Park et al. BMC Genomics 2012, 13:473 http://www.biomedcentral.com/1471-2164/13/473 related to horse racing performance: six previously iden- tified exercise-associated genes and 91 early regulated transcription factors that are differentially expressed by exercise, three genes that display high SNV density, and four alternatively expressed splicing variants. In addition, all 1,258 differentially expressed genes could be import- ant candidate genes for further research. Methods The muscle and blood samples from six retired thoroughbred horses were taken before and after trotting (30 minutes). 90-bp pair-end sequences were obtained with an Illumina HiSeq2000, San Diego, US, from the samples. These sequences were mapped against the horse reference genome (Ensembl release 62) using TopHat 1.2.0 with two options (−−mate- inner-dist = 200 and –allow-indels) for paired-end sequences and identified unigenes using the Cuf- flinks program without genome annotation data. From the results of 24 samples, novel genes were clustered based on the genomic coordination to de- fine unigene clusters (UCs). The generated UCs were subjected to the filter, which extracted UCs overlap- ping with the genome annotation. The expressed genes annotated by the pipeline and the filtered UCs were merged as the final set of UCs. In addition, unmapped sequences were assembled by the SOAP- denovo [28] program and were subjected to an ORF length filtering process. In-house bioinformatics pipelines were used to identify SNVs under several stringent conditions, especially for the exon-intron boundary misalignment filter (See Additional file 1: Supplementary Methods). Differentially expressed genes were selected by comparing the expression profiles of the six horses, with a selection criterion of more than two fold up- or downregulation in four or more horses. Data access All raw sequences of the horse transcriptomes are open- freely available at http://www.horsegenome.net/. Additional file Additional file 1: Figure S1. Six thoroughbred horses. Table S1. 24 sample names from six thoroughbred horses used in this study. Table S2. The statistics of RNA-Seq raw data from 24 different samples. Table S3. The mapping results against the horse reference genome (Ensembl 62). Figure S2. Procedure for identifying horse unigenes. Figure S3. Distribution plot of the exons identified without gene models which contain ORFs. Figure S4. Reverse transcript PCR (RT-PCR) confirmation of the 8 novel unigene clusters (UCs) Table S4. Primer information for the RT-PCR experiment. Table S5. Statistics of the filtered de novo transcripts identified from cufflink. Table S6. de novo assembly results from one sample with various k-mer values. Table S7. de novo assembly of unmapped sequences originated from 24 samples. Table S8. Filtered Page 6 of 8 and clustered unigenes from the scaffolds assembled from unmatched sequences. Figure S5. The whole process of identifying SNVs. Table S9. The proportion of the scaffolds from the unmapped sequences which were matched against the human genome. Table S10. The statistics of total SNVs identified from 24 samples. Table S11. The number of total SNVs identified in thoroughbred horses. Table S12. The number of individual-specific SNVs. Table S13. Conformation of the SNPs identified from the mouse sample [2]. Table S14. Distribution of SNP locations in three datasets. Table S15. The list of transcripts which have ten or more non-synonymous SNPs. Figure S6. GO classification of all expressed genes in human, mouse, and horse muscle tissue. Figure S7. GO classification of all expressed horse genes in blood and muscle tissue. Figure S8. Correlation matrix of the 24 samples. Figure S9. Correlation matrix of three human samples from kidney and liver tissues. Figure S10. Histogram of average expression level of the unigene clusters in the 24 samples. Table S16. List of DEGs in muscle and blood tissues. Table S17. Expression profiles of known exercise-related horse genes. Table S18. Comparison between DEGs in muscle tissue and the DEGs which are responsible to exercise training [25]. Table S19. List of transcription factors differentially expressed in muscle and blood tissues. Table S20. RT-PCR primers for seven transcription factors. Table S21. RT-PCR results of differentially expressed transcription factors in muscle tissue. Table S22. The list of four genes of which alternative splicing forms showed reversed expression patterns before and after exercising. Figure S11. Expression profiles of the genes of which alternative splicing forms showed reversed expression patterns before and after exercising. Table S23. Number of filtered de novo transcripts identified by Cufflink. Supplementary methods. Competing interests The authors declare that they have no competing interests. Authors’ contributions KDP, THK, SL, HKL, BWC, and JB designed and supervised the experiments and analyses. KDP, JP, BCK, HKL, BWC, and JB supervised the progress of the project. KDP, BWC, KTD, YMY, HSK, CK, and THK prepared blood and muscle samples from the six horses. CK, BCK, and THK generated sequences from the samples. JP, HC, HMK, SS, and SJ conducted the bioinformatics analyses. HSK, JP, and JB designed the validation experiments, and KA and KTD conducted the experiments. JP, BCK, SL, SH, JK, and JB wrote the manuscript and BCK, HC, SL, SH, and YMY participated in improving the manuscript. All authors read and approved the final manuscript. Acknowledgements This work was supported by a grant from the Next-Generation BioGreen 21 Program (No.PJ0081062011), Rural Development Administration, Republic of Korea. The Theragen team was supported by the Industrial Strategic Technology Development Program, 10040231, "Bioinformatics platform development for next generation bioinformation analysis", funded by the Ministry of Knowledge Economy (MKE, Korea). SH was supported by a grant from KRIBB Research Initiative Program. We thank Maryana Bhak for editing. Author details 1Department of Biotechnology, Hankyong National University, Anseong 456-749, Republic of Korea. 2Personal Genomics Institute, Genome Research Foundation, 443-270 Suwon, Republic of Korea. 3Theragen BiO Institute, TheragenEtex, 443-270 Suwon, Republic of Korea. 4Department of Biological Sciences, College of Natural Sciences, Pusan National University, Busan 609-735, Republic of Korea. 5Department of Animal Science, College of Life Sciences, Pusan National University, Miryang 627-702, Republic of Korea. 6Department of Pathology, School of Medicine, and Institute of Biomedical Science and Technology, Konkuk University, Seoul 143-701, Republic of Korea. 7Department of Nanomedical Engineering, College of Nanoscience and Nanotechnology, Pusan National University, Miryang 627-702, Republic of Korea. 8Korean Bioinformation Center, Korea Research Institute of Bioscience and Biotechnology, 305-806 Daejeon, Republic of Korea.