our computational data predicted 83 interactions for the
same genes at a stringent threshold, and 325 interactions at a lower threshold
(Zhong and Sternberg, 2006).
Had Lehner et al. tested only these 83 (or 325 using the lower threshold)
pairs instead of the original 65,000 pairs, they would have recovered 14 (or
41 using the lower threshold) genetic interactions. The efficiency under this
strategy is thus 14/83=16.9% (or 41/325=12.6% using the lower threshold). The
efficiency improvement in using data integration is thus 32-fold (16.9/0.53)
or 24-fold (12.6/0.53) for the two thresholds. Therefore, data integration can
greatly decrease the screening and increase the efficiency of discovery, which
would be highly desirable when studying complex phenotypes. However, this
example also illustrates one limitation of this technique: only a subset of
all real interactions (41/345=11.9%) would be recovered. In this section, we
discuss what data integration can and cannot do.
Data integration can fill in missing data
Although ongoing literature curation by model organism databases is
constantly extending our knowledge of new gene functions, most genome-wide
data sets do not cover every gene in multicellular animals and plants. For
example, in C. elegans, only 20% (3802/20,000) genes are
associated with an anatomical expression pattern in WormBase (WormBase WS166).
Genes that are not associated with an annotation constitute a missing data
problem. This problem severely limits the scope of our research if we rely on
only one data set to conduct the computation (as in the example of C.
elegans expression, we will miss out 80% of genes). A quick and effective
solution to the missing data problem is to increase genome coverage by
combining multiple data sets that cover different groups of genes. For
example, in our study of genetic interaction predictions, we noticed that only
292 C. elegans genes were annotated for all three data types of
phenotype, anatomical expression and GO process
(Zhong and Sternberg, 2006).
By adding cross-species data, we were able to expand our scope and predict
genetic interactions for over 2200 genes. Integrating other data sets enabled
us to `borrow' information from other experiments and other species to piece
together a more complete picture. Each type, source and quality of data will
carry a different weight. For example, in our study, not surprisingly,
anatomical-level expression data from C. elegans was more informative
than similar data from D. melanogaster; however, Drosophila
phenotypic information was more informative than similar data from C.
elegans because there were only a limited number of phenotypic
annotations in WormBase at that time.
Data integration can reduce noise
Data sets are often noisy, containing false positives and false negatives.
Also, a data set may be a good predictor of one type of prediction, but a weak
predictor for another type of prediction. Data integration improves data
reliability by confirming a conclusion with several independent experiments.
Therefore, we can filter out erroneous information and increase the overall
predictive strength by combining several weak predictions.
In one example, Natarajan et al.
(Natarajan et al., 2006)
integrated a set of data obtained by the Alliance for Cell signaling in which
they treated a macrophage cell line with single and multiple ligands and
measured multiple readouts, including calcium and cAMP dynamics, cytokine
expression and phosphorylation of signaling proteins. The data for each cell
biological readout was converted to a z-score, which is essentially the number
of standard deviations from the relevant control, and then the z-scores summed
to obtain an integrated view of each ligand treatment. This integration
allowed them to compare treatments, learning, among other things, that
receptor-stimulated calcium mobilization increases cAMP production stimulated
by a distinct signal transduction pathway.
It is, however, debatable whether increasing the number of data sets will
necessarily improve data quality in terms of reducing false positives and
false negatives. When predicting protein-protein interactions, a study of
yeast genomic data integration has suggested that there is a limit to
improving performance by the integration of more data sets
(Lu et al., 2005). At some
point, the utility of adding more data sets saturates and thus clustering
additional data sets, especially those constituting weak predictors and data
of poor quality, introduces confusion instead of further reducing noise
(Lu et al., 2005). With
current multicellular organism data sets, we are probably still far from this
saturation point, and the benefit of reducing missing data might outweigh the
concern of data saturation.
