Commit 2df3988c authored by Laros's avatar Laros
Browse files

Finalised melanoma lecture.

parent 82bc1c6f
../../submodules/presentation-pics/pics/cnv.png
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../../submodules/presentation-pics/pics/exome_genome.jpg
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\documentclass[slidestop]{beamer}
\author{Jeroen F.J. Laros}
\title{Whole genome sequencing in Dutch melanoma families}
\title{Whole genome sequencing of Dutch melanoma families}
\providecommand{\mySubTitle}{Experiences and challenges}
\providecommand{\myConference}{MTG work discussion}
\providecommand{\myConference}{MTG meeting}
\providecommand{\myDate}{3-dec-2015}
\providecommand{\myGroup}{Leiden Genome Technology Center}
\providecommand{\myDepartment}{Department of Human Genetics}
......@@ -21,21 +21,55 @@
% First page of the presentation.
\section{Introduction}
\subsection{Samples}
\subsection{Full genome sequencing}
\begin{pframe}
X families, about Y members per family.
In contrast to \emph{exome sequencing}, where we only the \emph{coding
regions} are sequenced, we sequence everything.
\bigskip
\pause
Advantages of \emph{full genome sequencing}:
\begin{itemize}
\item Full genome.
\item $z\times$ coverage.
\item Introns.
\begin{itemize}
\item Branch points.
\item Intronic splicing enhancers.
\end{itemize}
\item Promoters.
\item Transcription factor binding sites, insulators, etc.
\item Consistent coverage.
\begin{itemize}
\item Copy number variation.
\end{itemize}
\end{itemize}
\end{pframe}
\begin{pframe}
\begin{figure}[]
\begin{center}
\includegraphics[trim=0.3cm 1.5cm 0 3.4cm, clip, width=\textwidth]
{exome_genome}
\end{center}
\caption{Exome- versus genome sequencing.}
\end{figure}
\end{pframe}
\subsection{Copy number variation}
\begin{pframe}
\begin{figure}[]
\begin{center}
\includegraphics[trim=0 8.4cm 0 2.2cm, clip, width=\textwidth]{cnv}
\end{center}
\caption{Coverage pattern over a whole chromosome.}
\end{figure}
\end{pframe}
\section{Data generation}
\subsection{Sequencing}
\begin{pframe}
\begin{minipage}[t]{0.47\textwidth}
\begin{figure}
\includegraphics[width=\textwidth]{hiseq_2000}
\includegraphics[trim=0 1cm 0 0.5cm, clip, width=\textwidth]{hiseq_2000}
\caption{HiSeq 2500.}
\end{figure}
\end{minipage}
......@@ -53,8 +87,18 @@
\end{pframe}
\begin{pframe}
Sequencing was one at the Sanger institute.
% 938528037 mapped reads
\begin{figure}[]
\begin{center}
\includegraphics[trim=0 1cm 0 23cm, clip, width=\textwidth]
{single_paired_end}
\end{center}
\caption{Paired end sequencing.}
\end{figure}
\end{pframe}
\begin{pframe}
Sequencing was done at the Sanger institute.
% 938,528,037 mapped reads
% 1672174 unmapped reads
\begin{itemize}
......@@ -62,11 +106,13 @@
\item $1,\!000,\!000,\!000$ reads.
\item $100,\!000,\!000,\!000$ nucleotides.
\item $150$GB of data per sample (compressed).
\item $35 \times$ coverage.
\end{itemize}
\bigskip
\pause
A grand total of $4.5$TB was used, which completely filled up the storage at
Sanger.
A grand total of $4.5$TB was generated, which completely filled up the
storage at Sanger.
\vfill
\permfoot{\url{http://www.sanger.ac.uk/}}
......@@ -75,13 +121,25 @@
\subsection{Data transfer}
\begin{pframe}
We need to make sure the data is transfered in a \emph{secure} way.
\bigskip
Sending disks?
Data carrier:
\begin{itemize}
\item Disks.
\item Network.
\end{itemize}
\bigskip
\pause
Public server, GPG encryption.
\begin{itemize}
\item Encrypted with my \emph{public key}.
\item The encrypted data is public.
\item Can only be decrypted with my \emph{private key}.
\end{itemize}
\end{pframe}
\section{Data analysis}
\subsection{Pipelines}
\begin{pframe}
\begin{figure}[]
......@@ -116,7 +174,7 @@
\end{pframe}
\begin{frame}
\frametitle{Principle of variant calling}
\frametitle{Variant calling}
\begin{figure}[]
\begin{center}
......@@ -151,46 +209,6 @@
\end{itemize}
\end{pframe}
\section{Annotation}
\subsection{Effect prediction}
\begin{pframe}
In most cases we are still left with a lot of variants.
\bigskip
Variant annotation.
\begin{itemize}
\item Frequency within a population.
\item Location of the variant.
\begin{itemize}
\item Gene panels.
\item Location within a gene.
\end{itemize}
\item Conservation.
\end{itemize}
\end{pframe}
\subsection{Variant Effect Predictor}
\begin{pframe}
A selection of VEP annotation:
\begin{itemize}
\item Affected genes and transcripts.
