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Pipeline publication

[…] b'brary. A gel purification procedure was carried out to select the fragments sized from 250 to 350 bp to produce the library for cluster generation and sequencing. The library were qualified by Agilent 2100 bioanalyzer and quantified by Qubit and qPCR. The cluster formation and sequencing were performed on the HiSeq2000 platform following the manufacturer\xe2\x80\x99s standard cBot and sequencing protocols. For the multiplexing sequencing, a 100 cycles of single read 1 is used to sequence the RNA, following by a 7 cycles of index identification and 100 cycles of single read 2., A Perl program was written to remove low quality sequences from all sequences. Then the high quality reads were assembled with Velvet_1.2.10 to construct unique consensus sequences [] The trimed solexa transcriptome reads were mapped onto the unique consensus sequences using Bowtie Version 0.12.8 []. Unigenes were compared with the NCBI Non-redundant nucleotide database and Non-redundant protein database (http://www.ncbi.nlm.nih.gov/) using BLASTN and BLASTX respectively, and with the same E-value cutoffs < = 1e-5 [], identified by sequence similarity comparison against the SWISS-PROT (ftp://ftp.ebi.ac.uk/pub/databases/swissprot) with BLAST at E values < = 1e-10. The Clusters of Orthologous Groups (COG) of proteins database was used for functional annotation [], Kyoto Encyclopedia of Genes and Genomes (KEGG) database was used to retrieve KO information from blast result and then established pathway associations between unigene and databases []., The aim of our research was to learn more about the salt tolerance mechanisms and sought out the functional genes participated in, so we employed edgeR to identify differentially expressed mRNAs based on their relative abundance which was reflected by counting individual read between the two libraries [,]. With the help of the empirical Bayes estimation, tests based on the negative binomial distribution and differential signal analysis with other types of genome scale count data, genes with a P value < = 0.01 expression ratio > = 2 or expression ratio < = 0.5 were deemed to be significantly different between the two samples. In addition, both total expressed and differentially expressed genes were used to generate cluster diagrams by Cluster3.0 (http://bioservices.capitalbio.com/xzzq/rj/3885.shtml) using the hierarchical meth' […]

Pipeline specifications

Software tools Velvet, Bowtie, BLASTN, BLASTX, edgeR