Transcriptomics,Genomics

Dataset Information

45

Microarray validation of an alternative splicing database


ABSTRACT: Alternative splicing generates functional diversity in higher organisms through alternative first and last exons, skipped and included exons, intron retentions and alternative donor and acceptor sites. In large-scale microarray studies in human and mouse, emphasis so far has been placed on exon-skip events, leaving the prevalence and importance of other splice types largely unexplored. Using a new human splice variant database and a genome-wide microarray to probes thousands of splice events of each type, we measured differential expression of splice types across 6 pairs of diverse cell lines and validated the database annotation process. Results suggest that splicing in human is more complex than simple exon skip events, which account for a minority of splicing differences. The relative frequency of differential expression of the splice types correlates with what is found by our annotation efforts. In conclusion, alternative splicing in human cells is considerably more complex than the canonical example of the exon-skip. The complementary approaches of genome-wide annotation of alternative splicing in human and design of genome-wide splicing microarrays to measure differential splicing in biological samples provide a powerful high-throughput tool to study the role of alternative splicing in human biology. Keywords: alternative splicing Overall design: Identification of alternative splicing changes in pairwise sample comparisons with technical replicates as control. Array Design: The annotated database of human splice isoforms, SpliceExpress Human Spliceome 1 (SEHS1), was created by combining RefSeq, Build 35 of the NCBI genome assembly and Unigene-derived EST alignments from Alternative Splicing Database (ASD). For each gene, exonic regions and exonic portions from 5' to 3' were indexed. Using the in-dexes as well as sequence coordinates, a splice type was assigned to every splice event as exon-skips/includes, intron retentions, alternative first exons, alternative last exons, alter-native donor sites, and alternative acceptor sites. A second database of alternative splicing in human, SpliceExpress Human Spliceome 2 (SEHS2), was generated independently using UCSC genome assembly hg17 with genomic alignments of 3,493,559 cDNA/ESTs and 24,297 RefSeq mRNAs. Data were filtered to eliminate likely spurious introns of less than 15 bases and remove questionable spliceoforms based on the number of sequences providing evidence and based on donor and acceptor motifs. High-confidence splice events contributed to the microarray platform SEHS1.5. All events in SEHS1.5 were common to both SEHS1 and SEHS2 databases. To avoid mismatched probes arising from low quality EST sequences, the respective genomic sequences were used. To detect alternative splicing events, the probe set included 3 exon-exon junction probes per exon-skip event; two exon-exon junction probes per alternative first or last exon; one exon-exon junction probe plus two exon-intron junction probes per intron retention; and two exon-exon junction probes per alternative donor or acceptor site; and at least one probe per single exon gene or gene without splice isoforms. Probes had an average length of 38 nucleotides. Since fixed-length probes repre-senting junctions may vary widely in GC content and melting temperature, probe lengths were varied from 34 to 42 bases so that the total melting temperature was optimized to 68 degrees. Junction probes were isothermally balanced across the splice junction to mini-mize disparities in melting temperature between the two portions. Sense strand oligonucleotides with an additional 10-base polyT linker at the 3' end as spacer were synthesized in situ by NimbleGen Systems. Samples: Four human cell lines were tested: Capan 1 (pancreas), HEK 293 (Kidney), MCF7A (breast) and CaCO2 (colon) using an expression protocol. Cell lines were obtained from the American Tissue Culture Collection (Manassas, VA) and cultured according to ATCC specifications. Replicate data were generated for each cell line. For HEK293, two additional replicates were generated for use as a control to assign statistical significance when comparing replicate samples.

INSTRUMENT(S): SpliceExpress Human Spliceome 1.5

SUBMITTER: Jonathan L Bingham  

PROVIDER: GSE9734 | GEO | 2007-12-08

SECONDARY ACCESSION(S): PRJNA103653

REPOSITORIES: GEO

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Publications

Extent and diversity of human alternative splicing established by complementary database annotation and microarray analysis.

Bingham Jonathan L JL   Carrigan Patricia E PE   Miller Laurence J LJ   Srinivasan Subha S  

Omics : a journal of integrative biology 20080301 1


Alternative splicing generates functional diversity in higher organisms through alternative first and last exons, skipped and included exons, intron retentions and alternative donor, and acceptor sites. In large-scale microarray studies in humans and the mouse, emphasis so far has been placed on exon-skip events, leaving the prevalence and importance of other splice types largely unexplored. Using a new human splice variant database and a genome-wide microarray to probes thousands of splice even  ...[more]

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