2009 Vienna - European Society of Human Genetics

Genomics, Genomic technology and Epigenetics
of mappable sequence data per run. Currently, novel ligation protocols
have been developed to support increased read lengths of 75 bases to
100 bases, as well as reverse ligations to facilitate paired end reads.
These longer read lengths increase throughput per run, facilitate resequencing
efforts of large genomes, and aid in the identification of
SNPs, indels, and other structural variations. Longer reads also lend
themselves to novel applications of SOLiD such as RNA expression
analysis and de novo sequencing.
P11.086
targeted sequence enrichment for the sOLiD tm system
K. J. Li 1 , T. D. Sokolsky 2 , A. A. Antipova 2 , C. R. Clouser 2 , E. T. Dimalanta 2 ,
C. Kosnopo 2 , C. C. Lee 2 , S. S. Ranade 2 , L. Zhang 2 , C. L. Hendrickson 2 , A. P.
Blanchard 2 , K. J. McKernan 2 ;
1 Applied Biosystems, part of LIFE Technologies, Foster City, CA, United States,
2 Applied Biosystems, part of LIFE Technologies, Beverly, MA, United States.
The SOLiD TM system can acquire several gigabases of sequence within
a single run, allowing for accurate resequencing of large genomes.
The ultra high throughput of the SOLiD TM system also facilitates deeper
sequencing of targeted genomic regions of interest for specific applications.
Methods to enrich DNA in targeted regions include hybridization
with custom oligonucleotides in-solution or on microarrays. For smallscale
studies, microarray enrichment is more cost-effective, while insolution
enrichment is preferred for larger studies due to its scalability
and potential for automation. Here, we report coupling of both enrichment
techniques with SOLiD TM sequencing, using Agilent HD-CGH microarrays
and in-solution probes to extract target regions from Yoruba
DNA. We present an optimized SOLiD TM workflow for both enrichment
strategies and provide deep sequencing results from these methods.
The workflow from enrichment to SOLiD TM sequencing is streamlined
by library construction prior to probe hybridization. Post-enrichment
material can be used directly for downstream steps including emulsion
PCR and sequencing on the SOLiD TM system. We demonstrate that
combining these hybridization-based enrichment methods with the
SOLiD TM system platform provides useful solutions for targeted resequencing
applications.
P11.087
Whole Genome microarray and Real-time PcR to detect genes
involved in non-syndromic ascending aortic aneurysms
A. Pasquali1 , C. Patuzzo1 , M. Iafrancesco2 , A. Zamboni3 , F. Santini2 , G. Faggian2
, A. Mazzucco2 , P. F. Pignatti1 , E. Trabetti1 ;
1 2 Section of Biology and Genetics, Verona, Italy, Section of Cardiovascular
Surgery, Verona, Italy, 3Dpt. Biotechnology, Verona, Italy.
The non-syndromic ascending aortic aneusysm is a complex disease
that involves a lot of people. The media coat is principally involved in
the disease. Aim of this study is to identify gene expression differences
between aneurysmal and normal ascending aortic media coats. A total
of 41 aneurysmal aortic samples (cases) and 22 aortic samples without
aneurysm (controls), had been harvested from patients undergoing
aneurysmectomy and heart transplantation, respectively. After separation
of the three coats, RNA from the media coats has been extracted,
amplified and labelled according to standard protocols. Competitive
hybridization of 3 single RNA cases vs a pool of 10 RNA controls has
been performed on microarray platforms, consisting of 21,329 70mer
oligonucleotides. Differentially expressed genes are validated by Taqman
Real Time PCR. From the microarray analysis the following eight
differentially expressed genes have been selected: Decorin, Receptor-interacting
serine-threonine kinase 3, Osteoblast specific factor 2,
Period homolog 2, EPH receptor A8, Adaptor protein with pleckstrin
homology and src homology 2 domains, Resistin, CD2 antigen. Presently,
semiquantitative analysis has confirmed up-regulation of Period
homolog 2, and down-regulation of Decorin genes. Both these genes
are involved in the integrity of the aortic wall, and in extracellular matrix
remodelling.
