2009 Vienna - European Society of Human Genetics

Genomics, Genomic technology and Epigenetics
AB’s next generation sequencing platform. We have determined several
conditions that result in high coverage and low bias libraries. These
libraries enable the ability to read 50 bases on each tag, allowing rapid
and precise mapping of structural variations, including translocations,
across the entire genome of complex organisms.
P11.110
improving the quality of DNA libraries in the Next Generation
Sequencing workflow
R. Salowsky1 , K. Gromadski1 , S. Glueck1 , N. Bontoux2 ;
1 2 Agilent Technologies, Waldbronn, Germany, Agilent Technologies, Massy,
France.
Next-generation sequencing technologies play an important role in investigating
complete cancer genomes and transcriptomes. To further
increase productivity of this compelling technique, the quality of DNA
libraries plays an important role. One important step in the Illumina sequencing
workflow is the amplification of the generated libraries for determining
the exact sequencing cluster concentration. The drawback
of this step is that amplification artefacts and errors will be introduced
into the target sequence.
An on-chip electrophoresis instrument has become a standard tool for
implementing DNA library quality control and quantification in the Illumina
workflow. The microfluidic device monitors the size and quantification
of the amplified libraries and also helps to detect contaminating
artefacts. With an optimized protocol and newly developed electrophoresis
chemistry, the sensitivity could be increased by a factor of 20-30,
down to the pg/µl concentration range. This improved detection sensitivity
allows for the significant reduction of amplification cycles thereby
reducing the target sequence error rate.
P11.111
mapreads: a tool that rapidly maps short reads to a genome
E. N. Spier1 , S. Katzman2 , N. Mullaken1 , Y. Sun1 , J. Ni1 , Z. Zhang1 ;
1 2 Applied Biosystems, Foster City, CA, United States, University of California,
Santa Cruz, Santa Cruz, CA, United States.
Mapreads is a bioinformatics tool that is optimized to rapidly map short
SOLiD (color space) or sequence reads to the genome. Mapreads
indexes the genome for a specific word size (W=14/15 is default for
mapreads), but unlike BLAST can use both continuous and discontinuous
word patterns called “schemas”. An example of a discontinuous
schema for a 25-mer will be 7 matches in the beginning of the
read followed by 11 charcters followed by 7 matches (effective W=14).
Only seven W=14 schemas are required to map a 25-mer with up to
two mismatches (25-2) enabling much quicker searchers than BLAST
W=7. A new feature of mapreads allows to specify the number of mismatches
for each schema and report “k-best” hits in the genome. A
single base difference between a read and the reference leads to two
color differences - so called valid-adjacent (VA) mismatches. Mapreads
can count these as a single (VA=1) or two (VA=2) mismatches. In
addition mapreads supports IUB codes in the reference sequence enabling
to avoid “non-reference allele bias” when mapping short reads
to known SNPs. We present results how mapping and false-positive /
false-negative SNP-calling rates for human paired 25-mers depend on
mapping parameters. We use both artificially generated color-reads
with “spiked errors” and SOLiD sequences from a HapMap individual
with known genotypes. Performance for mapping to human genome
ispresented. The source code of mapreads is available under GPL
from http://solidsoftwaretools.com/gf/project/mapreads/
P11.112
multiplex sequencing on the sOLiD Platform with 10, 16, or 96
Barcodes
L. Zhang1 , G. Silfwerbrand2 , A. Rico2 , J. Stuart1 , J. Bodeau2 , C. Hendrickson1 ,
E. Dimalanta1 , J. Manning1 , H. Peckham1 , A. Blanchard1 , G. Costa1 , T. Sokolsky1
, K. McKernan1 ;
1 2 Applied Biosystems, Beverly, MA, United States, Applied Biosystems, Foster
City, CA, United States.
The SOLIDTM DNA sequencing system utilizes stepwise ligation of
oligonucleotide probes and enables high fidelity, high throughput sequencing.
In order to maximize sequencing capacity and reduce workflow
of sample preparation, a single sequencing run containing multiple
biological samples is sometimes preferred. To this end, a multiplexing
method with barcodes has been developed for the SOLIDTM platform.
