Nucleic Acids Research Advance Access originally published online on October 8, 2008
Nucleic Acids Research 2009 37(Database issue):D49-D53; doi:10.1093/nar/gkn694
Nucleic Acids Research, 2009, Vol. 37, Database issue D49-D53
© 2008 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
MachiBase: a Drosophila melanogaster 5'-end mRNA transcription database
Budrul Ahsan1,
Taro L. Saito1,2,
Shin-ichi Hashimoto3,
Keigo Muramatsu4,
Manabu Tsuda4,
Atsushi Sasaki1,
Kouji Matsushima3,
Toshiro Aigaki4 and
Shinichi Morishita1,2,*
1Department of Computational Biology, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa 277-0882, 2Japan Science and Technology Agency (JST), Tokyo 102-8666, 3Department of Molecular Preventive Medicine, School of Medicine, The University of Tokyo, Tokyo 113-0033 and 4Department of Biological Sciences, Tokyo Metropolitan University, Hachioji, Tokyo, Japan
*To whom correspondence should be addressed. Tel: +81 47 136 3984; Fax: +81 47 139 3977; Email: moris{at}k.u-tokyo.ac.jp, moris{at}cb.k.u-tokyo.ac.jp
Received August 15, 2008. Accepted September 25, 2008.
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ABSTRACT
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MachiBase (
http://machibase.gi.k.u-tokyo.ac.jp/) provides a
comprehensive and freely accessible resource regarding
Drosophila melanogaster 5'-end mRNA transcription at different developmental
states, supporting studies on the variabilities of promoter
transcriptional activities and gene-expression profiles in the
fruitfly. The data were generated in conjunction with the recently
developed high-throughput genome sequencer Illumina/Solexa using
a newly developed 5'-end mRNA collection method.
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INTRODUCTION
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Characterization of the complete repertoire of expressed messenger
RNA (mRNA) is central to the functional analysis of a genome.
To date, several studies have been undertaken to achieve a better
understanding of the
Drosophila melanogaster genome (
1–4).
The technical approaches used in these studies included in-depth,
full-length cDNA cloning and tiling microarrays. However, despite
the absence of prior knowledge of the locations of previously
identified genes, the 5'-end SAGE (
5) method has demonstrated
efficacy in cataloging high numbers of expressed genes. Following
the simple modification of adopting the recently developed high-throughput
genome sequencer Illumina/Solexa, 5'-end SAGE has become a potent
tool for elucidating transcriptional mechanisms. To achieve
a deeper insight into transcriptional activity, we collected
approximately 25 million 25–27 nt 5'-end mRNA tags from
the embryos, larvae, young males, young females, old males,
old females and S2 (culture cell line) of
D. melanogaster with
high mechanical reproducibility. After aligning these tags to
unique positions in the fly genome while allowing three mismatches,
2.87–4.05 million uniquely mapped tags were amassed for
each of the seven samples. These data constitute the most substantial
transcriptional start site (TSS) and gene-expression database
for
D. melanogaster currently available.
MachiBase is designed to assist fly biologists in their analyses of gene expression and in placing expression data in the context of functional genomics through genomic orientation. Thus, information on differentially expressed genes can be accessed by either inputting the gene name as a keyword or selecting a chromosomal location. Aside from providing information on gene expression, these data constitute a potent resource for analyses of transcriptional regulation. The core promoter, which is the region surrounding the TSS of a gene required for recruitment of the transcription apparatus, warrants analysis. However, TSSs and core promoters have previously been identified on a gene-by-gene basis. With the help of this database, biologists can explain transcriptional initiation mechanisms by combining additional information on chromatin structure and DNA methylation. In addition, these data allow accurate predictions of gene structures, particularly of the 5'-untranslated region (5'-UTR).
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METHODS
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The newly developed 5'-end mRNA collection method extends the
range of the original 5'-end SAGE technique developed by Hashimoto
et al. (
5). This method initially profiles 25–27 nt tags
using a novel strategy that incorporates the oligo-capping method
(
6). The 5'-end tags are then ligated directly to the Illumina/Solexa
linker, to prepare for sequencing with the Illumina/Solexa system.
