SSHscreen and SSHdb : software for microarray-based screening and sequence management of cDNA libraries

A pipeline was developed for the quantitative screening and sequence management of clones from suppression subtractive hybridization (SSH) cDNA libraries. The pipeline is particularly useful for gene discovery in non-sequenced organisms, and was illustrated with SSH library data from pearl millet (Pennisetum glaucum) and cowpea (Vigna unguiculata) and Arabidopsis (Arabidopsis thaliana) ecotype Kil-0. The objective of each library was to identify stress-response genes. cDNA microarrays provide a high-throughput screening method. Accordingly, these SSH libraries were amplified by PCR and spotted onto glass microarray slides. Subtracted and un-subtracted cDNA samples, that were used to construct the SSH libraries were prepared as Cy3- and Cy5-labeled targets and hybridized to the microarrays. The R package SSHscreen version 2.0.0, available from http://microarray.up.ac.za/SSHscreen/, was developed to analyze the resulting microarray data using limma (linear models for microarray data) functions. Commonly, loess normalization is used for within-slide normalization, however this is based on the assumption that most of the genes on the array are not differentially expressed. This is legitimate for most whole genome microarray experiments, however it is not appropriate when the array is constructed from an SSH library which is enriched for differentially expressed genes. Therefore, control spot-based normalization was used in the SSHscreen analysis. Empirical Bayes methods were employed to calculate the moderated t-statistic using functions from the limma package. This procedure in effect borrows information from the ensemble of genes to aid with inference about individual genes, taking advantage of the parallel structure whereby the same model is fitted to the data for each gene. In the Arabidopsis, pearl millet and cowpea forward libraries, 18%, 58% and 58% of the clones were identified as significantly up-regulated (adjusted p-value < 0.05) and in the reverse libraries, 18%, 30% and 28% significantly down-regulated, respectively. SSHscreen analysis was used to assist in selection of clones for sequencing. The SSHscreen data output (ranked gene lists in terms of differential expression), as well as the selected sequences in FASTA format were uploaded to SSHdb. For the Arabidopsis library, 114 out of the 262 sequenced clones (55%) were identified as unique/non-redundant; and for the pearl millet and cowpea libraries respectively, 37% and 33% of the sequenced clones were unique. SSHdb was developed as a web-based tool for sequence management and annotation of clones in SSH libraries and can freely be accessed at http://sshdb.bi.up.ac.za. BLAST analysis that was carried out when sequences were uploaded to SSHdb was used to combine clones with the same sequence into redundant partner groups, as well as identify putative annotations for each group. Individual clones from the abovementioned SSH libraries were selected and an independent technique, quantitative PCR, was used to validate the microarray/SSHscreen results. The pipeline was applied successfully to Arabidopsis, pearl millet and cowpea SSH cDNA libraries. Interesting genes in each case were identified for further study. CopyrightDissertation (MSc)--University of Pretoria, 2010.Biochemistryunrestricte

Maize microarray annotation database

<p>Abstract</p> <p>Background</p> <p>Microarray technology has matured over the past fifteen years into a cost-effective solution with established data analysis protocols for global gene expression profiling. The Agilent-016047 maize 44 K microarray was custom-designed from EST sequences, but only reporter sequences with EST accession numbers are publicly available. The following information is lacking: (a) reporter - gene model match, (b) number of reporters per gene model, (c) potential for cross hybridization, (d) sense/antisense orientation of reporters, (e) position of reporter on B73 genome sequence (for eQTL studies), and (f) functional annotations of genes represented by reporters. To address this, we developed a strategy to annotate the Agilent-016047 maize microarray, and built a publicly accessible annotation database.</p> <p>Description</p> <p>Genomic annotation of the 42,034 reporters on the Agilent-016047 maize microarray was based on BLASTN results of the 60-mer reporter sequences and their corresponding ESTs against the maize B73 RefGen v2 "Working Gene Set" (WGS) predicted transcripts and the genome sequence. The agreement between the EST, WGS transcript and gDNA BLASTN results were used to assign the reporters into six genomic annotation groups. These annotation groups were: (i) "annotation by sense gene model" (23,668 reporters), (ii) "annotation by antisense gene model" (4,330); (iii) "annotation by gDNA" without a WGS transcript hit (1,549); (iv) "annotation by EST", in which case the EST from which the reporter was designed, but not the reporter itself, has a WGS transcript hit (3,390); (v) "ambiguous annotation" (2,608); and (vi) "inconclusive annotation" (6,489). Functional annotations of reporters were obtained by BLASTX and Blast2GO analysis of corresponding WGS transcripts against GenBank.</p> <p>The annotations are available in the Maize Microarray Annotation Database <url>http://MaizeArrayAnnot.bi.up.ac.za/</url>, as well as through a GBrowse annotation file that can be uploaded to the MaizeGDB genome browser as a custom track.</p> <p>The database was used to re-annotate lists of differentially expressed genes reported in case studies of published work using the Agilent-016047 maize microarray. Up to 85% of reporters in each list could be annotated with confidence by a single gene model, however up to 10% of reporters had ambiguous annotations. Overall, more than 57% of reporters gave a measurable signal in tissues as diverse as anthers and leaves.</p> <p>Conclusions</p> <p>The Maize Microarray Annotation Database will assist users of the Agilent-016047 maize microarray in (i) refining gene lists for global expression analysis, and (ii) confirming the annotation of candidate genes before functional studies.</p

