Nucleic Acids Research Advance Access originally published online on September 10, 2008
Nucleic Acids Research 2009 37(Database issue):D567-D570; doi:10.1093/nar/gkn583
Nucleic Acids Research, 2009, Vol. 37, Database issue D567-D570
© 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.
FLYSNPdb: a high-density SNP database of Drosophila melanogaster
Doris Chen1,2,*,
Jürg Berger1,
Michaela Fellner1,2 and
Takashi Suzuki1
1Research Institute of Molecular Pathology (IMP), Dr Bohr-Gasse 7 and 2Institute of Molecular Biotechnology (IMBA), Dr Bohr-Gasse 3, A-1030 Vienna, Austria
*To whom correspondence should be addressed. Tel: +43 1 79044 4513; Email: doris.chen{at}univie.ac.at
Received August 15, 2008. Accepted August 28, 2008.
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ABSTRACT
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FLYSNPdb provides high-resolution single nucleotide polymorphism
(SNP) data of
Drosophila melanogaster. The database currently
contains 27 367 polymorphisms, including
>3700 indels (insertions/deletions),
covering all major chromsomes. These SNPs are clustered into
2238 markers, which are evenly distributed with an average density
of one marker every 50.3 kb or 6.6 genes. SNPs were identified
automatically, filtered for high quality and partly manually
curated. The database provides detailed information on the SNP
data including molecular and cytological locations (genome Releases
3–5), alleles of up to five commonly used laboratory stocks,
flanking sequences, SNP marker amplification primers, quality
scores and genotyping assays. Data specific for a certain region,
particular stocks or a certain genome assembly version are easily
retrievable through the interface of a publicly accessible website
(
http://flysnp.imp.ac.at/flysnpdb.php).
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INTRODUCTION
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Drosophila melanogaster is one of the most well-studied model
organisms due to its short generation time and ease of genetic
manipulation. Hence, it is continuously providing major insights
into biological processes which are conserved in multicellular
organisms. Single nucleotide polymorphisms (SNPs) are widely
used as genetic markers in mapping experiments, quantitative
trait loci (QTL) analyses, population genetic or evolutionary
studies, since they are frequent, mostly phenotypically neutral
and molecularly defined. FLYSNPdb contains data of a polymorphism
map with an unprecedented resolution of
50 kb between SNP markers,
which is significantly higher than the density of previous
Drosophila SNP maps (
1–4). Polymorphisms >1 nt were also counted,
including indels, which are particularly useful for genotyping
assays based on PCR-product length polymorphisms [PLP; (
2)]
or denaturing high performance liquid chromatography [DHPLC;
(
5)] or also for evolutionary analyses (
6,
7). The map comprises
SNPs from five different
D.
melanogaster stocks (
Supplementary Table 1).
Since polymorphisms in
Drosophila are generally bi-allelic and
randomly distributed among the utilized strains, we anticipate
that most of our SNP markers can be used to discriminate almost
any other pair of
Drosophila stocks. FLYSNPdb is part of the
FLYSNP website (
http://flysnp.imp.ac.at/), which provides detailed
information on the practical aspects of SNP mapping and genotyping
in
Drosophila (
8) as well as a user guide for the database,
a glossary and protocols. With this database, we want to provide
a versatile SNP data resource, which is easy to use and has
a user-friendly web interface.
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DATA SOURCE
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For SNP identification, we designed primer pairs to amplify
fragments which are
1 kb long (
9), equally distributed along
each major chromosome arm (X, 2L, 2R, 3L and 3R), and which
preferentially lie in unique, non-protein coding regions (
Figure 1).
Genomic DNA of up to five standard laboratory stocks per amplicon
served as template: besides the wild-type stocks Canton S and
Oregon R, we selected for each chromosome arm one strain that
carries visible recessive markers, one stock with a Flp recombinase
target (FRT) element (
10) close to the centromere, and one stock
with an enhancer-promoter P- (EP) element at the chromosome
tip and a visible
white+ marker (
11) (see also
Supplementary Table 1).
The wild-type and FRT stocks are commonly utilized in mutagenesis
screens, and the recessive marker as well as EP stocks are useful
for identification of recombination events in defined chromosomal
regions (
2,
4). PCR products were sequenced in both orientations,
each using one of the amplification primers as sequencing primer.
In total, >2.3 Mb (1.7%) of the 117 Mb long euchromatic region
of the
D. melanogaster genome were resequenced and analysed.
After sequencing the PCR fragments, the Phred/Cross_match/PolyBayes
software package (
12–14) was used for trace quality assessment,
alignment to the reference genome (strain y; cn bw sp) (
15,
16),
and automated SNP discovery. In order to obtain high-quality
data, SNPs at the first and last 75 bases of an amplicon or
below Phred score 20 were omitted. In addition,
27% of the alignments
were visually inspected [with the help of Consed 11.0 (
17)],
which was particularly necessary for detection of long indels
(>6 bases). If multiple sequence reads from the same stock
were available at one site, the allele with the highest Phred
score was selected. Moreover, SNPs located at adjacent loci
were considered as a single polymorphic site. Of the analysed
amplicons, 86.9% contained at least one polymorphism in any
of the examined stocks. The SNP positions were updated to Release
5 (FB2006_01) of the
D. melanogaster genome by aligning 40 bp
of the sequences (from Release 3 or 4) flanking each SNP site
to the new reference sequence using Blastn (
18). Prediction
of restriction fragment length polymorphism (RFLP) sites was
accomplished with the help of Remap [EMBOSS software suite (
19,
20)]
and the REBASE list of commercially available restriction enzymes
with cut sites
4 bp (
21,
22).
