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Alignment of mRNAs to genomic DNA Sequence

Alignment of mRNAs to genomic DNA Sequence. Martin Berglund Khanh Huy Bui Md. Asaduzzaman Jean-Luc Leblond. Objectives. To examine how ESTs collected in the public EST division of Genbank/EMBL compare to the known structure of a gene.

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Alignment of mRNAs to genomic DNA Sequence

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  1. Alignment of mRNAs to genomic DNA Sequence Martin Berglund Khanh Huy Bui Md. Asaduzzaman Jean-Luc Leblond

  2. Objectives • To examine how ESTs collected in the public EST division of Genbank/EMBL compare to the known structure of a gene. • The analysis is used to identify exons and to show evidence of alternative splicing • The NCBI alignment tool Spidey is used for the analysis.

  3. Outline • ESTs and gene prediction • Alternative Splicing • UniGene and Spidey • HIP2 • Analysis HIP2’s ESTs

  4. Expressed Sequence Tags (ESTs) • To determine which genes (or parts of genes) that are expressed in a particular cell type or tissue, mRNAs are isolated and reverse transcribed into cDNA. • Short fragments of these cDNAs (ESTs) are then sequenced. • The resulting EST sequences are compared with the nucleotide sequence of the entire genome (or the sequence of a single gene) to locate the gene (or parts of a gene) that contains each EST.

  5. ESTs and Gene Prediction • Unlike DNA, cDNA contains only expressed DNA sequences. • If a region of sequence matches ESTs with high stringency then the region is probably an exon of a gene.

  6. NCBI UniGene Database • UniGene partitions GenBank sequences into a non-redundant set of gene-oriented clusters. Each UniGene cluster contains sequences that represent a unique gene, as well as related information such as the tissue types in which the gene has been expressed and map location. Features: • Clusters in UniGene database are generated automatically. • It’s not true that sequences in one cluster can be always assembled into one contiguous sequences. • UniGene clusters are only available for model organisms.

  7. SPIDEY • Aligns one or more mRNA sequences to a single genomic sequence. • Try to determine the exon/intron structure, returning one or more models of the genomic structure, including the genomic/mRNA alignments for each exon.

  8. Alternative Splicing

  9. ESTs and RNA Splicing ESTs cDNA Recombination of the gene Intron Exon

  10. HIP2 • Huntingtin Interacting Protein 2 • This protein has been implicated in the degradation of huntingtin and suppression of apoptosis. • Huntingtin appears to play a critical role in nerve • For human, located on chromosome: 4

  11. Alternative Splicing of HIP2 • HIP2 xx--------------xx-------x--------xx----xx-xxxxxxxxxxxxxxxxxxxxx Different human mRNAs for this protein show alternative splicing • mRNA 1 xx------------xx----------------xx----xx-xxxxx • mRNA 2 -x------------xx-------x--------xx----xx-xx-xxxxxxxxxxxxxxxxxxxx • mRNA 4 xx------------xx-------x--------xx----xx-xx-xxxx • mRNA 5 xx------------xx-------x--------xx----xx-xx-xxxx • mRNA 7 ------------------------------------------------xxxxxxx • mRNA 8 xx------------xx-------x--------xx----xx-xxxxxxxxxxxxxxxxxxxx • mRNA 10 xx-----------xx-------x---------xx----xx-xx-xxxxxxxxxxxxxxxxxxx

  12. Analysis HIP2’s ESTs • Download 614 ESTs of HIP2 from GenBank using Bioperl module • Align all ESTs to HIP2 using Spidey locally • Parse Spidey results using a Perl script

  13. Analysis HIP2’s ESTs

  14. Conclusion • Alternative splicing is evident from the EST data • Some of the ESTs show different coding regions other than the exons specified in HIP2’s annotation.

  15. Thank you for listening

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