Transcript Alignment of mRNA 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.
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