2009 Vienna - European Society of Human Genetics

Concurrent Sessions
c01.1
mRNA-seq Whole transcriptome Analysis of a single cell
K. Q. Lao 1 , F. Tang 2 , C. Barbacioru 1 , Y. Wang 1 , E. Nordman 1 , C. Lee 1 , N. Xu 1 ,
X. Wang 1 , J. Bodeau 1 , A. Surani2 3 ;
1 Molecular Cell Biology Division, Foster City, CA, United States, 2 2Wellcome
Trust/Cancer Research UK Gurdon Institute of Cancer and Developmental Biology,
University of Cambridge, Cambridge, United Kingdom, 3 Wellcome Trust/
Cancer Research UK Gurdon Institute of Cancer and Developmental Biology,
University of Cambridge, Cambridge, United Kingdom.
We developed a digital gene expression profiling assay at single cell
resolution by combining a modified single cell whole transcriptome
amplification method with the next generation sequencing technique,
SOLiD System. Using only a single mouse blastomere, our mRNA-
Seq assay can detect the expression of 74% (5,270) more genes than
microarray techniques. Moreover, 8 - 19 % of the genes with multiple
known transcript isoforms express at least two isoforms in the same
blastomere or mature oocyte, which unambiguously demonstrated the
complexity of the transcript variants at whole genome scale. Finally,
for Dicer and Ago2 knockout oocytes, we also showed that in Dicer
knockout and Ago2 knockout mature oocytes, 1,924 and 1,687 genes
respectively were abnormally upregulated, and 1,343 and 987 transcripts
respectively were downregulated compared to wildtype controls,
which proves the global importance of small RNAs (including microRNAs
and endogenous siRNAs) for oogenesis. The main technical
novelty of this work is the combination of an improved unbiased amplification
of cDNAs from single cells with well over a 100 million reads,
or a few gigabases of cDNAs on SOLID TM . This not only allowed us to
discover many novel transcripts that have been overlooked but also
to get a quantitative estimate of their abundance in the cell by the frequency
with which the sequence occurs in the mRNA-Seq reads. This
single cell mRNA-Seq assay will greatly enhance our ability to analyze
transcriptome complexity during mammalian development, especially
for early embryonic development and for stem cells.
c01.2
integrated analysis of high-resolution transcriptomics data
reveals new insights into the differentiation state-dependent
control of transcript isoform abundance
P. A. C. ‘t Hoen 1 , M. S. Hestand 1 , Y. Ariyurek 1 , A. Klingenhoff 2 , M. Scherf 2 , M.
Harbers 3 , W. van Workum 4 , G. J. B. van Ommen 1 , J. T. den Dunnen 1 ;
1 Center for Human and Clinical Genetics, Leiden, The Netherlands, 2 Genomatix
Software GmbH, München, Germany, 3 DNAFORM, Yokohama, Japan, 4 ServiceXS
B.V., Leiden, The Netherlands.
Around 90% of human genes have been estimated to undergo alternative
splicing. Apart from switching genes on and off, switching between
transcript isoforms can be used for fine-tuning and orchestration
of cellular differentiation. Using the myoblast cell line C2C12 as
a well-controlled model for cell differentiation, we applied a variety of
high-resolution genomics technologies to study gene transcription in
undifferentiated and differentiated cells. We applied CAGE-Seq (cap
analysis of gene expression followed by Illumina deep sequencing) to
quantitatively identify the 5’-ends of transcripts, SAGE-Seq to quantify
the 3’-ends of transcripts, and assayed mRNA degradation rates
on Affymetrix exon arrays. We found 1400 transcription start sites not
previously annotated. Around 50% of the expressed genes demonstrated
use of multiple polyadenylation sites. We observed extensive
qualitative and quantitative differences in use of transcription start
sites, internal exons, 3’-UTRs, and polyadenylation sites between differentiated
myotubes and undifferentiated myoblasts. Splice variants
from some genes were produced at comparable levels, but degraded
with different efficiencies; a transcript from the Itga7 gene with an additional
internal exon was much more abundant in differentiated than in
undifferentiated cells, mainly because of specific and extremely rapid
degradation of transcripts lacking this exon. Since it is thought that the
abundance of different splice isoforms is mainly controlled by tissuespecific
splicing factors, this represents a new mechanism to regulate
the ratio between different splice isoforms. We conclude that promoter
usage, alternative splicing and RNA degradation must be tightly coupled
through yet unknown mechanisms.