Caveats of data integration
As shown by our example of comparing brute force and prediction-guided
tests, although data integration can promote discovery, it often can recover
only a subset of all hits. The scope of data integration is limited to the
genes that exist in data sets. As a result of the biased genome coverage of
some data sets (especially those compiled from small-scale studies), data
integration tends to favor genes that have been well studied and genes that
are conserved in multiple organisms. Therefore, data integration cannot
replace genetic screens for predicting gene function and interactions because
genetic screens provide unbiased coverage of the genome: genes can be
identified without any prior assumption or knowledge.
|
Because of these limitations, we consider data integration as a means of prioritizing experiments rather than dictating which experiments to pursue.
Data integration: how?
We explain in this section, with a few examples, the basic steps of data integration. In brief, the steps are: to obtain data in a consistent, structured form that can then be fed into a computer program; to define orthologs when data are to be integrated across species; to select a statistical model with which to integrate data; and to choose a threshold by which to interpret the output of the computer program.
Make data computable
Although numeric data are desirable because they can be used directly, data
in the format of annotated text can be converted into a form that is amenable
to information extraction and mathematical computation. One simple way to
convert text annotations to numbers is to use a binary code (0, 1) to denote
the absence and presence of an annotation term. Piano et al.
(Piano et al., 2002) used this
method to convert C. elegans embryonic phenotype annotations into a
string of binary numbers. This enabled them to compare the similarity in
phenotype of two genes by computing the correlation of the two strings of 0s
and 1s (shown as yellow and blue in Fig.
3).
Annotation statistics can provide additional information, especially when
an ontology is used. For example, in GO, the general term `developmental
process' is associated with as many as 17,281 genes, whereas the more specific
term `embryonic development' is associated with only 4826 genes
(http://www.geneontology.org,
as of July 2007). The number of genes associated with an annotation term thus
indicates how specific the term is. Such information can prove to be very
useful when predicting functional interactions because if two genes share a
more specific term then they are more likely to interact than genes that share
a general term (Jansen et al.,
2003; Zhong and Sternberg,
2006).
Another method is to convert heterogeneous data into weighted scores. A
popular scoring scheme uses likelihood ratios (see glossary,
Box 1)
(Jansen et al., 2003;
Lee et al., 2004;
Rhodes et al., 2005;
Zhong and Sternberg, 2006).
This requires a training set (see glossary,
Box 1) with known positives and
negatives. The likelihood ratio is the value of the frequency of a feature
appearing in the positive training set divided by the frequency in the
negative training set. A high likelihood ratio indicates that more positives
than negative have the feature. If an unknown gene has this feature, a high
likelihood ratio is awarded indicating that the gene is more likely to have a
positive outcome.
Map orthologs
Orthologs are homologs that diverged concomitant with the divergence of
species. When performing cross-species data integration, one important step is
to select a good method to identify orthologs among species. Most of us are
familiar with assessing orthology for individual genes of interest, but when
the process is scaled up to 20,000 genes, some automation is
necessary.
A number of automated strategies to detect orthologs have been proposed,
and several databases have been devoted to ortholog mapping
(Fig. 4). Many methods detect
reciprocally best-matching proteins from BLAST searches
(Altschul et al., 1997) as
orthologs; for example, NCBI KOG [euKaryotic Orthologous Groups,
http://www.ncbi.nlm.nih.gov/COG/
(Tatusov et al., 2003)],
InParanoid
[http://inparanoid.sbc.su.se
(Remm et al., 2001)] and its
multi-species extensions, such as OrthoMCL
[http://orthomcl.cbil.upenn.edu/cgi-bin/OrthoMclWeb.cgi
(Li et al., 2003)] and
MultiParanoid
[http://www.sbc.su.se/~andale/multiparanoid/html/index.html
(Alexeyenko et al., 2006)].
Using a different approach, TreeFam
(http://www.treefam.org)
constructs phylogenetic trees and uses manual curation to annotate orthologs
(Li et al., 2006).