\item Location of the variant.
\begin{itemize}
\item Upstream of a transcript, in coding sequence, in non-coding RNA,
in regulatory region.
\end{itemize}
\item Consequence on the protein sequence.
\begin{itemize}
\item Stop gained, missense, stop lost, frameshift.
\end{itemize}
\item Minor allele frequencies from the 1000 Genomes Project.
\item SIFT and PolyPhen scores for changes to protein sequence.
\end{itemize}
\vfill
\permfoot{\url{http://www.ensembl.org/info/docs/tools/vep/index.html}}
\end{pframe}
\subsection{Databases}
\begin{pframe}
In most cases we are not interested in common variants.
......@@ -201,7 +219,7 @@
\end{itemize}
\medskip
A cut-off of $1\%$ is usually fine.
A cut-off of $1$\% is usually fine.
\bigskip
\pause
......@@ -220,26 +238,39 @@
\bs{\url{http://www.hgmd.cf.ac.uk/}}
\end{pframe}
\subsection{Inheritance based filtering}
\begin{pframe}
% iets moet in alle familieleden voorkomen, maar mag niet in een deel van een
% andere familie voorkomen maar wel in de gehele familie
We used the following intersection method.
\bigskip
{\it ``A variant should be present in all affected members of a family, but
it may not occur in part of the affected members of an other family.''}
\begin{table}[]
\begin{center}
\begin{tabular}{ccl}
Family one & Family two & Filter result \\
\hline
$5/5$ & $4/4$ & Passed \\
$0/5$ & $4/4$ & Passed \\
$5/5$ & $4/4$ & Pass \\
$0/5$ & $4/4$ & Pass \\
$3/5$ & $4/4$ & Filtered \\
$3/5$ & $3/4$ & Filtered \\
\end{tabular}
\end{center}
\caption{The advanced intersection.}
\caption{Advanced intersection.}
\end{table}
\vfill
\bs{P.J. van 't Hof}
\end{pframe}
\subsection{Filters}
\begin{pframe}
We first did some general filtering based on allele frequencies found in
databases and inheritance patterns.
\begin{table}[]
\begin{center}
\begin{tabular}{lr}
......@@ -258,6 +289,8 @@
\end{pframe}
\begin{pframe}
By combining filters, we are left with a reasonable amount of variants.
\begin{table}[]
\begin{center}
\begin{tabular}{lr}
......@@ -274,7 +307,48 @@
\caption{Combining filters.}
\end{table}
(25.127 in all families)
Note: $25,\!127$ variants are present in every individual.
\end{pframe}
\section{Annotation}
\subsection{Effect prediction}
\begin{pframe}
We are still left with a lot of variants.
\bigskip
Variant annotation.
\begin{itemize}
\item Frequency within a population.
\item Location of the variant.
\begin{itemize}
\item Gene panels.
\item Location within a gene.
\item Regulatory regions.
\end{itemize}
\item Conservation.
\end{itemize}
\end{pframe}
\subsection{Variant Effect Predictor}
\begin{pframe}
A selection of VEP annotation:
\begin{itemize}
\item Affected genes and transcripts.
\item Location of the variant.
\begin{itemize}
\item Upstream of a transcript, in coding sequence, in non-coding RNA,
in regulatory region.
\end{itemize}
\item Consequence on the protein sequence.
\begin{itemize}
\item Stop gained, missense, stop lost, frameshift.
\end{itemize}
\item Minor allele frequencies from the 1000 Genomes Project.
\item SIFT and PolyPhen scores for changes to protein sequence.
\end{itemize}
\vfill
\permfoot{\url{http://www.ensembl.org/info/docs/tools/vep/index.html}}
\end{pframe}
\subsection{Conservation}
......@@ -295,11 +369,15 @@
% Make the acknowledgements slide.
\makeAcknowledgementsSlide{
\begin{tabular}{lll}
\bf Dermatology & \bf SASC & \bf Sanger \\
Mijke Visser & Peter van 't Hof & Thomas Keane \\
Nelleke Gruis & Sander van der Zeeuw & Kim Wong \\
Nienke van der Stoep & Leon Mei & Daniela Espinoza \\
Remco van Doorn & & David Adams \\
\bf Dermatology & \bf SASC & \bf Sanger \\
Mijke Visser & Peter van 't Hof & Thomas Keane \\
Nelleke Gruis & Sander van der Zeeuw & Kim Wong \\
Remco van Doorn & Leon Mei & Daniela Espinoza \\
Remco van Doorn & & David Adams \\
\\
\bf Clinical Genetics & \bf Medical Statistics \\
Nienke van der Stoep & Ramin Monajemi \\
& Jeanine Houwing \\
\end{tabular}
}
......
../../submodules/presentation-pics/pics/single_paired_end.png
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Subproject commit 63b5792d877b40e3ffeca84ece106e63159439cb
Subproject commit ed45c13b1e4237f49053420d0e47c1ca3fe545f2
Subproject commit 57047ad1fa7d5f013e2af50c306329e4abca9db7
Subproject commit 42ef031c5797a73435d9c4eda433f54bc47e26f3
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