title: [Agilent-024550] USP Schistosoma mansoni 4x44K oligo-array version 2.0
status: Public on Dec 16 2014
contact.city: São Paulo
contact.first: Katia
contact.middle: Cristina Pereira
contact.last: Oliveira
contact.state: O
contact.address: Av Prof lineu prestes 748
contact.department: Centro de Parasitologia e Micologia
contact.country: Brazil
contact.institute: Instituto Adolfo Lutz
organism: Schistosoma mansoni
manufacture_protocol: This platform has the same oligos that GPL8606 just a diferent arranjment. All transcript information for S. mansoni available at both nr and dbEST databases at GenBank as of March, 2006 were filtered for contaminants and assembled with the CAP3 program. BLASTx were performed against the nr, with a cutoff of 1x10-10. The highest score alignment was used for annotation and assignment of the coding strand, in which the probe was designed. Those without significant hits against nr were annotated as no match, and probes for both strands were designed. In order to include possible new S. mansoni genes that have orthologous expressed sequences in S. japonicum but have not yet been cloned and sequenced in S. mansoni, we adopted the following strategy: S. japonicum RNA sequences from GenBank were assembled and submitted to a tBLASTx search against our assembled S. mansoni sequence set, and those with e-value lower than 1x10-20 were discarded. The remaining sequences were considered as S. japonicum transcripts without a clear corresponding S. mansoni transcript in the public database, thus being a candidate for a new S. mansoni gene. In order to obtain the S. mansoni sequence corresponding to these transcripts, the S. japonicum sequences were submitted to a BLASTn search against the S. manoni genome assembly (version 3), downloaded from the Wellcome Trust Sanger Institute. S. mansoni genomic loci that produced an alignment with at least 100 bases and had an identity higher than 70% were selected. Alternatively, tBLASTx was used, and S. mansoni genomic sequences were selected when they matched at least two high-scoring segment pairs (HSPs) each with e-value below 0.005. [NOTE added on October 2009: The criteria used for mapping S. japonicum evidence of transcription to the S. mansoni genome were poor; besides the set of bona fide probes, there were 521 spurious probes designed from japonicum information; all of these 521 probes were marked with a note *probe has no match to prediction=>abandon*, in the updated annotation table as of 28_October_2009. Expression data obtained with these 521 probes should be used with extreme caution.] These conserved regions in the S. mansoni genome were subsequently used in the probe design process. For each contig or singlet from the assembly, we selected all 60-mer sequences that satisfied a series of conditions that have been established by Agilent Technologies as follows: probes should not have 8 or more bases derived from low complexity regions; they should have a GC content of 35-55% and a melting temperature (Tm) of 68-76 Celsius degrees. In order to reduce cross-hybridization, each 60-mer sequence was searched by BLASTn against the S. mansoni genome sequence, downloaded from the Wellcome Trust Sanger Institute. In case that no hits were produced in this first search, the probes were used in a BLAST search against the CAP3 assembled mRNA/EST sequences. Probes were selected that had 100% match to the target sequence, which translates into a best-match alignment bit-score of 119. Those selected 60-mer sequences were checked for a low probability of cross-hybridization to non-specific targets by selecting probes for which the alignment bit-score of the second-best match was equal to or lower than 42.1. The selected probes were carried forward to the last step, which consisted of picking the probe closest to the 3-end for eachh target.