c01.3
Genomic variation detection by DNA selection and high
throughput sequencing
S. Nikolaev 1 , C. Iseli 2 , D. Robyr 1 , A. Sharp 1 , J. Rougemont 3 , C. Gehrig 1 , L.
Farinelli 4 , S. Antonarakis 1 ;
1 University of Geneva, Geneva, Switzerland, 2 Swiss Institute of Bioinformatics,
Lausanne, Switzerland, 3 Ecole Polytechnique Fédérale de Lausanne, Lausanne,
Switzerland, 4 FASTERIS SA, Geneva, Switzerland.
The resequencing of a targeted region of the genome has become a
major goal in order to understand the correlation between genomic
and phenotypic variability. We have optimized a genomic selection
method for high throughput sequencing. The repeat-masked contiguous
region of 8.9Mb was targeted on human chromosomes 21 and
7. We used DNA from an individual from the International HapMap
Project for which the genotype data are available. After microarraybased
enrichment and sequencing of genomic DNA from chromosome
21 we observed a 260-fold enrichment with 41% of reads from the
targeted region. The median coverage of the targeted region using
two lines of an Illumina GA2 sequencing instrument was 16-fold. We
also observed that regions with low sequence coverage are AT rich
and are close to low-complexity DNA stretches. 83% of SNPs have at
least 4-fold coverage, and 80% of the SNPs identified were already
listed in dbSNP. For these dbSNP positions our sequence genotypes
are concordant in 92% of cases with previously obtained genotypes of
NA12782. 54% of all dbSNP positions had at least 15-fold sequence
coverage, the coverage previously estimated as minimal for rigorous
SNP search. At this threshold, 98.8% of dbSNP genotypes are concordant
between sequencing and HapMap data for NA12872. Validation
experiments using Sanger sequencing after PCR amplification were
done for 46 SNPs covered 15-20 fold; the confirmation rate obtained
was 96%.
We conclude that DNA selection method could provide an accurate
and inexpensive search for genomic variability.
c01.4
Adult human brain samples deep sequencing of small-RNAs
reveals specific expression profiles in different brain areas
E. Martí 1,2 , L. Pantano 1,2 , M. Bañez-Coronel 1,2 , E. Miñones 1,2 , E. Mateu 1,2 , S.
Porta 1,2 , X. Estivill 1,2,3 ;
1 Center for Genomic Regulation (CRG), Barcelona, Catalonia, Spain, 2 Public
Health and Epidemiology Network Biomedical Research Center (CIBERESP),
Barcelona, Catalonia, Spain, 3 Pompeu Fabra University (UPF), Barcelona,
Catalonia, Spain.
Small RNAs are non-coding RNAs of 20-30 nt in length, associated
with members of the Argonaute family of proteins. Small RNAs are
involved in the guidance of diverse types of gene regulation, tipically
resulting in reduced expression of target genes. In the central nervous
system miRNAs are key in developmental processess, contributing to
neuronal cell identity and synapse formation. miRNAs also play a role
in mature neurons, participating in synaptic plasticity and neuronal survival.
Alterations in miRNA-related pathways have been associated to
several neurological and neurodegenerative diseases. Here we have
used Illumina/Solexa deep sequencing to extensivelly characterize
and profile small RNA libraries of three adult brain areas: frontal cortex,
striatum and amygdala. In all the samples the majority of reads
corresponded to previously annotated miRNAs. The most abundant
sequences in all libraries included members of the let-7 family, mir-
29a, mir-1, mir-101 and mir-103 miRNAs. Selective miRNAs were specifically
enriched in each brain area. We have found strong variability
in the mature miRNA reference sequence, mainly in the form of length
modifications, that match the precursor sequence of the miRNA. Variability
was also detected as nucleotide substitutions in the different
positions of the reference mature miRNA, which was clearly reduced
in the 5’-seed region. These results suggest a miRNA-signature in the
different brain areas that may be related with the maintance of the transcriptome
in each brain structure. The present results further highlight
the possible importance of the modified mature miRNA sequences in
the physiology and pathology of the adult brain.