Added lecture.

lectures/2018-09-17_hpc_analysis/2018-09-17_hpc_analysis.tex

+\documentclass[slidestop]{beamer}
+
+\author{Jeroen F.J. Laros}
+\title{High throughput NGS data analysis}
+\providecommand{\mySubTitle}{}
+\providecommand{\myConference}{SIG Bioinformatics in Medical Microbiology NL}
+\providecommand{\myDate}{17-09-2018}
+\providecommand{\myGroup}{}
+\providecommand{\myDepartment}{Research Software Engineering, LUMC}
+\providecommand{\myCenter}{Program manager bioinformatics, RIVM}
+
+\usetheme{lumc}
+
+\begin{document}
+\lstset{language=make, mathescape=false}
+
+\input{petrinet}
+
+% This disables the \pause command, handy in the editing phase.
+%\renewcommand{\pause}{}
+
+% Make the title slide.
+\makeTitleSlide{} %\includegraphics[height=3.5cm]{logos/lumc_logo_small}}
+
+
+% First page of the presentation.
+\section{Introduction}
+\makeTableOfContents
+
+\section{Pipelines}
+\subsection{Early NGS sequencing}
+\begin{pframe}
+  \begin{minipage}[t]{0.47\textwidth}
+    \begin{figure}[]
+      \begin{center}
+        \includegraphics[width=0.8\textwidth]{illuminagenomeanalyzer}
+      \end{center}
+      \pause
+      \caption{Genome Analyzer II.}
+    \end{figure}
+  \end{minipage}
+  \hfill
+  \begin{minipage}[t]{0.47\textwidth}
+    \begin{figure}[]
+      \begin{center}
+        \includegraphics[trim=1cm 1.5cm 1cm 1, clip, width=0.5\textwidth]
+          {k_marjolein}
+      \end{center}
+      \caption{Marjolein Kriek.}
+    \end{figure}
+    Sequencing took nine months.
+    \begin{itemize}
+      \item Data analysis mostly by hand.
+    \end{itemize}
+  \end{minipage}
+\end{pframe}
+
+\begin{pframe}
+  Routine data analysis:
+  \begin{itemize}
+    \item First pipelines were written in Perl.
+    \item Pipelines were executed on a single machine.
+  \end{itemize}
+  \bigskip
+
+  Drawbacks:
+  \begin{itemize}
+    \item Single threaded.
+    \item Use of \lstinline{system()} calls.
+  \end{itemize}
+  \bigskip
+
+  Later pipelines in Bash.
+  \begin{itemize}
+    \item Easier to work with external programs.
+  \end{itemize}
+\end{pframe}
+
+\subsection{NGS for routine diagnostics}
+\begin{pframe}
+  \begin{minipage}[t]{0.47\textwidth}
+    \begin{figure}
+      \includegraphics[width=\textwidth, trim=0 40 0 0, clip]{hiseq_2000}
+      \caption{HiSeq 2500.}
+    \end{figure}
+  \end{minipage}
+  \hfill
+  \begin{minipage}[t]{0.47\textwidth}
+    Characteristics:
+    \begin{itemize}
+      \item High throughput ($3$~genomes).
+      \item Paired end.
+      \item High accuracy.
+      \item Read length $2 \times 125$bp.
+      \item Run time ($6$~days).
+    \end{itemize}
+  \end{minipage}
+\end{pframe}
+
+\begin{pframe}
+  \begin{minipage}[t]{0.47\textwidth}
+    \begin{figure}
+      \includegraphics[width=0.95\textwidth]{DellBlade4}
+      \caption{Dell M610 blade server.}
+    \end{figure}
+  \end{minipage}
+  \hfill
+  \begin{minipage}[t]{0.47\textwidth}
+    Necessity to run multiple analyses at the same time.
+    \begin{itemize}
+      \item Linux SGE cluster.
+      \item Central (Isilon) storage.
+    \end{itemize}
+    \bigskip
+
+    Current pecifications:
+    \begin{itemize}
+      \item $51$ nodes providing $908$ cores.