A good ortholog mapping method should identify all members in an ortholog group and avoid large groups of paralogs. For example, in the hypothetical protein family of Fig. 4C, applying a two-species InParanoid analysis of species B and C will correctly identify two ortholog groups, (B1, C1) and (B2, C2, C3); but including a distant species A in a KOG analysis might erroneously lead to all the proteins being grouped together as one group. Based on its phylogenetic approach and manual supervision, TreeFam probably provides the highest data quality.
A good ortholog mapping method should also be updated with the latest gene model and annotation changes. Since gene models are updated continually as genome sequence, assembly and gene finding improves, ortholog analysis needs to be based on the latest sequence data. InParanoid, OrthoMCL and MultiParanoid provide open source codes at their websites and thus can be installed to analyze any desired genome sequences. However, it might require a substantial computational resource to conduct these analyses when a large number of genomes are included.
|
) and
embryogenesis (Gunsalus et al.,
2005) (see Box
2).
|
| Box 2. Using data integration to study C. elegans
embryogenesis: a success story In 2005, Gunsalus et al. integrated RNAi phenotypic data, microarray profiles and large-scale yeast two-hybrid results from C. elegans to predict a gene function network that regulates C. elegans early embryogenesis. In this study, they constructed a graph in which each of 661 genes is a node. Edges are drawn in this graph if the protein products of genes interact, or if the spectrum of phenotypes associated with the perturbation of two genes is highly correlated, or if the expression of two genes is highly correlated. By doing this, they obtained 31,173 connections among the 661 genes. A multiple-support graph was then developed in which two genes were linked if there were two or more edges linking them in the previous graph. This graph contains 305 nodes and 1,036 edges. It was this multiple-support graph that was analyzed computationally to identify highly interconnected sets of genes (sub-graphs), which can be interpreted as sets of gene products that act together, possibly as macromolecular machines. Indeed, the tight sub-graphs identified in this study correspond to known machines, such as the anaphase promoting complex (APC). Gunsalus et al. tested the predictive power of their approach by examining the subcellular localization dynamics of ten novel proteins using GFP translational fusions. In most cases, the localization was highly consistent with their predictions. For example, proteins connected to those involved in DNA synthesis licensing factors localized to condensed chromosomes at metaphase. The success of this study is due in part to its focus on one aspect of C. elegans development, and the authors' great expertise in the biological detail of this process.
|
When training sets are available, more-sophisticated statistical models can
be used. S. cerevisiae has been a test-bed for such bioinformatic
methods. For example, yeast protein and genetic interactions have been
inferred by Bayesian networks (Troyanskaya
et al., 2003), decision trees
(Wong et al., 2004) and kernel
methods (Ben-Hur and Noble,
2005) (see below). Whereas some of these methods require
substantial amounts of programming, most of the statistical models can be
constructed with the assistance of freely available software packages. The
computational principle behind these methods is to formalize different data
types into a number of attributes, to detect the patterns of these attributes
in known positives and negatives, and to define a process (function) to
differentiate positives from negatives based on their attribute values.
For example, if there are a total of n attributes, then each data point in the training set is a vector of n values, and is associated with an outcome. Kernel methods, in particular support vector machines (SVMs), use a kernel function (often non-linear) to transform these attribute values so that they are linearly separable, and then use a regression (often a simple linear regression) to derive a hyperplane that achieves the maximum separation between positives and negatives (Fig. 5B). We can then use this hyperplane to classify an unknown item into a positive or a negative outcome based on its attribute values.
A decision tree judges one condition at a time and reduces the problem at each step (Fig. 5C). Starting from the root node, a condition (e.g. do the two genes have the same phenotype?) is applied, and the unknown item is classified into one of the daughter nodes based on its values. The decision-making step continues until a leaf node is reached where the unknown item is classified into a final category.
