State-of-the-art epigenomics using NGS-based BiSulfite
Sequencing (BSAS) and targeted Allele-Specific RNAseq
Amplicon
Matthias Linke
Johannes Gutenberg University Mainz, Institute of Human Genetics, Mainz,
Germany
Background: DNA methylation is the most stable epigenetic mark known so far.
It modulates gene expression and is involved in a large number of physiological
key processes like X-inactivation, genomic imprinting, suppression of
transposable elements as well as carcinogenesis. Currently, Sanger- and
pyrosequencing-based analyses of bisulfite-treated genomic DNA represent the
gold standard of hypothesis-driven examinations of DNA methylation. Both
methods have their drawbacks with pyrosequencing hampered by short read
length and Sanger Sequencing requiring a disproportionate amount of time and
cost effort. Quantitative allele-specific RNA expression analysis is an ideal
method for studying not only the impact of differential DNA methylation but also
the effects of rare promoter variants on allele-specific gene expression. Several
methods have been developed such as pyrosequencing, real-time PCR,
microarrays and primer extension assays, but suffer from the difficulty to
quantify an allele with a transcript abundance of less than 1-2%.Alternatively,
NGS-based methods of DNA methylation and allele-specific RNA expression
quantification offer much more information in a single run and are thus
considerably more time- and cost-effective..
Methods & Results: Illumina-based Next-Generation-Sequencing was adopted
to quantify the DNA methylation of up to 23 amplicons in 12 samples (BiSulfite
Amplicon Sequencing; BSAS; Masser et al, 2013) within a single MiSeq run.
Library preparation started with Nextera XT-based Tagmentation of pooled
Bisulfite PCR amplicons of 1 sample and was followed by introduction of the
index primers for sample multiplexing. Sequencing was performed with MiSeq
300 cycle cartridges v2 that yield about 10 – 11 Mio. reads per run. Technical
validation of BSAS by comparison with bisulfite pyrosequencing data was
performed for regulatory regions of the Bdnf, Crh, Nr3c1 and Nrsf genes as part of
a neuroepigenetics research project. Both methods showed a remarkable
consistency in absolute DNA methylation values. Targeted Allele-specific RNAseq
started with cDNA synthesis of RNA derived from mouse intersubspecific F1
hybrids. Illumina TruSeq Adapters were modified in a way, that they contain the
specific PCR primer sequences amplifying a region including a coding SNP
between the two mouse strains and a six base long index sequence for
multiplexing different samples. The samples were sequenced on an Illumina
MiSeq 150 cycle cartridge v3 giving a total of 25 Mio. reads.
Discussion: BiSulfite Amplicon Sequencing and targeted Allele-Specific RNAseq
provided far more information compared to their respective standard
techniques. Assuming proper read counts per Amplicon, BSAS allows for
unprecedented resolution of differential DNA methylation. Both methods are
ideally suited for hypothesis-driven epigenetic research and functional follow-up
studies.
p53 isoforms combinatorics: is there a p53 code?
Jean-Christophe Bourdon
University of Dundee, College of Medicine, Ninewells Hospital, Centre for Oncology
and Molecular Medicine, UK
The human TP53 gene encodes at least 12 different p53 protein isoforms.
Altering expression of a few p53 protein isoforms, in vivo, is sufficient to trigger
different or opposite cell responses to a same cell signal, suggesting that the
balance between p53 isoforms define cell responses.
However, because of its apparent high expression level, it is currently
understood that the p53-mediated responses are exclusively dependent of
canonical full-length p53 protein (p53α).
Here we investigated by manipulating endogenous expression of a few p53
isoforms using siRNAs whether endogenous p53 isoforms regulate cell response
to treatment in cells devoid of p53α expression. In addition, we explored the
molecular mechanisms of p53 isoforms in presence and in absence of p53α in
cells treated or not treated with UV.
