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De novo sequencing and transcriptome analysis.pdf

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De novo sequencing and transcriptome analysis

Pang et al. BMC Genomics 2013, 14:488 /1471-2164/14/488RESEARCH ARTICLE Open AccessDe novo sequencing and transcriptome analysis of the desert shrub, Ammopiptanthus mongolicus, during cold acclimation using Illumina/Solexa Tao Pang, Chu-Yu Ye, Xinli Xia* and Weilun Yin*Abstract Background: Ammopiptanthus mongolicus (Maxim. ex Kom.) Cheng f., an evergreen broadleaf legume shrub, is distributed in Mid-Asia where the temperature can be as low as ?30°C during the winter. Although A. mongolicus is an ideal model to study the plant response to cold stress, insufficient genomic resources for this species are available in public databases. To identify genes involved in cold acclimation (a phenomenon experienced by plants after low temperature stress), a high-throughput sequencing technology was applied. Results: We sequenced cold-treated and control (untreated) samples of A. mongolicus, and obtained 65,075,656 and 67,287,120 high quality reads, respectively. After de novo assembly and quantitative assessment, 82795 all- unigenes were finally generated with an average length of 816 bp. We then obtained functional annotations by aligning all-unigenes with public protein databases including NR, SwissProt, KEGG and COG. Differentially expressed genes (DEGs) were investigated using the RPKM method. Overall, 9309 up-regulated genes and 23419 down- regulated genes were identified. To increase our understanding of these DEGs, we performed GO enrichment and metabolic pathway enrichment analyses. Based on these results, a series of candidate genes involved in cold responsive pathways were selected and discussed. Moreover, we analyzed transcription factors, and found 720 of them are differentially expressed. Finally, 20 of the candidate genes that were up-regulated and known to be associated with cold stress were examined using qRT-PCR. Conclusions: In this study, we identified a large set of cDNA unigenes from A. mongolicus. This is the first transcriptome sequencing of this non-mode