2008 Barcelona - European Society of Human Genetics

Genomics, technology, bioinformatics 0
will present data from enrichment studies of the human genome of
sequence areas related to different diseases . The paired-end method
for the technologies provides additional information about large scale
variations in the genome allowing an accuracy approaching classical
Sanger sequencing . Comparison of the next-gen sequencing data to
a reference genome provides a mapping that takes advantage of the
high coverage to identify SNPs and other structural differences and
variations . The results can be imported into other assembly programs
(e .g . SeqMan of the Lasergene suite from DNASTAR Inc ., USA) .
P08.59
A novel approach for miRNA profiling and discovery using
massively parallel ligation-based dibase sequencing technology
R. R. Samaha, C. Barbacioru, J. Gu, S. Kuersten, R. Setterquist, M. Barker, R.
Wicki;
Applied Biosystems, Foster City, CA, United States.
Next-generation sequencing (NGS) platforms produce 10-100’s of millions
of short reads (25-50bp) in a single run which makes them particularly
suited for tag counting application including gene expression
profiling. With Applied Biosystem’s new platform, sequencing is carried
out via dibase sequential rounds of ligation with high fidelity and high
read quality . Using a newly developed library protocol which requires
low sample input and results in sequence ready samples in less than
a day, we explored the expression profiles of small non-coding RNAs
in two normal tissues, using the SOLiD system . The frequency and
distribution of miRNAs, isomiRNAs and miRNA* were evaluated and
the fold changes generated from these tissues were compared to
those of 380 TaqMan® miRNA assays. Significant correlation levels
were observed confirming the applicability of this approach for small
RNAs expression profiling. Moreover, more than 3000 potentially novel
miRNAs or non-coding RNAs were discovered . These potential novel
small RNAs are currently being further validated .
This new library approach coupled with the SOLiD System provides
a high throughput method for digital gene expression that enables the
discovery of novel small ncRNAs and miRNAs as well as profiling their
expression levels, without the probe bias of microarrays . Because of
the SOLiD System’s throughput which is greater than 100M reads
per slide, it is particularly suited for the analysis of gene expression
being able to deliver the dynamic range required to detect genes expressed
at very low levels and to accurately measure fold changes at
the same low expression levels .
P08.60
cloning and construction of mouse PEP cDNA chimera with
EGFP under regulation of cmV promoter
M. Ostadsharif1,2 , K. Ghaedi2,3 , S. Tanhaei2 , K. Karbalaei2 , M. Nasr-e-Esfahani2
;
1Islamic Azad University, Research& science campus, Tehran, Islamic Republic
of Iran, 2Royan,Isfahan Research Campus, Isfahan, Islamic Republic of Iran,
3Biology Dept., School of Sciences, The University of Isfahan, Isfahan, Islamic
Republic of Iran.
Peroxisomes are tiny organelles in almost all eukaryotic cells . They
exhibit various functions including β-oxidation of very long chain fatty
acids and metabolite peroxidation which is essential for the cell function
and differentiation . We have cloned (Peroxisomal Protein) PEP
cDNA in a mammalian expression vector in a chimeric cDNA type,
encompassing PEP with EGFP cDNA . Amino acid alignment analysis
revealed two hydrophobic domains . The First Comprises twenty amino
acid residues between 12-31 residues and the second one, is located
at 152-169 residues . There is a tripeptide (SKI) at carboxy terminus responsible
for sorting of this protein to the matrix of peroxisome . There
is a fibronectin type III domain between residues 31-114 in pep. In order
to see the importance of above sorting signal, we performed a sitedirected
mutagenesis to delete SKI tripeptide. Amplified Pep cDNAs
either containing SKI or deleted ones were constructed downstream
of EGFP cDNA under regulation of CMV promoter in pEGFP-C1 vector
and were send for sequence . Transfection of plasmids containing
chimera of EGFP-PEP cDNAs in to the CHO-K1 showed several punctuate
structures presumably peroxisomes while, SKI deletion showed
a cytosolic pattern like EGFP-C1 . Taken together, these data strongly
suggest that SKI, which is located at the C- terminus of protein is required
for sorting of this protein .