The data leads us to realise that a p53-mediated cell response, which involved
coordination of numerous complex biological pathways, is not orchestrated by
only one p53 protein isoform but by all p53 protein isoforms. None of the p53
isoforms, including canonical p53α, is able to abolish the activity of the other coexpressed p53 isoforms. Thus, a p53-mediated cell response is the sum of the
activities of co-expressed p53 isoforms, which can be controlled by manipulating
p53 isoform expression and/or post-translational modifications. It suggests that
p53 isoforms work in combination and would compose a cellular code.
High throughput screening of phage display libraries for production of fully
human antibodies challenged to cells expressing native claudin-1
Emanuele Sasso1,2, Alfredo Nicosia1,2, Claudia De Lorenzo1,2, Nicola
Zambrano1,2
1Dipartimento
di Medicina Molecolare e Biotecnologie Mediche, Università degli
Studi di Napoli Federico II, Via S. Pansini, 5 - 80131 Napoli, Italy
2CEINGE Biotecnologie Avanzate S.C. a R.L., Via G. Salvatore, 486 - 80145 Napoli,
Italy
Monoclonal antibodies (mAbs) represent valuable tools in many biological fields.
Screening of antibody libraries by phage display allows for rapid selection of
single-chain variable fragments (scFvs). In the paradigm of viral hepatitis the
availability of mAbs preventing hepatitis C virus (HCV) infection of hepatocytes
is an active field of investigation within medical biotechnologies. We describe a
complete pipeline for high-throughput screening of libraries by next-generation
sequencing (NGS) approach, to select human scFv against native Claudin-1, a
tight-junction protein involved in hepatitis C virus infection. Our strategy allows
to rapidly identify the potential binders of a given antigen, based on the counts of
the corresponding scFv fragments, within a cycle, and on the kinetic of their
enrichments, within consecutive cycles. After their identification, the clones of
interest need to be recovered from the DNA sub-library of the relevant selection
cycle, for validation of binding. Thus, we also implemented a rapid and effective
method, for one-step recovery of scFv. The checked clones were successfully
converted to active IgG4 antibodies and produced in a scale-down process, thus
demonstrating the effectiveness of the whole procedure. This novel approach
provides rapid and cheap isolation of antibodies for virtually any native antigen
involved in human diseases, for therapeutic and/or diagnostic applications.
Effects of change-of-function mutations on helical propensity in the
intrinsically disordered τ1-core activation domain of the Glucocorticoid
receptor.
Evdokiya Salamanova, Alok Juneja, Anthony Wright, Lennart Nilsson
Karolinska Institutet, Department of Biosciences and Nutrition, Hälsovägen 7, SE141 83, Huddinge, Sweden
In recent years the physiological role of unstructured regions in proteins has
been proven. The intrinsically disordered proteins (IDPs) hold important
functions at cellular level, such as regulation of transcription and translation,
signal pathways and self-assembly of macromolecular units in active complexes.
The lack of compact 3D-structure or folding upon binding to their targets is
related to the specific role of the unstructured regions. The Glucocorticoid
receptor (GR) belongs to a family of ligand-inducible nuclear receptors. Two of
its domains (1 and 2) have shown a conserved activity after they have been
removed from the receptor entity1. The disordered core region of the 1-domain
consists of 58-amino-acids. It carries most of the activity and has shown a helical
propensity in hydrophobic solvent conditions (TFE). We have investigated the
effect of change-of-function point mutations in the 1-core transactivation
domain in GR on the helical propensity using an in silico model system. For our
goal we used CHARMM simulation package2 with Charmm36ff to perform
molecular dynamics simulations at different temperature regimes.
1. Almlöf T, Ford J, Gustafsson J A, Wright A P (1997) Role of hydrophobic amino
acid clusters in the transactivation activity of the human glucocorticoid receptor.
Mol Cell Biol. 17(2): 934–945.
