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Maize microarray annotation database.

Coetzer N, Myburg AA, Berger DK - Plant Methods (2011)

Bottom Line: 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).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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Plant Science, Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Private Bag X20, 0028, South Africa. Dave.Berger@fabi.up.ac.za.

ABSTRACT

Background: 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.

Description: 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.The annotations are available in the Maize Microarray Annotation Database http://MaizeArrayAnnot.bi.up.ac.za/, as well as through a GBrowse annotation file that can be uploaded to the MaizeGDB genome browser as a custom track.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.

Conclusions: 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.

No MeSH data available.


Related in: MedlinePlus

Strategy followed to assign genomic and functional annotations to the reporters on the Agilent-016047 maize microarray. Using BLASTN and exonerate software, the 42,034 60-mer reporters were matched to available EST sequences, the maize B73 RefGen v2 genome and the WGS predicted transcripts. BLASTN and exonerate results were filtered and compared to test agreement between EST, WGS transcript and gDNA hits. Based on the agreement analysis, one of six genomic annotation groups was assigned for each reporter. Functional annotations of reporters were based on the functional annotations of their corresponding WGS transcripts. The data has been made accessible from the Maize Microarray Annotation Database http://MaizeArrayAnnot.bi.up.ac.za/.
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Figure 1: Strategy followed to assign genomic and functional annotations to the reporters on the Agilent-016047 maize microarray. Using BLASTN and exonerate software, the 42,034 60-mer reporters were matched to available EST sequences, the maize B73 RefGen v2 genome and the WGS predicted transcripts. BLASTN and exonerate results were filtered and compared to test agreement between EST, WGS transcript and gDNA hits. Based on the agreement analysis, one of six genomic annotation groups was assigned for each reporter. Functional annotations of reporters were based on the functional annotations of their corresponding WGS transcripts. The data has been made accessible from the Maize Microarray Annotation Database http://MaizeArrayAnnot.bi.up.ac.za/.

Mentions: Figure 1 outlines the strategy that was followed to obtain genomic annotations for each reporter on the Agilent-016047 microarray. All nucleotide sequences were searched against target datasets using the BLASTN algorithm version 2.2.18 [18]. For BLASTN searches of the 60-mer reporter sequences against ESTs, the WGS transcripts and gDNA (B73 RefGen v2), the word size parameter was set to 23 and gaps were not allowed. This cut-off was chosen based on a study that showed that matches of ≥ 23 contiguous nucleotides yielded hybridization signals under stringent conditions in more than 90% of a set of Agilent reporters [19]. Thus, the identity out of 60, rather than the E-value, was used as the measure of similarity for BLASTN searches with the reporters. We also carried out BLASTN searches with EST sequences, and in these cases E-values were used. All BLAST results were stored in a relational database. The parameters used for the BLAST searches are shown in Additional file 1.


Maize microarray annotation database.

Coetzer N, Myburg AA, Berger DK - Plant Methods (2011)

Strategy followed to assign genomic and functional annotations to the reporters on the Agilent-016047 maize microarray. Using BLASTN and exonerate software, the 42,034 60-mer reporters were matched to available EST sequences, the maize B73 RefGen v2 genome and the WGS predicted transcripts. BLASTN and exonerate results were filtered and compared to test agreement between EST, WGS transcript and gDNA hits. Based on the agreement analysis, one of six genomic annotation groups was assigned for each reporter. Functional annotations of reporters were based on the functional annotations of their corresponding WGS transcripts. The data has been made accessible from the Maize Microarray Annotation Database http://MaizeArrayAnnot.bi.up.ac.za/.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3198759&req=5

Figure 1: Strategy followed to assign genomic and functional annotations to the reporters on the Agilent-016047 maize microarray. Using BLASTN and exonerate software, the 42,034 60-mer reporters were matched to available EST sequences, the maize B73 RefGen v2 genome and the WGS predicted transcripts. BLASTN and exonerate results were filtered and compared to test agreement between EST, WGS transcript and gDNA hits. Based on the agreement analysis, one of six genomic annotation groups was assigned for each reporter. Functional annotations of reporters were based on the functional annotations of their corresponding WGS transcripts. The data has been made accessible from the Maize Microarray Annotation Database http://MaizeArrayAnnot.bi.up.ac.za/.
Mentions: Figure 1 outlines the strategy that was followed to obtain genomic annotations for each reporter on the Agilent-016047 microarray. All nucleotide sequences were searched against target datasets using the BLASTN algorithm version 2.2.18 [18]. For BLASTN searches of the 60-mer reporter sequences against ESTs, the WGS transcripts and gDNA (B73 RefGen v2), the word size parameter was set to 23 and gaps were not allowed. This cut-off was chosen based on a study that showed that matches of ≥ 23 contiguous nucleotides yielded hybridization signals under stringent conditions in more than 90% of a set of Agilent reporters [19]. Thus, the identity out of 60, rather than the E-value, was used as the measure of similarity for BLASTN searches with the reporters. We also carried out BLASTN searches with EST sequences, and in these cases E-values were used. All BLAST results were stored in a relational database. The parameters used for the BLAST searches are shown in Additional file 1.

Bottom Line: 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).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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Plant Science, Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Private Bag X20, 0028, South Africa. Dave.Berger@fabi.up.ac.za.

ABSTRACT

Background: 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.

Description: 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.The annotations are available in the Maize Microarray Annotation Database http://MaizeArrayAnnot.bi.up.ac.za/, as well as through a GBrowse annotation file that can be uploaded to the MaizeGDB genome browser as a custom track.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.

Conclusions: 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.

No MeSH data available.


Related in: MedlinePlus