P08.61
the mouse Genome informatics (mGi) resource: translating
mouse phenogenomics into models of human disease
A. V. Anagnostopoulos, H. Dene, S. M. Bello, H. Onda, T. F. Meehan, M. N.
Knowlton, D. L. Burkart, I. Lu, L. L. Washburn, M. Tomczuk, R. P. Babiuk, B. A.
Richards-Smith, C. L. Smith, J. T. Eppig;
The Jackson Laboratory, Bar Harbor, ME, United States.
The mouse is universally considered a premier model system for studying
human development and disease . The MGI resource (http://www .
informatics .jax .org) provides free access to integrated data on the
genetics, genomics and biology of the laboratory mouse, facilitating
navigation through sequence, polymorphism, spatiotemporal expression,
mapping, biochemical function and process, sub-cellular topology,
mammalian homology, phenotype and disease model data . MGI
curates aberrant mouse phenotypes in the context of mutations (spontaneous,
induced or genetically engineered), strain variations, QTLs,
and complex traits that serve as credible models of human genetic
disorders, incorporating phenotype-related images as available . Robust
querying parameters include standardized terms from the Mammalian
Phenotype Ontology, a hierarchically-structured vocabulary
that supports morphophysiological annotations to background-specified
allelic genotypes at varying degrees of granularity. Parallel use
of key bio-ontologies, including the Anatomical Dictionary, GO, and
OMIM, fosters innovative approaches to peruse expression profiles,
map functional features of gene products to complex pathophysiological
states, and establish associations between observed mouse phenotypes
and orthologous human gene mutations or distinct nosological
entities for which defined mouse genotypes phenomimic the human
condition . MGI advances translational research through an integrated
data platform which optimizes retrieval and semantic interpretation of
multi-parametric, genome-scale datasets, and permits disease model
mining from a genotype, phenotype or computational perspective .
Recent enhancements include a redesigned homepage and site-wide
navigation paradigm, and an image-rich “Phenotype, Alleles & Disease
Models” portal, as one of several mini-homepages, each encapsulating
a different MGI biodomain along with content-specific access instructions,
statistics, FAQs, and news announcements . Supported by
NIH grants HG000330, HG002273, HD033745 .
P08.62
characterization of post-transcriptional regulation of the human
chromosome 21 transcriptome
S. I. Nikolaev1,2 , S. Deutsch1,2 , R. Genolet3 , C. Ucla1 , L. Parand1 , B. Conne1 , J.
Vassalli1 , J. Curran3 , S. E. Antonarakis1 ;
1Department of Genetic Medicine and Development, Geneva University,
Geneva, Switzerland, 2Authors contributed equally to the work, Switzerland,
3Department of Microbiology and Molecular Medicine, University of Geneva,
Geneva, Switzerland.
To characterize the post-transcriptional regulation of transcriptional
units in human chromosome 21 (HSA21), we performed a sucrose
gradient separation of RNA in 3 cell lines (GM06990, HelaS3 and
SKNAS) . We generated fractions according to ribosome content to
separate (i) RNA undergoing active translation (associated with at least
2 ribosomes) and (ii) a pool of all fractions representing total RNA .
Each pool was hybridized to a 22bp resolution tiling array comprising
the entire non-repetitive sequence of HSA21 (18 Mb) .
We observed that approximately 5% of HSA21 is transcribed in each
cell line, and a total of 8 .5% is transcribed in at least 1 line . On average
51 .6% of signals correspond to annotated regions, whereas the
remaining have been previously referred to as Txfrags .
We performed RT-PCR (with RT-minus control) and/or 5 and 3’ RACE
for 100 random Txfrags, and sequenced positive bands . In all cases
the sequences correspond to known exons elsewhere in the genome
but also with high homology to HSA21 . This strongly suggests that
these signals result from cross-hybridisation .
To identify genes with significant levels of post-transcriptional regulation,
we performed a paired-rank analysis of all detected exons . We
observed that 86 out of 247 HSA21 genes demonstrate a significant
shift in their rank distribution, suggesting post-transcriptional control .
Our data suggest that 1) there is a considerable posttranscriptional
control of gene expression and 2) a substantial fraction of Txfrags
result from cross-hybridization with exons mapping elsewhere in the
genome .