SSHscreen and SSHdb, generic software for microarray based gene discovery: application to the stress response in cowpea

<p>Abstract</p> <p>Background</p> <p>Suppression subtractive hybridization is a popular technique for gene discovery from non-model organisms without an annotated genome sequence, such as cowpea (<it>Vigna unguiculata </it>(L.) Walp). We aimed to use this method to enrich for genes expressed during drought stress in a drought tolerant cowpea line. However, current methods were inefficient in screening libraries and management of the sequence data, and thus there was a need to develop software tools to facilitate the process.</p> <p>Results</p> <p>Forward and reverse cDNA libraries enriched for cowpea drought response genes were screened on microarrays, and the R software package SSHscreen 2.0.1 was developed (i) to normalize the data effectively using spike-in control spot normalization, and (ii) to select clones for sequencing based on the calculation of enrichment ratios with associated statistics. Enrichment ratio 3 values for each clone showed that 62% of the forward library and 34% of the reverse library clones were significantly differentially expressed by drought stress (adjusted p value < 0.05). Enrichment ratio 2 calculations showed that > 88% of the clones in both libraries were derived from rare transcripts in the original tester samples, thus supporting the notion that suppression subtractive hybridization enriches for rare transcripts. A set of 118 clones were chosen for sequencing, and drought-induced cowpea genes were identified, the most interesting encoding a late embryogenesis abundant Lea5 protein, a glutathione S-transferase, a thaumatin, a universal stress protein, and a wound induced protein. A lipid transfer protein and several components of photosynthesis were down-regulated by the drought stress. Reverse transcriptase quantitative PCR confirmed the enrichment ratio values for the selected cowpea genes. SSHdb, a web-accessible database, was developed to manage the clone sequences and combine the SSHscreen data with sequence annotations derived from BLAST and Blast2GO. The self-BLAST function within SSHdb grouped redundant clones together and illustrated that the SSHscreen plots are a useful tool for choosing anonymous clones for sequencing, since redundant clones cluster together on the enrichment ratio plots.</p> <p>Conclusions</p> <p>We developed the SSHscreen-SSHdb software pipeline, which greatly facilitates gene discovery using suppression subtractive hybridization by improving the selection of clones for sequencing after screening the library on a small number of microarrays. Annotation of the sequence information and collaboration was further enhanced through a web-based SSHdb database, and we illustrated this through identification of drought responsive genes from cowpea, which can now be investigated in gene function studies. SSH is a popular and powerful gene discovery tool, and therefore this pipeline will have application for gene discovery in any biological system, particularly non-model organisms. SSHscreen 2.0.1 and a link to SSHdb are available from <url>http://microarray.up.ac.za/SSHscreen</url>.</p

Detoxification enzymes associated with insecticide resistance in laboratory strains of Anopheles arabiensis of different geographic origin

Background The use of insecticides to control malaria vectors is essential to reduce the prevalence of malaria and as a result, the development of insecticide resistance in vector populations is of major concern. Anopheles arabiensis is one of the main African malaria vectors and insecticide resistance in this species has been reported in a number of countries. The aim of this study was to investigate the detoxification enzymes that are involved in An. arabiensis resistance to DDT and pyrethroids. Methods The detoxification enzyme profiles were compared between two DDT selected, insecticide resistant strains of An. arabiensis, one from South Africa and one from Sudan, using the An. gambiae detoxification chip, a boutique microarray based on the major classes of enzymes associated with metabolism and detoxification of insecticides. Synergist assays were performed in order to clarify the roles of over-transcribed detoxification genes in the observed resistance phenotypes. In addition, the presence of kdr mutations in the colonies under investigation was determined. Results The microarray data identifies several genes over-transcribed in the insecticide selected South African strain, while in the Sudanese population, only one gene, CYP9L1, was found to be over-transcribed. The outcome of the synergist experiments indicate that the over-transcription of detoxification enzymes is linked to deltamethrin resistance, while DDT and permethrin resistance are mainly associated with the presence of the L1014F kdr mutation. Conclusions These data emphasise the complexity associated with resistance phenotypes and suggest that specific insecticide resistance mechanisms cannot be extrapolated to different vector populations of the same species