Further expression analyses will better indicate pathways involved in
thoracic aortic aneurysms development.
00
P11.088
improving signal strength consistency in capillary
electrophoresis for high resolution fragment sizing applications
R. N. Fish1 , S. R. Berosik1 , A. Chhibber1 , C. J. Davidson1 , S. Hung1 , B. F. Johnson1
, J. Lee1 , R. A. Padilla1 , D. Rodriguez1 , E. S. Nordman1 , J. D. Kyle1 , A. Y.
Spoonde1 , M. Yamazaki2 , Y. Lou1 , J. A. Benfield1 , A. M. Wheaton1 , A. A. Pradhan1
, A. C. Felton1 , L. K. Joe1 ;
1 2 Life Technologies, Foster City, CA, United States, Hitachi High Technologies,
Naka, Japan.
Capillary electrophoresis (CE) is an important technology used for
many DNA fragment sizing applications. Researchers have reported
that when the same DNA fragment is analyzed by CE, a certain degree
of variation in signal strength may be observed across multiple
CE instruments; within a single instrument among different capillaries;
or among different runs from the same capillary. For applications
that interrogate human polymorphisms associated with disease, such
as short tandem repeats, loss of heterozygosity, or single nucleotide
polymorphisms, minimal signal variation is desired. Quantitative analyses
such as quantitative fluorescent PCR or gene expression studies
would also be improved by reducing signal variation. We describe
the different sources of variation and elucidate two methods (one that
adjusts hardware and one that uses chemistry) to obtain more consistent
signal across the three levels of variation on CE: from run to
run, capillary to capillary, and instrument to instrument. In combination,
these methods have been observed to provide a significant reduction
in the range of peak heights obtained across several instruments. This
methodology would be useful to a single researcher or a consortium
of investigators with multiple instruments who require the most consistent
and comparable results possible from every instrument for high
resolution fragment sizing applications. We describe a workflow from
sample preparation to software analysis to quickly and simply obtain
consistent results.
P11.089
A high resolution comparison of human and mouse alternative
splicing patterns
J. M. Mudge1 , A. Frankish1 , J. Fernandez-Banet1 , T. Derrien2 , R. Guigó2 , J.
Harrow1 ;
1 2 Wellcome Trust Sanger Institute, Hinxton, United Kingdom, Centre for Genomic
Regulation, Barcelona, Spain.
The HAVANA group will manually annotate all protein-coding genes,
transcripts and pseudogenes on the human reference genome assembly
as part of the ENCODE project, providing a significant resource for
genome-science and medicine. Manual annotation is advantageous in
the classification of alternative splicing, a process to which the majority
of human genes are subjected. Transcription and splicing are error-prone
processes, however, and GenBank contains immature ESTs
and mRNAs; the proportion of the transcriptome which represents
functional splice variation is therefore unclear. Here, we compare our
annotation of the splicing patterns of 310 human coding genes within
the ENCODE pilot regions against their mouse orthologs. We (1) investigate
the conservation of alternative splicing at the transcriptional
and splice site level, (2) classify conserved and non-conserved variants
into 41 categories of alternative spliceform stucture, and (3) categorise
each variant as potentially protein coding or nonsense-mediated
decay inducing. Conserved and nonconserved alternative splice
events are seen to display distinct - perhaps unexpected - enrichments
for different structural categories as well as functional potential. In line
with previous reports, we observe that conserved alternative splice
junctions are frequently (not always) embedded within near identical
blocks of genomic sequence; we can now compare the presence
of such elements with our inferred functionality of the corresponding
splicing events. Overall, manual annotation generates a significantly
higher resolution dataset than available to previous studies, affording
a deeper view into the function and evolution of the alternative splicing
process.
All HAVANA annotation is presented in the Vega genome browser:
http://vega.sanger.ac.uk.