title: Agilent-035550 UniversityPadova_Ruditapes decussatus_8x15K_v4
status: Public on May 25 2017
contact.city: Legnaro
contact.first: Massimo
contact.last: Milan
contact.email: massimo.milan@unipd.it
contact.state: PD
contact.address: Viale dell'Università 16
contact.department: Dept. of Department of Public Health, Comparative Pathology,and Veterinary Hygiene
contact.country: Italy
contact.institute: University of Padova
contact.phone: +39 0498272941
organism: Ruditapes decussatus
description: A total of 41,119 contigs of R.decussatus obtained in the previous study (see Leite et al. 2013 for details) were reannotated to design a new version of R. decussatus DNA microarray. The Basic Local Alignment Search Tool (BLAST) was used to perform annotation of R. decussatus contigs. Batch Blast similarity searches for the entire set of contigs were locally conducted against NCBI (National Centre for Biotechnology Information) amino acidic non redundant (nr) database using Blastx option. To improve the number of annotated contigs five different approaches were attempted: i) blastx searches (cut off e-value of < 1.0 E-3) against protein database UniProtKB/SwissProt, ii) blastx (cut off e-value of < 1.0 E-3) and blastn (cut off e-value of < 1.0 E-5) searches against proteins and high quality draft trascriptomes of Danio rerio, Gasterosteus aculeatus, Oryzias latipes, Takifugu rubripes, Tetraodon nigroviridis, Homo sapiens, Bos taurus, Mus musculus, Xenopus tropicalis, Drosophila melanogaster, Aedes aegypti, Aedes mellifera, Schistosoma mansoni, Caenorhabditis elegans, Ciona intestinalis, Ciona savignyi, Culex quinquefasciatus available on Ensembl Genome Browser, iii) blastx (cut off e-value of < 1.0E-3) and blastn (cut off e-value of < 1.0 E-5) searches against proteins, transcripts and assembly scaffolds of Lottia gigantea v1.0 database and Crassostrea gigas. The Gene Ontology (GO) terms associations for BP, MF and CC were performed using Blastx algorithm against the NCBI amino acid nr database implemented in Blast2GO software.   Probe design started with the selection of target sequences to be represented onto the R. decussatus   microarray. All annotated entries (13,161 corresponding to the 32% of total contigs) were included. As most sequence reads were obtained from a non directional cDNA library, sense strand orientation was inferred putatively from that of homologuous protein sequences of other species. Accordingly, one probe for annotated transcripts with known orientation was designed to construct a high-density oligo-DNA microarray, while two probes with both orientations were designed for contigs with ambiguous orientation. Probe design was carried out using the Agilent eArray interface, which applies proprietary prediction algorithms to design 60 mer oligoprobes. Microarrays were synthesized in situ using the Agilent ink-jet technology with a 8 × 15 K format. Each array included default positive and negative controls.  A total of 14,003 probes, representing 13,161 R. decussatus transcripts were successfully obtained.  Independent probes are named withunivocal ID; the anti-sense probes are distinguished by suffix _AS. Oligonucleotides, 60-mer plus a “linker”, are synthesized directly on glass slide using the Agilent’s SurePrint Tecnology.
manufacture_protocol: See SurePrint Technology at manufacturer's web site http://agilent.com/