Barcodes are unique 5-7 base sequences that are added at the 3’ end
of the template along with a barcode priming region. Sets of 10, 16,
and 96 barcodes have been designed and can be assigned to up to
96 individual samples. Data presented shows the sequencing results
of all three sets of barcodes, as well as the results of an alternative
design of 20 barcodes. For all sets of barcode analysis, over 96% of
matching sequenced tags contain a barcode. The multiplexing system
coupled with SOLID TM ’s ability to process two slides, accommodating
two to sixteen depositions, enables researchers to sequence over a
thousand patients or unique biological samples within a single run.
P11.113
the Detectable Genome: How much of the human genome is
accessible to variant discovery by next-generation sequencing?
L. He 1 , S. Thoraval 2 , H. E. Peckham 3 , Y. Fu 3 , S. F. McLaughlin 3 , E. F. Tsung 3 , S.
S. Ranade 4 , C. C. Lee 3 , C. R. Clouser 3 , J. M. Manning 3 , C. L. Hendrickson 3 , L.
Zhang 3 , E. T. Dimalanta 3 , T. D. Sokolsky 3 , J. K. Ichikawa 3 , J. B. Warner 3 , M. W.
Laptewicz 3 , B. E. Coleman 3 , B. Li 4 , A. P. Blanchard 3 , J. A. Malek 5 , G. L. Costa 3 ,
K. J. McKernan 3 , J. Mangion 6 ;
1 Applied Biosystems, Sweden, 2 Applied Biosystems, France, 3 Applied Biosystems,
MA, United States, 4 Applied Biosystems, CA, United States, 5 Weill Cornell
Medical College in Qatar, Qatar, 6 Applied Biosystems, United Kingdom.
The human genome is being vigorously sequenced in an effort to
understand the extent of normal human variation as well as disease
causing variants. This initiative brings with it the challenge of assessing
the areas of the human genome that are accessible to variant detection.
We illustrate the amount of the human genome that is covered
with uniquely placed single tags and uniquely placed mate pairs and
demonstrate how both larger insert sizes and read lengths increase
the portion of the genome that is uniquely mappable by paired-end
tags. We use various human genomes (NA18507 - 10x Yoruban male,
NA19240 - 26x Yoruban female) sequenced with SOLiD TM sequencing
to illustrate the amount of SNPs and indels that are detected at various
levels of average sequence coverage. We also demonstrate the
sequence and clone coverage needed to identify indels of any size
between paired-end reads. We use libraries with an assortment of
insert sizes to show that larger libraries increase the accessibility of
the genome by spanning larger insertions. We show that the bisulfite
converted human genome is less uniquely mappable than the normal
human genome but significantly less signature is lost in color space
than in base space. We also illustrate that a significant portion of large
segmental duplications are accessible to sequence and clone coverage
by paired-end reads. These principles are applicable to all nextgeneration
sequencing platforms and are essential to comprehend the
amount and location of variability in the human genome.
P11.114
Tissue-specific forkhead protein FOXA2 regulates SOX gene
expression
J. Popovic, M. Stevanovic;
Institute of molecular genetics and genetic engeenering, Belgrade, Serbia.
Sox14/SOX14 is a member of B2 sub-group of Sox/SOX gene super-family
that functions as transcriptional repressor. Its expression is
restricted to a limited population of neurons in the developing brain and
spinal cord. In spinal cord explants, expression of Sox14 was found to
be regulated by Sonic hedgehog (SHH). Foxa2 (previously named Hepatic
nuclear factor-3-beta: HNF3β) displays a remarkable functional
diversity and is involved in a wide variety of biological processes during
development and adulthood. In the developing nervous system,
Foxa2 can be detected in the floor plate of the spinal cord and in periventricular
areas of the midbrain and diencephalons.
We have proceeded with investigation of transcriptional regulation of human
SOX14 gene expression. Our gel-shift and super-shift experiments
demonstrated that FOXA2 interacts directly with predicted binding site
within the SOX14 enhancer region. Using mutated oligonucleotide probe
we further confirmed specificity of the FOXA2 binding to the predicted
site. Results obtained with Foxa2 over-expression in co-transfection experiments
confirmed that SOX14 enhancer region possesses regulatory
element activated by this protein in HepG2 and U87MG cells.
In conclusion, here we present the first evidence that transcription factor
FOXA2 is involved in the up-regulation of human SOX14 expression
in HepG2 and U87MG cells by direct interaction with the binding
site within its enhancer region.
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