+      \item $1$PB HPC storage.
+      \item $1.5$PB archive.
+    \end{itemize}
+  \end{minipage}
+\end{pframe}
+
+\begin{pframe}
+  Limitations of conventional scripting languages.
+  \begin{itemize}
+    \item Manual identification of parallel steps.
+    \item Explicit job submission.
+    \item Explicit job monitoring.
+  \end{itemize}
+  \bigskip
+
+  Pipelines became large and unmanageable.
+  \bigskip
+
+  Need for a framework that takes care of parallelisation.
+  
+  Separation of the workflow from the computational infrastructure.
+\end{pframe}
+
+\begin{pframe}
+  %\frametitle{A different viewpoint}
+  What if\ldots
+  \begin{itemize}
+    \item All commands are atomic.
+    \begin{itemize}
+      \item We describe input and output.
+    \end{itemize}
+    \item We build a \emph{dependency graph}.
+    \item Trace a path in this graph to find a workflow.
+  \end{itemize}
+  \bigskip
+  \pause
+
+  This way we do not need to:
+  \begin{itemize}
+    \item Design a workflow.
+    \item Figure out which parts can be run in parallel.
+  \end{itemize}
+\end{pframe}
+
+\begin{pframe}
+  \begin{figure}
+    \petrinet{\bs{fastq}}{\bs{fq.f}}{\bs{stats}}{\bs{bam}}{\bs{report}}{
+      \underline{\bsi{fastx}}}{\underline{\bsi{bwa}}}{\underline{\bsi{latex}}}{
+      red}
+    \caption{A parallel workflow.}
+  \end{figure}
+\end{pframe}
+
+\begin{pframe}
+  \lstinline{make} is an implementation of this idea.
+  \begin{itemize}
+    \item Works on a single machine as well as on a cluster.
+    \item Used for clinical genetics pipeline until a few months ago.
+  \end{itemize}
+  \bigskip
+  \pause
+
+  Drawbacks:
+  \begin{itemize}
+    \item Old language with awkward syntax.
+    \item No scoping.
+    \item Difficult to debug.
+  \end{itemize}
+  \bigskip
+  \pause
+
+  We now use \lstinline{Snakemake} for all of our diagnostics pipelines.
+
+  \vfill
+  \permfoot{\url{https://www.gnu.org/software/make/}}
+
+  \permfoot{\url{https://snakemake.readthedocs.io/}}
+\end{pframe}
+
+\section{Stable pipelines}
+\subsection{Virtual environments}
+\begin{pframe}
+  To have stable pipelines in a shared environment, some form of isolation is
+  needed.
+  \bigskip
+
+  Package manager / environment management system.
+  \begin{itemize}
+    \item Conda.
+    \item GNU Guix.
+  \end{itemize}
+  \bigskip
+
+  Compartimentalisation:
+  \begin{itemize}
+    \item Docker.
+    \item Singularity.
+  \end{itemize}
+  \bigskip
+
+  Separate the pipeline and its dependences from the computational
+  infrastructure.
+\end{pframe}
+
+\subsection{Version control}
+\begin{pframe}
+  Our pipelines are stored in Git.
+  \bigskip
+
+  Configuration of virtual environments.
+  \begin{itemize}
+    \item Fixed version numbers.
+  \end{itemize}
+  \bigskip
+
+  For reproducibility:
+  \begin{itemize}
+    \item Use releases.
+    \item Store git checkout hash for every pipeline run.
+  \end{itemize}
+
+  \vfill
+  \permfoot{\url{https://git.lumc.nl/}}
+\end{pframe}
+
+\subsection{Summary}
+\begin{pframe}
+  Manageable pipelines:
+  \begin{itemize}
+    \item Separate the workflow from the computational infrastructure.
+  \end{itemize}
+  \bigskip
+
+  Stable pipelines:
+  \begin{itemize}
+    \item Separate the pipeline and its dependencies from the computational
+      infrastructure.
+    \item Full control of dependencies.