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Figure 1. Data source pipeline for SNP identification, data retrieval and curation. Software tools are displayed below each task (for references please see text); if not otherwise stated, costum-made scripts were used.
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DATABASE CONTENT
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The FLYSNPdb data set currently comprises >81 700 SNP alleles
at 27 367 sites in 2238 amplicons of about 1 kb length (
Table 1).
One SNP marker contains in average 12 polymorphisms, the maximal
SNP count per marker is 73. The average distance between SNP
markers is 50.3 kb, a region in which one can find in average
6.6 genes [according to the FlyBase Release 5.10, FB2008_07
annotation (
23)]. The biggest gap between markers is 360 kb
long and lies at the tip of chromosome arm 3R, between cytological
region 82A1 and 82C3. Only 169 polymorphic loci (0.6% of total
SNPs) are tri-allelic, the rest is bi-allelic. A total of 13.7%
(3743) of the SNPs are indels, which are up to 360 bp long,
but predominantly (96.4%) <10 bp (46.6% of the indels are
1 nt long). For any given stock-pair, the average percentage
of SNP markers with a sequence divergence between these two
stocks is 76.6%, ranging from 35.3% to 92.0%. Furthermore, the
database provides information on the molecular and cytological
SNP locations for three genome assembly versions [Release 3–5;
(
15)], together with the 30 bp flanking sequences as additional
site identification feature. For data quality assessment, PolyBayes
probability scores (
14), Phred trace quality scores (
12), as
well as the number of sequence reads per alignment are available,
and manually curated SNPs are indicated. Since non-coding regions
are more polymorphic, we have put our focus on non-exonic regions.
If SNPs lie within an intron or exon (according to FlyBase Release
5.10), the corresponding gene name is also retrievable. In addition,
information on SNP marker amplification primers is available
for genotyping assays which are based on sequencing. Polymorphisms
that are suitable for RFLP assays (SNPs which result in differential
restriction enzyme sites) or for which verified PLP or tag-array
mini-sequencing (TAMS) assays (8) are available, are also indicated,
including further information like verified primers or suitable
restriction enzymes.
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IMPLEMENTATION, USAGE AND ACCESS
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All data are organized and stored in a relational database.
For increasing the speed of web queries, several summarizing
tables were precomputed and put into a MySQL database which
is accessed through PHP scripts.
The form on the first page asks the user to specify the chromosomal region and two stocks for which data on differential polymorphisms will be retrieved (Figure 2). The region can be indicated as molecular coordinates (position 1 – position 2 or position 1 + length) or as cytological segment (region 1 – region 2). Furthermore, it is possible to select whole chromosome arms by leaving the ‘Location’ field blank, or getting all data by selecting the ‘Browse all’ option. SNP data can be viewed as list of SNP markers (including SNP count, amplification primer sequences) or as table of SNP sites (with alleles, flanking sequences, etc.). Additional information concerning quality scores, genotyping assay suitability or coding information (genic, intronic or exonic) can be optionally selected. For users of the previous FLYSNP database version, old identifiers (ids) are retrievable and a link to this version is provided. On each query result page, sub-selections can be made by clicking on the checkboxes at the left side of each row, or by entering search parameters in the fields below each column (Figure 2). The tables are downloadable, e.g. as tab-separated text files which can be easily imported into commonly used databases or Excel spreadsheets, or as track files which can be uploaded to the FlyBase genome viewer [GBrowse; (24)]. As an additional feature, a link to FlyBase GBrowse is provided for the graphical display of the region previously specified by the user.
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Figure 2. Screenshots of FLYSNPdb input form and query result. On the first page, the user selects chromosomal region and stocks as well as different view options. On the search result page, further features such as table download or sub-queries are available.
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RECENT AND FUTURE DEVELOPMENTS
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The FLYSNPdb data were recently submitted to dbSNP (NCBI, Release
129;
http://www.ncbi.nlm.nih.gov/projects/SNP/) so that direct
linkage to the FlyBase data repository is feasible. Furthermore,
sequence traces and alignments will be provided for users who
would like to see the raw data for detailed quality assessment.
We are open to help users with their individual needs and will
implement suggestions of common use.
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SUPPLEMENTARY DATA
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Supplementary data are available at NAR Online.
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FUNDING
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European Union Fifth Framework Programme (QLRI-CT-2001-00004);Boehringer
Ingelheim GmbH; Japan Society for the Promotion of Science.
Funding for open access charge: IMP.
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ACKNOWLEDGEMENTS
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We thank the whole FLYSNP Consortium, especially Barry Dickson
(IMP) for initiation and coordination of the FLYSNP project;
Montserrat Aguadé and Dorcas Orengo (Univ. of Barcelona)
for providing primers. We are also grateful to Cerebrum Web
Consulting for initial setup of the first FLYSNP database version,
Werner Kubina and Christian Brandstaetter (IMP) for IT support,
Gotthold Schaffner (IMP) for sequencing and Angela Graf (IMP)
for stock keeping.
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Footnotes
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Present addresses: Doris Chen, Department of Biochemistry, University
of Vienna, Max F. Perutz Laboratories (MFPL), c/o IMBA, Dr Bohr-Gasse
3, A-1030 Vienna, Austria
Jürg Berger, Roche Austria GmbH, Engelhorngasse 3, A-1210 Vienna, Austria
Michaela Fellner, Vienna Drosophila Research Center (VDRC), Dr Bohr-Gasse 3, A-1030 Vienna, Austria
Takashi Suzuki, Max Planck Institute of Neurobiology, Am Klopferspitz 18, D-82152 Martinsried, Germany
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