Prior to construction of the Illumina/Solexa libraries, we confirmed
the integrity of the cDNA using the Agilent 2100 Bioanalyser.
Collection of numerous 5'-end tags from seven libraries and testing the reproducibility of the method used
To characterize the transcriptional activity patterns of the D. melanogaster genome, we collected 25–27 nt 5'-end mRNA tags from embryos, larvae, young males, young females, old males, old females and the S2 cell line. Table 1 presents the results of this process. The second column shows more than five million raw tags collected from each of the seven libraries. As most of these tags were redundant, they were grouped into non-redundant representative tags, the statistics for which are shown in the third column. Each non-redundant tag represents a duplicated occurrence and is therefore associated with its frequency, i.e. the number of times that it occurs.
The frequency is expected to be reproducible, in that the frequency
of each non-redundant tag is proportional to the total number
of tag occurrences in independent experiments. To test for reproducibility,
we performed an additional collection of 5'-end tags from the
same young female
Drosophila library.
Figure 1A reveals a strong
correlation between the two independent experiments. Furthermore,
in a comparison with a quantitative PCR analysis, the employed
method has been validated as a means to quantify the expression
level of a transcript as the number of 5'-end tags (
7).
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Figure 1. Statistical analysis of the TSS information. (A) A dot represents one non-redundant 5'-end tag, such that the values on the x-axis and y-axis indicate the frequencies of the focal tags in the respective experiments. (B) A case in which the known representative TSS is not consistent with the most frequent TSS. Note that the most abundant TSSs coincide across the seven different libraries. (C) The distribution of distances between the known representative TSSs and most frequent TSSs. Overall, 1033 (8.8%) of the 11 725 known representative TSSs coincide with the most frequent TSSs.
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Identification of transcription start sites by millions of 5'-end tags
For the identification of TSSs, non-redundant tags were aligned
to the genome of
D. melanogaster (R5.3) in FlyBase (
8). We observed
that 5'-end tags tended to contain read errors, especially towards
their termini. To correct these read errors, the tags were aligned
to the genome while allowing, at most, three mismatches. The
efficient mapping of millions of tags was an issue that needed
to be resolved. We developed and used a parallel version of
BLAT (
9), which operates on massive parallel clusters. Another
major technical issue involved the fact that a single 5'-end
tag could be mapped to multiple locations, making it difficult
to determine the original location of the tag. To eliminate
false-positive positional data, these ambiguous tags were simply
excluded from our analysis, so that only uniquely aligned tags
were considered. A tag was considered to be
uniquely aligned if, for a non-negative number
k (
3), the tag was mapped to a
unique location with
k mismatches, although it could be mapped
to multiple positions with more than
k mismatches. The number
of uniquely aligned and redundant tags in each library, and
their ratios to the number of raw redundant tags, are shown
in the fourth and fifth columns of
Table 1, respectively. A
uniquely aligned 5'-end tag identified a TSS in the genome.
Distinct tags could be mapped to the same TSS, since the alignment
step tolerated mismatches and replaced erroneous nucleotides
with the correct nucleotides in the genome. From all seven libraries,
a total of 25 083 481 tags were mapped to unique locations,
thereby identifying 1 773 851 TSSs; the data breakdown in terms
of chromosomes is presented in
Table 2.
Discrepancy between the known representative TSS and the most frequent TSS
In attempting genome annotation, it is usual to choose the longest
cDNA sequence in a specific locus to define the representative
cDNA. To examine the level of agreement between the newly collected
5'-end tags and the known representative cDNA sequences, we
calculated how many of the uniquely aligned, redundant tags
were located in the promoters and 5'-UTRs of the representative
sequences, and found 96.2% of the 5'-end tags in the UTR regions
(
Table 3).
Figure 1B illustrates the 5'-end tag expression patterns
surrounding a representative TSS in the seven libraries. It
was intriguing to observe that the representative TSS was not
necessarily the most frequent TSS, but that another TSS slightly
downstream of the representative was the most abundant, which
motivated us to examine this discrepancy. We calculated the
distances between the representative TSSs and the most frequent
TSSs in the promoters and 5'-UTRs of the 11 725 longest cDNA
sequences in FlyBase.