+  \end{itemize}
+  \bigskip
+\end{pframe}
+
+
+\section{Fast analysis: NIPT}
+\subsection{Detection of fetal chromosomal aberrations}
+\begin{pframe}
+  \begin{minipage}[t]{0.47\textwidth}
+    Large events:
+    \begin{itemize}
+      \item Trisomies.
+      \begin{itemize}
+        \item 13, 18 and 21.
+      \end{itemize}
+      \item Large deletions or duplications.
+    \end{itemize}
+    \bigskip
+
+    Primary targets:
+    \begin{itemize}
+      \item Patau syndrome (13).
+      \item Edwards syndrome (18).
+      \item Down syndrome (21).
+    \end{itemize}
+  \end{minipage}
+  \hfill
+  \begin{minipage}[t]{0.47\textwidth}
+    \begin{figure}[]
+      \begin{center}
+        \includegraphics[width=0.8\textwidth]{down_syndrome}
+      \end{center}
+      \caption{Down syndrome.}
+    \end{figure}
+  \end{minipage}
+\end{pframe}
+
+\begin{pframe}
+  \begin{figure}[]
+    \begin{center}
+      \includegraphics[height=0.7\textheight]{NIPT_story}
+    \end{center}
+    \caption{Free floating DNA in the maternal bloodstream.}
+  \end{figure}
+\end{pframe}
+
+\subsection{Sample handling}
+\begin{pframe}
+  Isolation:
+  \begin{itemize}
+    \item From blood plasma instead of white blood cells.
+  \end{itemize}
+  \bigskip
+
+  Sample preparation:
+  \begin{itemize}
+    \item Whole genome sequencing.
+  \end{itemize}
+  \bigskip
+
+  Sequencing on HiSeq 4000:
+  \begin{itemize}
+    \item Low pass ($\pm15$M reads).
+    \item Batches of $96$ samples.
+    \item One or two batches on one flowcell.
+  \end{itemize}
+\end{pframe}
+
+\subsection{Visualisation}
+\begin{pframe}
+  \begin{figure}[]
+    \begin{center}
+      \includegraphics[height=0.7\textheight, trim=25 0 0 0, clip]{NIPT_visualisation}
+    \end{center}
+    \caption{WISECONDOR CNV calls.}
+  \end{figure}
+\end{pframe}
+
+\subsection{Original pipeline}
+\begin{pframe}
+  The pipeline is straightforward:
+  \begin{itemize}
+    \item Alignment to the reference genome (GRCh37, hg19).
+    \item Deduplication.
+    \item CNV calling.
+  \end{itemize}
+  \bigskip
+
+  Original run time: $6$ hours, but with up to $500$ samples per week.
+  \begin{itemize}
+    \item $20$ minutes per sample.
+  \end{itemize}
+  \bigskip
+  \pause
+
+  Additional constraint: $8$ (office) hours turnaround time.
+  \begin{itemize}
+    \item $2.5$ minutes per sample ($2$ batches of $96$ samples).
+    \pause
+    \item Implementation time of two months.
+  \end{itemize}
+\end{pframe}
+
+\subsection{Optimisation}
+\begin{pframe}
+  Improvements on the pipeline:
+  \begin{itemize}
+    \item Other aligner (\lstinline{BWA mem}).
+    \item Other computational framework (\lstinline{Snakemake}).
+  \end{itemize}
+  \bigskip
+
+  These improvements brought the runtime back to around $40$ minutes per sample.
+  \bigskip
+  \pause
+
+  Parallel processing on the LUMC cluster.
+  \begin{itemize}
+    \item Up to $200$ cores for this project.
+    \item Between $90$ and $180$ minutes per batch.
+    \begin{itemize}
+      \item Depending on the number of batches per flowcell.
+      \pause
+      \item One batch in $1.5$ to $3$ hours.
+    \end{itemize}
+  \end{itemize}
+\end{pframe}
+
+\section{Production data analysis}
+\subsection{Fully automatic pipeline runs}
+\begin{pframe}
+  Prerequisites:
+  \begin{itemize}
+    \item Input data.
+    \item Metadata of the input data.
