ORIGINAL ARTICLE
Large-scale correlation of DNA accession numbers to thecDNAs in the FANTOM full-length mouse cDNA clone set
Itsuki Ajioka,1 , 2 Takuya Maeda,1 , 2 and Kazunori Nakajima2 , 3
1
Equal contributors in this work
Department of Anatomy, School of Medicine, Keio University
Tokyo, Japan;
3
Department of Molecular Neurobiology, Institute of DNA Medicine, Jikei University
School of Medicine, Tokyo, Japan
2(Received for publication on February 2, 2006)(Revised for publication on June 1, 2006)(Accepted for publication on June 1, 2006)

Abstract. Oligonucleotide-based microarrays, such as GeneChip, are widely used to determine the large-scale gene expression profiles. However, GeneChip only provides information on the identity of the molecules, and the investigator must obtain each cDNA clone forfurther analyses. In this study, we devised a program which enables us to correlate a large
number of DNA accession numbers to the FANTOM (functional annotation of the mouse)full-length mouse cDNA clone set, and made a correlative table between mouse GeneChipclones and FANTOM clones. This allows easy identification of the corresponding FANTOM
clone for each GeneChip clone, even if the sequence of the GeneChip clone does not directly
match the FANTOM clone. Using this table, for example, a large number of in situ hybridization probes can be synthesized easily, because the FANTOM clones are flanked by T3/T7
promoters on both ends. In addition, we further developed a program which retrieves theamino acid sequence (AA Seq) for each clone, even for the FANTOM clones that lack theAA Seq description, and classifies the proteins automatically. As an example, we devised acorrelation table with predictions of the secretory or transmembrane molecules. The correlation table is useful for a large-scale screening of molecules involved in cell-cell communication in various biological processes. The full correlation table for the GeneChip clones
is available at http://www.kjm.keio.ac.jp/past/55/3/correlation_table1.html (Keio J Med 55 (3):107－110, September 2006)
Key words: GeneChip, FANTOM, screening, secretory protein, transmembrane protein

Introduction
As various databases of gene expression profiles havebecome available, it has become increasingly necessaryfor biologists to have to deal with a long list of DNA accession numbers. Oligonucleotide-based microarrays,
such as GeneChip, for example, are widely used to determine the large-scale expression profiles of numerous
genes. However, GeneChip analysis only provides information on the identity of the expressed molecules, andthe investigator must obtain each cDNA clone for further

analyses, including in situ hybridization and analysis byectopic overexpression. Thus, from this point of view,
although the GeneChip is very powerful for large-scale
analyses of gene expression, use of this database is rather
impractical for determining the expression profiles ofgenes at the cellular level or performing functional assays of genes at a large scale. The emerging strategy
of in silico screening on the internet, that is, the digital
differential display, has the same problem. The mouse
cDNAs clone set, namely, FANTOM clones, comprisedof 60,770 full-length cDNAs (FANTOM2 1), is com-Reprint Requests to: Kazunori Nakajima, M.D., Ph.D., Department of Anatomy, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku,
Tokyo160-8582, Japan, E-mail: kazunori@sc.itc.keio.ac.jp
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mercially available (although we used the FANTOM2clones 1 in this study, the updated FANTOM3 clones
have been reported recently2). After the GeneChip assay, investigators often search for the cDNAs such as
FANTOM clones by using world wide web services, andalso use other web services to obtain additional information for further assays. However, manual operation of theinternet services by means of a GUI-based web browserpractically confines the investigators to analysis of only arelatively small number of molecules. Thus, in this study,
we report on our development of a Perl script program
which enables us to correlate a large number of DNA
accession numbers to the FANTOM clones. The program accesses various web-based databases and services
imitating the manual operation, so the investigators neednot to construct and maintain enormous local databases.
As an example, we devised a correlative table between
mouse GeneChip clones and FANTOM clones, which allows easy identification of the corresponding FANTOM
clone for each GeneChip clone, even if the sequence ofthe GeneChip clone does not directly match the FANTOM clone. In addition, since most GeneChip experiments are being performed for screening molecules, itwould be useful if the clones could be further classified
by the type of proteins encoded. Thus, we further developed a program which retrieves the amino acid sequence(AA Seq) for each clone, even for the FANTOM clones
that lack the AA Seq description in the flat files, andclassifies the proteins automatically. As an example, wedevised a correlation table with predictions of the secretory or transmembrane molecules (S/TMs).
Methods and Results
Hyper text transfer protocol (HTTP) was applied to thenetwork program, and a Perl module, LWP, was downloaded from the Comprehensive Perl Archive Network(CPAN) to enable the program to communicate at HTTP
level (http://www.perl.com/CPAN/). First, we collected
HTML-formatted files that were displayed while performing manual operations through websites, and researched the interconnection by investigating the HTMLfiles. Since the query data themselves change according
to the series of HTML-formatted files as responses fromeach web service, our programs extract proper data thatmatch patterns predetermined by our investigations of theserver’s responses, and communicate with the server bysubmitting proper query data. The first program preparedflat files of the Affymetrix GeneChip U74A, B, andC clones by submitting their accession numbers to thewebsite of DDBJ (http://getentry.ddbj.nig.ac.jp/getstarte.html) 3 and receiving all the flat files via FTP. The
program executed an FTP program, wget, and ‘zgip’
program to expand a compressed file. The second
program converted the flat files into a multi-FASTA