Bayesian networks (see glossary, Box
1) use a directed acyclic graph to represent variables and their
relationships. The network is composed of nodes that represent variables,
directed edges that link two dependent nodes, and probability tables
(Fig. 5D). The training set
tells us the conditional probability of an outcome given the values of all
variables. Bayesian networks can use this information together with the prior
probabilities to compute the inverse problem, that is, given a set of values,
what the probability of an outcome is
(Eddy, 2004).
| Box 3. Putting it together: an application of advanced data integration
techniques
Jansen et al. (Jansen et al.,
2003
|
A simplified Bayesian network model, naïve Bayes classifier, has
become one of the most popular methods for integrating data to predict gene
functions (Jansen et al.,
2003; Lee et al.,
2004; Rhodes et al.,
2005) (see example in Box
3). This method assumes all variables to be independent, and thus
simplifies the computation down to an easy product calculation: the final
score is the product of the likelihood ratios of all features of the input
gene(s). The higher the scores, the more likely that the genes are positives.
A cut-off value is then applied to the final score.
Although numerous approaches have been developed, there has been no
systematic study of the different statistical models. However, the bottleneck
in data integration is likely still to be the data collection step. The impact
from statistical models seems relatively minor considering the severity of the
missing data problem and the unreliable nature of the data quality in
multicellular organisms. When the performance of a logistic regression model
to predict C. elegans genetic interactions was compared with that of
a simple naïve Bayes classifier, only minor improvements were observed
(Zhong and Sternberg, 2006),
suggesting that improving data quality should be of higher priority than
finding better statistical models.
Choose a threshold
The threshold stringency directly affects the ratios of false positives and
false negatives in the final computational predictions. As developmental
biologists, we care more about individual genes and proteins than about
statistical properties of the genome and data sets, but such properties
provide important clues. False positives can only be eliminated by experiment.
If 80% of inferences are false positives, then on average we have to test five
inferences to obtain one true result. If the experiments are relatively
straightforward (double mutants; examining gene expression), then such rates
might be acceptable. If the experiments are less straightforward (making a
conditional knockout mouse; determining a crystal structure), then those rates
are probably not acceptable.
The extent of false negatives depends on the threshold set for predictions. If one followed up on all predictions, then there would be no false negatives, but then one would gain nothing from the computation. There is no correct threshold, but rather it depends on the value and costs placed on following predictions.
Conclusions
We have discussed how data integration, pioneered in yeast, has been extended to metazoans, especially C. elegans. Data integration is likely to be especially important in studies of mammalian development, where the interest, complexity and cost of experiments are typically higher than in invertebrate systems. The desire to maximize the yield per experiment should lead a developmental biologist to want to use automated data integration.
This review has focused on integrating large-scale data sets to learn about
individual genes, but the analyses of features of whole interaction networks
promises additional, global insights (reviewed by
Albert, 2005). Features of
networks, such as the degree of sub-structure and the average connectedness of
genes, are likely to be related to functional features. In addition,
stereotyped features of networks (network motifs) raise general questions
about relationships among genes. Protein-protein interactions can be analyzed
at the level of individual domains, and many genetic interactions can be
analyzed in terms of specific alleles that affect parts of proteins. Taken
together, these data might provide a higher resolution view of interaction
networks.
Data integration is not an isolated field. Further improvements in data integration are closely tied to the progress of source databases, such as the synchronization of data annotation with gene model changes, the standardization of ortholog mapping techniques and the development of bio-ontologies and their consistent compilation, often by a professional curator.
Negative data are relatively underreported in the published literature. As discussed above, sophisticated data integration methods often use positive and negative training sets. Authors, reviewers and editors could help the cause by encouraging the inclusion of negative observations (`gene A does not interact with gene B for phenotype C') in papers, for example as supplemental data.
ACKNOWLEDGMENTS
Many of the model organism databases and the Gene Ontology Consortium that are crucial to data integration are supported by the National Human Genome Research Institute (e.g. WormBase grant HG02223 to P.W.S. and Gene Ontology Consortium grant HG002273 to Judith Blake). We thank Min Han for communicating unpublished results, and Alok Saldanha, Erich Schwarz, Xiaodong Wang and anonymous reviewers for comments on the manuscript. Relevant genetics research in our laboratory is supported by the Howard Hughes Medical Institute, with which P.W.S. is an Investigator and W.Z. an Associate.
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