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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. The analysis is used to identify exons and to show evidence of alternative splicing The NCBI alignment tool Spidey is used for the analysis. Outline ESTs and gene prediction Alternative Splicing UniGene and Spidey HIP2 Analysis HIP2’s ESTs 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. 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. NCBI UniGene Database UniGene partitions GenBank sequences into a nonredundant 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. 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. Alternative Splicing ESTs and RNA Splicing ESTs cDNA Recombination of the gene Intron Exon 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 Alternative Splicing of HIP2 HIP2 xx--------------xx-------x--------xx----xx-xxxxxxxxxxxxxxxxxxxxx Different human mRNAs for this protein show alternative splicing mRNA mRNA mRNA mRNA mRNA mRNA mRNA 1 xx------------xx----------------xx----xx-xxxxx 2 -x------------xx-------x--------xx----xx-xx-xxxxxxxxxxxxxxxxxxxx 4 xx------------xx-------x--------xx----xx-xx-xxxx 5 xx------------xx-------x--------xx----xx-xx-xxxx 7 ------------------------------------------------xxxxxxx 8 xx------------xx-------x--------xx----xx-xxxxxxxxxxxxxxxxxxxx 10 xx-----------xx-------x---------xx----xx-xx-xxxxxxxxxxxxxxxxxxx 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 Analysis HIP2’s ESTs 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. Thank you for listening