2. Brooks B, Brooks III C, Mackerell Jr A, Nilsson L, Petrella R, Roux B, Won Y,
Archontis G, Bartels C, Boresch S, Caflisch A, Caves L, Cui Q, Dinner A, Feig M,
Fischer S, Gao J, Hodoscek M, Im W, Kuczera K, Lazaridis T, Ma J, Ovchinnikov V,
Paci E, Pastor R, Post C, Pu J, Schaefer M, Tidor B, Venable R, Woodcock H, Wu X,
Yang W, York D, Karplus M (2009) CHARMM: The biomolecular simulation
program. J. Comput. Chem. 30 (10): 1545-1614.
Bioinformatics Business Services in 2015: culture, good practices, and
structure
Jorge Bouças,
Max Planck Institute for Biology of Ageing, Cologne, Germany
In the era of digital information, high-throughput systems, and the emerging
Internet of Things (IoT), the need for data-centric services in biomedical
research has dramatically spiked. Still obscured between informatics, statistics,
and biology the role of a bioinformatician has further expanded into computer
science and IT. The exponential growth of physical and logical tools and the
mingle between research and service at the eyes of wet-bench biomedical
researchers has made Bioinformatics Business Services a complex environment
dwelled only by highly structured and dynamic multidisciplinary teams. This talk
will cover the current needs for service offers, the dos and do nots, structure, and
the Agile Manifesto in Bioinformatics Business Services.
The Proteasix Ontology
Mercedes Arguello Casteleiro1, Julie Klein2 and Robert Stevens1
1School
of Computer Science, University of Manchester, Oxford road, Manchester,
United Kingdom.
2Institut National de la Santé et de la Recherche Médicale (INSERM), U1048,
Toulouse, France. And Université Toulouse III Paul-Sabatier, Toulouse, France.
We describe the Proteasix Ontology (PxO) a new application ontology that
supports the Proteasix tool (http://www.proteasix.org/). Proteasix is an opensource peptide-centric tool that can be used to predict automatically and in a
large-scale fashion in silico the proteases involved in the generation proteolytic
cleavage fragments (peptides). Body fluids (e.g. serum, urine, cerebrospinal
fluid) contain thousands of peptides. However, there is a lack of knowledge about
the proteolytic mechanisms leading to the generation of these fragments, which
may hold the key to a better understanding of molecular mechanisms of disease,
define new biomarkers, and therapeutic targets for a variety of disorders such as
kidney, cardiovascular, autoimmune and infectious diseases as well as certain
types of cancer. Proteasix addresses this by using the N- and C-terminal
sequences of peptides which are reconstructed using information from
UniprotKB and knowledge about cleavage sites to predict the possible proteases
that were involved in their generation. This is a knowledge intensive task and the
new version of Proteasix uses an ontology to provide this knowledge.
PxO re-uses parts of the Protein Ontology, the three Gene Ontology subontologies, the Chemical Entities of Biological Interest Ontology, the Sequence
Ontology, the NCBI Taxonomy Ontology and bespoke extensions in support of a
series of roles:
1. To describe the known proteases, their target cleaveage sites, observed and
predicted proteolytic cleaveage fragments resulting from proteolysis.
2. To enable the description of data within and produced by Proteasix to facilitate
analysis.
3. To use knowledge about the function and location of a protease to support the
prioritisation of proteases in the prediction.
4. To facilitate the visualisation of data in Proteasix.
In this paper we will describe the motivation for the PxO and how it supports
and improves Proteasix in its analysis. In doing so we will describe the
competencies PxO will need to support Proteasix; the axiom patterns that
capture the appropriate knowledge and then the use of the ontology in satisfying
those competencies. At the core of PxO is the modelling of the proteolytic process
and its various participants, such as the protease and proteins together with
their cleaveage sites (observed and predicted), and then the output proteolytic
cleaveage fragments.
The Proteasix Ontology may be found at: http://swproteasix.cs.man.ac.uk . This
ontology is free and open for use by everyone.