Figure 1C shows the numbers of representative
TSSs in terms of distances, highlighting that only 1033 (8.8%)
of the 11 725 known representative TSSs were the most frequent
TSSs. Our analysis indicates that the common practice of selecting
the longest cDNA sequence as the representative one needs to
be revised, and demonstrates the efficacy of 5'-end tag collection
for detecting the most abundant TSS as an alternative to the
representative TSS.
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Table 3. Ratios of uniquely aligned, redundant tags located in the promoters and 5'-UTR of the representative sequences
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Database features and applications
We visualized the numbers of 5'-end tags for each position in
a vertical bar (
Figure 2). This arrangement of 5'-end data provides
an insight into fly transcription, in combination with other
annotated genomic information. In the MachiBase database server,
users can browse the TSSs and frequencies of individual genes
by querying the FlyBase gene ID, FlyBase transcription ID, etc.
In addition to the gene-specific view, it is also possible to
generate an overview of all the expressed transcripts for an
assigned position on a chromosome. Furthermore, all these genes
are linked with the FlyBase annotation server, which contains
Gene Ontology (GO), orthologue information, etc. In addition
to revealing differentially expressed genes, genome-wide TSS
discovery is a valuable resource for biologists studying flies.
This high-throughput study has revealed a surprisingly large
number of novel genic (intron–exon regions) and intergenic
TSSs, which has prompted a rethink of the relationships between
gene transcription and promoter architecture. For example, if
we display the location (2L: 2 391 450–2 391 850) by inputting
2L into the ‘Target’ box, 2 391 450 into the ‘Start’
box, 2 391 850 into the ‘End’ box, we can see the
existence of an a new transcript supported by a significant
number of 5'-end tags in the un-annotated intergenic region.
Thus, the precise locations of the TSSs enable an in-depth analysis
of
cis-acting elements that are bound by transcription factors.
This data resource provides a starting point for elucidating
novel molecular details of transcription by reliably integrating
TSS location data with related functional data, such as histone
methylation and acetylation states (
10,
11), the positions of
nucleosomes (
12–14) and the occupancy of transcription
factor binding sites (
15), each of which, as features, can now
be examined on a genome-wide basis.
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Figure 2. Snapshot of the MachiBase genome browser. The frequencies of the 5'-end mRNA tags mapped to individual positions on the fruitfly genome in the seven libraries are displayed as histograms in the bottom seven tracks. In the histograms, the vertical bars in log scale indicate the numbers of 5'-end mRNA tags aligned to each position on the x-axis. The upper track shows the exon–intron structures of two alternative splice variants. Observe that the peaks for the 5'-end tags are around the 5'-end of the longer splice variant in all of the seven libraries. In addition, note that many 5'-end tags are expressed from the second, second-to-last, and last exons in four adult samples (young/old and male/female).
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DISCUSSION
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The vast transcriptional datasets have been used to characterize
differentially expressed genes, especially in relation to age
and sexual development. Using these datasets, we have confirmed
that the representative TSSs, the abundantly expressed TSSa
flanking FlyBase-annotated TSSs, differ from many of the known
FlyBase-annotated TSSs. It has become evident that the rules
for start site selection are fundamentally different for different
promoters, and large-scale studies have given us the tools to
partition promoters into functional classes with respect to
TSS information in future studies. As a novel and high-quality
data resource, MachiBase is a valuable tool for experimental
biologists who are working on
D. melanogaster. In future, we
will empower this database with various annotated data on the
fly genome.
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FUNDING
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Scientific Research on Priority Areas (C) from the Ministry
of Education, Culture, Sports, Science and Technology of Japan
partially; Bioinformatics Research and Development (BIRD); the
Japan Science and Technology Agency (JST). Funding for open
access charge: JST.
Conflict of interest statement. None declared.
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ACKNOWLEDGEMENTS
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Computational time was provided by the Information Technology
Center and the Human Genome Center, at the University of Tokyo.
All 5'-end sequence data are deposited at NCBI Short Read Archive
under the accession number SRA002200.
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Footnotes
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The authors wish it to be known that, in their opinion, the
first two authors should be regarded as joint First Authors.
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INTRODUCTION