European Human Genetics Conference 2007 June 16 – 19, 2007 ...

Genomics, technology, bioinformatics
sequence searching for putative ncRNAs at this region. Among 86
stable hairpin candidates, of 100 nucleotides and not overlapping with
repeats, we selected 30 to check their expression by northern blot and
primer extension and we identified 21 primate-specific new ncRNAs.
Two correspond to new microRNAs and 19 to a new class and family
of ncRNAs. Computational analysis based on conservation of the
secondary structure supports that 15 of them are real ncRNAs. Most
predicted genes targeted by these two miRNAs were common to both,
and 11% of the targets encode proteins that participate in the development
of the central nervous system, suggesting a role of 15q11.2
miRNAs in neurological functions and disorders
P1283. High throughput genomics using Applied Biosystem’s
SOLiD System
M. D. Rhodes, G. Costa, J. Ichikawa, J. Malek, A. Sheridan, L. Apone, C. Hendrickson,
H. Peckham, S. Ranade, J. Sorenson, K. McKernan, A. Blanchard;
Applied Biosystems, Foster City, CA, United States.
Massively parallel sequencing systems are making genetic analysis
cheaper and enabling experiments capable of answering increasingly
complex biological questions. The SOLiD system, is a new platform
using either fragment or mate paired libraries to generate >1-2 GB of
data/run with >99.99% consensus accuracy. We will present a summary
of the current chemistry of the SOLiD system together with
data generated to demonstrate sequence quality and coverage of a
number of increasingly complex targets, including bacteria, yeast, C.
elegans and mammals. The SOLiD chemistry and instrumentation
can be readily adapted to a number of applications by modification of
the ways in which the input nucleic acids are prepared and the output
data is analyzed. Some of the applications under development with
collaborators will be presented as well as data demonstrating the utility
of the system
P1284. Next generation sequencing - Opportunities and
constraints in pooling and quantitative sequencing
F. M. De La Vega1 , F. C. L. Hyland1 , J. Sorenson1 , E. Cuppen2 , K. McKernan3 ;
1 2 Applied Biosystems, Foster City, CA, United States, Hubretch Lab, Utrecht,
The Netherlands, 3Applied Biosystems, Beverly, MA, United States.
Next generation sequencing promises to address a range of applications
including: identification of somatic mutation profiles, gene expression
by tag counting, and measurement of allele frequencies in
pooled samples of cases and controls in association studies. We developed
a model to simulate digital sequencing with pooled samples,
in the presence of error. We discover that for pooling and quantitative
sequencing, the number of samples that can be pooled and the minor
allele frequency of variants that can be detected is critically dependant
on the threshold for SNP calling, which in turn is strongly influenced by
the measurement error rate. As next generation sequencing platforms
typically produce short reads (25-35bp), coverage needs to increase
over 20x to compensate. Increasing the coverage improves the estimate
of the error rate, but cannot overcome problems with detecting
very low frequency variants with large numbers of pooled samples. We
validated this model through empirical sequencing by oligonucleotide
ligation and detection (Applied Biosystems SOLiD(TM) system) of 81
PCR amplicons from exons of EMS-mutagenized C. elegans worms
encompassing ca. 25kb of sequence with over 1500x coverage. Amplicons
were pooled down to a 1:100 ratio (1:200 ratio for alleles). The
results were compared with di-deoxy sequencing data carried out independently.
Our results suggest that even if coverage needs to increase
significantly when using short reads as compared with di-deoxy
sequencing, low platform error rate is the most critical factor for detecting
allele variants in pooled samples or mixtures by next generation
sequencing platforms.
P1285. Measuring the quality of computer tools used in
diagnostic genetic testing
A. Devereau1,2 , N. Walker2 ;
1 2 National Genetics Reference Laboratory, Manchester, United Kingdom, University
of Manchester, Manchester, United Kingdom.
As part of the EuroGentest project (www.eurogentest.org) we investigated
quality measurement of computer tools used within diagnostic
laboratories. A literature survey highlighted the need to provide specific
information, such as what the tool does, who it is for, etc.; information
about data used by the tool; information about tool operation; and in-
formation on its performance, such as sensitivity and specificity, or the
completeness of its data coverage. A survey of laboratories found that
most tools used are either for particular pieces of equipment or are databases.
It also showed that quality assessment is often missing or unstructured.
Furthermore, there is no trustworthy source to validate the
quality of the clinically most critical features of the tools. We propose
that tools can be categorised according to their purpose, with a specific
list of features developed for each category. Assessment of performance
requires standardised tests for parameters specific to each
tool category. Expert groups for each tool type are needed to propose
and review the features and performance measures. One important
category of tool that we found in laboratories was sequence analysis
tools. We have used previous work to propose a list of specific features
and performance measures for these tools. We have investigated presentation
of this data using a ‘wiki’, i.e. a web site editable by its users.
This allows groups of experts to be formed for the particular tool category
and to collaboratively enter and review data about the tool.
P1286. In search of tissue specific regulators in periodontium - a
bioinformatic approach.
A. M. Lichanska, N. Pham;
School of Dentistry, Brisbane, Australia.
Tissue specific gene expression can be regulated by tissue specific
promoters, enhancers, silencers, transcription factors, differential
methylation, tissue specific alternative splicing, as well as other transcriptional
and post-transcriptional factors. The methods used for
studying the regulatory elements are multiple, however, they are mostly
useful in cases where some information about the promoters active
in a given tissue is available.
While the tooth development is well described, the regulation of gene
expression in the tissues maturing after the tooth development is
complete is unclear. Periodontal ligament tissue (PDL) is essential for
structural support of the teeth (attaches root to the bone). The understanding
of what makes it so special is essential for the development
of regenerative treatments of this tissue.
Expression profiling data of the primary cell cultures of periodontal
ligament tissue and outer gum tissue (gingiva) was performed using
Affymetrix HU133A arrays. The analysis has identified 333 genes differentially
regulated in these tissues. This set of genes was then subjected
to promoter analysis to identify the CpG islands and promoter
binding sites. We have used a number of tools, such as Promoter-Express,
TRES, TFSEARCH, PAINT, CpGProD, CpG islands searcher,
Methylator and MethCGI to generate an overview of the promoters of
the differentially regulated genes. As a result we identified signature
promoter features of these differentially expressed genes.
Currently, we are analyzing the role of the differential methylation between
the two tissues and the role of some selected transcription factors
identified in this screen.
P1287. Direct Sequencing Quality Control: a Novel Software
Approach to Reducing Variant Review Time and Labor
S. Jankowski, E. Vennemeyer, K. Hunkapiller, P. Shah;
Applied Biosystems, Foster City, CA, United States.
With the completion of the Human Genome Project, the shift from de
novo sequencing to direct sequencing (resequencing) has created the
need for more accurate variant detection for medical research and clinical
diagnostics. The bottleneck in the workflow (from DNA extraction
to result data analysis) has been cited as taking up to 70% of researcher’s
time per project, due to manual review of individual nucleotide
bases. This review has been required due to the necessity of having
confidence in the variant result.
Increasing confidence can come from applying diligent quality control
metrics, including use of Quality Values for DNA trace value and confidence
values for variant validity. Based on Applied Biosystems experience,
this system will filter out low quality data. The software will then
direct users to review only low confidence variants.
A flexible workflow based system is being built to enable researchers
to obtain their high confidence results in less time. Methods for filtering
low quality data based on optimal settings and quality visualization
tools will be integrated into the system along with simpler variant
review and reporting tools to allow researchers to quickly analyze their
data.
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