Fig. 1 Correlation of each GeneChip clone with FANTOM
clonesWe developed the four Perl programs to correlate each
GeneChip Clone with FANTOM clones as described in the maintext. Acc: accession. E: E-value.

formatted file of DNA Seqs. Then, we executed thestand-alone BLAST program downloaded from the National Center for Biotechnology Information (NCBI)(http://www.ncbi.nlm.nih.gov/Ftp/index.html)4 by using
the FASTA file as queries and by setting the FANTOM
database (http://genome.gsc.riken.jp)5 as a targeted database. Then, the third program correlated each accession
number of the GeneChip clones to that of the highestscored FANTOM clones in the obtained BLAST searchresults (Fig. 1). As a result, while 27,533 of the 35,813
clones (76.9%) were found to match with the FANTOM
clones, 8,280 clones did not match. Some of the nonmatching clones of GeneChip were due to low BLAST
scores, because we set the threshold of E-value at 0.0001.
The other non-matching ones might have correspondedto a part of the untranslated region (UTR) that was notcontained in the FANTOM clones. To obtain the longer
cDNA Seq corresponding to the GeneChip clones, thefourth program submitted the multi-FASTA formatted
file of the 8,280 DNA Seqs to the DDBJ BLAST sitewith a ‘Rodents’ database as the targeted database, and7,589 of the 8,280 clones were identified as the rodent
cDNA clones. Then, we correlated them to the FANTOM clones again and found that 2,458 of the 7,589
clones (32.3%) matched with the FANTOM clones, although they did not have any overlapping sequences withthe GeneChip clones. Finally, we combined these 2,458
clones with the above 27,533 clones and obtained a correlation table of 29,991 of the 35,813 clones (83.7%)of GeneChip U74. The full list is available as Supplementary table 1 at http://www.kjm.keio.ac.jp/past/55/3/
correlation_table1.html This “2-step correlation program”
allows easy development of correlation tables between
independent cDNA clone sets in general, even if the twoKeio J Med 2006; 55 (3): 107－110

clones in each set derived from the same mRNA andcontained only partial sequences without any overlap.
To predict S/TMs in the FANTOM clones, we applied
the first program to the accession numbers of the FANTOM clones and obtained their flat files. Our second
program generated not only a multi-FASTA file of DNA
Seqs but also that of AA Seqs from flat files. By examining the multi-FASTA file of the AA seqs, we foundthat while 17,487 clones had the AA Seq description,
12,504 clones did not (Fig. 2). We carefully read theflat files without the AA Seq description and found thatthe FANTOM clones occasionally had sequence errorsthat prevented translation into an AA, as reported previously.6 To obtain the AA Seq of such clones, we appliedthe “2-step correlating program” to the 12,504 clones.
As a result, 11,359 clones were found to match with therodent cDNA clones, and 5,187 clones were identified
as clones with an AA Seq description (Fig. 2). We combined these 5,187 clones with the 17,487 clones that had
the AA Seq description in the FANTOM flat files, andobtained the AA Seqs of a total of 22,674 clones (Fig. 2).
To determine whether or not they were S/TMs, we used
2 independent prediction programs. One was a simple
program predicting a signal peptide in the N-terminus of
the proteins based on an argorithm of the iPSORT program7 and the other was a web client program that communicates with a web service of TMHMM that accurately predicts transmembrane helices in proteins more accurately as compared with the other prediction programs.8 , 9Using iPSORT-based programs, 4,485 clones were identified as having a signal sequence, while the TMHMM
programs identified 4,841 clones which had a transmembrane region (Fig. 2, Supplementary table 1 at http://www.kjm.keio.ac.jp/past/55/3/correlation_table1.html
Thus, the “2-step correlation program” used herein isalso useful for generating correlation tables of clones forany types of proteins by using the respective prediction
programs.
Discussion
The correlation table devised in this study can be generally applied to the analyses of the stage-, region- andcell type-specific molecules involved in both normal andpathological events. Since the FANTOM cDNA clones
are inserted between T3/T7 promoters on the vector
plasmids, a large number of in situ hybridization probes
can be synthesized at once easily, for example.1 0 Thus,
the correlation table, which combines the large-scale
microarray analysis and the full-length cDNA clone set,
will effectively facilitate the large-scale analyses of themolecules involved in various biological events.
Acknowledgement: We thank members of DDBJ for their valu-

able advice on the utilization of the DDBJ web services via FTP,
and members of the Nakajima laboratory for the valuable dis-109Fig. 2 Screening of predicted secretory or transmembrane
molecules
By using the “2-step correlation program”, we obtained theAA Seq of a total of 22,674 clones as described in the main text.
To determine whether or not they were S/TMs, we developed asimple program predicting a signal peptide in the N-terminus of
the proteins based on an algorithm of the iPSORT program7 anda web client program that communicates with a web service of
the TMHMM that accurately predicts transmembrane helices.9cussions. We also thank Genome Exploration Research Groupof RIKEN Genome Sciences Center for the use of FANTOM
database in this study. This work was supported by grants from
the Ministry of Education, Culture, Sports, and Science andTechnology of Japan, Japan Society for the Promotion Science, aGrant-in-Aid for the 21st Century Center of Excellence Program
entitled “Understanding and Control of Life’s Function via Systems Biology”, a Keio University Special Grant-in-Aid for Innovative Collaborative Research Projects, the Novartis Foundation,
the Casio Science Promotion Foundation, the Okawa Foundation,and the Japan Brain Foundation.

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