DDT and pyrethroid resistance in Anopheles arabiensis from South Africa

BACKGROUND: Pyrethroid resistance has been well documented in Anopheles arabiensis, one of the major African malaria vectors, and the predominant malaria vector in South Africa. METHODS: In this study, the genetic basis of pyrethroid resistance in a selected laboratory strain of An. arabiensis from South Africa was investigated using a custom-made microarray, known as the An. gambiae detoxification chip. RESULTS: A large number of P450 genes were over-transcribed, as well as a suite of redox genes and glutathione S-transferases. The five genes that showed the highest level of gene transcription when compared with an insecticide susceptible strain were: CYP6AG2, CYPZ1, TPX2, CYPZ2 and CYP6P1. CONCLUSIONS: Permethrin resistance in South African An. arabiensis is associated with increased transcription of multiple genes, and a large proportion of these genes were also previously recorded as over-transcribed in another An. arabiensis strain selected for resistance to DDT with cross-resistance to deltamethrin. The deltamethrin resistance developed de novo in the DDT-selected strain and is most likely due to increased transcription of those genes associated with DDT resistance. However, of particular interest was the fact that the strain selected for resistance to pyrethroids did not develop de novo resistance to DDT. These differences are compared and discussed.National Research Foundation Scarce Skills Scholarships and DAAD (LN), National Research Foundation and the National Health Laboratory Service-Research Trust to LLK, partial funding from the Department of Science and Technology, National Research Foundation Research Chair Initiative grant to Maureen Coetzee.http://www.parasitesandvectors.com/content/6/1/229am201

DDT and pyrethroid resistance in Anopheles arabiensis from South Africa

BACKGROUND: Pyrethroid resistance has been well documented in Anopheles arabiensis, one of the major African malaria vectors, and the predominant malaria vector in South Africa. METHODS: In this study, the genetic basis of pyrethroid resistance in a selected laboratory strain of An. arabiensis from South Africa was investigated using a custom-made microarray, known as the An. gambiae detoxification chip. RESULTS: A large number of P450 genes were over-transcribed, as well as a suite of redox genes and glutathione S-transferases. The five genes that showed the highest level of gene transcription when compared with an insecticide susceptible strain were: CYP6AG2, CYPZ1, TPX2, CYPZ2 and CYP6P1. CONCLUSIONS: Permethrin resistance in South African An. arabiensis is associated with increased transcription of multiple genes, and a large proportion of these genes were also previously recorded as over-transcribed in another An. arabiensis strain selected for resistance to DDT with cross-resistance to deltamethrin. The deltamethrin resistance developed de novo in the DDT-selected strain and is most likely due to increased transcription of those genes associated with DDT resistance. However, of particular interest was the fact that the strain selected for resistance to pyrethroids did not develop de novo resistance to DDT. These differences are compared and discussed.National Research Foundation Scarce Skills Scholarships and DAAD (LN), National Research Foundation and the National Health Laboratory Service-Research Trust to LLK, partial funding from the Department of Science and Technology, National Research Foundation Research Chair Initiative grant to Maureen Coetzee.http://www.parasitesandvectors.com/content/6/1/229am201

De Novo sequencing, assembly, and analysis of the root transcriptome of Persea americana (Mill.) in response to Phytophthora cinnamomi and flooding

Avocado is a diploid angiosperm containing 24 chromosomes with a genome estimated to be around 920 Mb. It is an important fruit crop worldwide but is susceptible to a root rot caused by the ubiquitous oomycete Phytophthora cinnamomi. Phytophthora root rot (PRR) causes damage to the feeder roots of trees, causing necrosis. This leads to branchdieback and eventual tree death, resulting in severe losses in production. Control strategies are limited and at present an integrated approach involving the use of phosphite, tolerant rootstocks, and proper nursery management has shown the best results. Disease progression of PRR is accelerated under high soil moisture or flooding conditions. In addition, avocado is highly susceptible to flooding, with even short periods of flooding causing significant losses. Despite the commercial importance of avocado, limited genomic resources are available. Next generation sequencing has provided the means to generate sequence data at a relatively low cost, making this an attractive option for non-model organisms such as avocado. The aims of this study were to generate sequence data for the avocado root transcriptome and identify stress-related genes. Tissue was isolated from avocado infected with P. cinnamomi, avocado exposed to flooding and avocado exposed to a combination of these two stresses. Three separate sequencing runs were performed on the Roche 454 platform and produced approximately 124 Mb of data. This was assembled into 7685 contigs, with 106 448 sequences remaining as singletons. Genes involved in defence pathways such as the salicylic acid and jasmonic acid pathways as well as genes associated with the response to low oxygen caused by flooding, were identified. This is the most comprehensive study of transcripts derived from root tissue of avocado to date and will provide a useful resource for future studies.The Technology and Human Resources Programme (THRIP, grant number TP2011060300010) as an initiative of the National Research Foundation (NRF), the Hans Merensky Foundation, and the Genomics research institute (GRI) at the University of Pretoria.www.plosone.orgam201