+    \begin{itemize}
+      \item Which files belong to which sample.
+      \item Read pair information.
+    \end{itemize}
+    \item Additional sample information (LIMS).
+    \begin{itemize}
+      \item Pedigree information.
+      \item Gene panel.
+    \end{itemize}
+    \item Pipeline.
+  \end{itemize}
+  \bigskip
+
+  
+\end{pframe}
+
+\subsection{Computational infrastructure}
+\begin{pframe}
+  Observation:
+  \begin{itemize}
+    \item Much of the automated data analysis is extremely complex.
+    \begin{itemize}
+      \item Many implicit dependencies and workflows.
+    \end{itemize}
+    \item Virtually impossible to transfer to other people.
+    \begin{itemize}
+      \item Problematic in diagnostics.
+    \end{itemize}
+  \end{itemize}
+  \bigskip
+
+  Our approach:
+  \begin{itemize}
+    \item Highly specialised microservices.
+    \begin{itemize}
+      \item Do only one thing and do it well.
+    \end{itemize}
+  \end{itemize}
+\end{pframe}
+
+\begin{pframe}
+  Reproducibility and automation.
+  \bigskip
+
+  Computational infrastructure:
+  \begin{itemize}
+    \item Transfer: gatekeeper for \emph{production data}.
+    \item Cerana: Project conductor.
+    \item Florea: API adapter for the HPC cluster.
+    \item Amegilla: API adapter for legacy / proprietary systems.
+  \end{itemize}
+
+  \vfill
+  \permfoot{\url{https://git.lumc.nl/groups/apis}}
+\end{pframe}
+
+\subsection{Transfer server}
+\begin{pframe}
+  Previous situation:
+  \begin{itemize}
+    \item We receive data on hard disks or via an sFTP server.
+    \item Frequently missing or wrong metadata, mixups.
+  \end{itemize}
+  \bigskip
+
+  Current situation:
+  \begin{itemize}
+    \item The \emph{consumer} of the data decides what is to be sent.
+    \begin{itemize}
+      \item Sample IDs and grouping, QC metrics, \ldots
+    \end{itemize}
+    \item The data is only accepted when this metadata is valid.
+  \end{itemize}
+  \bigskip
+  \pause
+
+  Gain:
+  \begin{itemize}
+    \item $24$\% rejected transfers.
+  \end{itemize}
+\end{pframe}
+
+\subsection{Cerana, the project conductor}
+\begin{pframe}
+  Gather data needed to run a data analysis.
+  \begin{itemize}
+    \item Files (e.g., from the transfer server).
+    \item Metadata (e.g., trio information from a LIMS system).
+  \end{itemize}
+  \bigskip
+
+  About the metadata:
+  \begin{itemize}
+    \item The order is not relevant.
+    \item The data can come from any (authorised) source.
+  \end{itemize}
+  \bigskip
+
+  When all data is available, a signal is sent to an actor that runs the
+  analysis.
+\end{pframe}
+
+\subsection{Florea, the pipeline runner}
+\begin{pframe}
+  Start a pipeline on the cluster.
+  \begin{itemize}
+    \item Get the pipeline configuration from our GitLab system.
+    \item Start the pipeline.
+    \item Monitor the pipeline progress.
+    \item Keep track of the status (query via the API).
+  \end{itemize}
+  \bigskip
+
+  Once the pipeline is finished, a status update is reported back to the
+  submitter of the pipeline run.
+\end{pframe}
+
+\subsection{Underlying infrastructure}
+\begin{pframe}
+  Security:
+  \begin{itemize}
+    \item Encryption and identity management with X.509.
+  \end{itemize}
+  \bigskip
+
+  Interfaces:
+  \begin{itemize}
+    \item Fully documented APIs.
+    \begin{itemize}
+      \item Only open standards.
+    \end{itemize}
+  \end{itemize}
+  \bigskip
+
+  All actions are stored in a database that can be queried via the API.
+\end{pframe}
+
+\begin{pframe}
+  Any of these services may fail.
+  \bigskip
+
+  Well defined interface:
+  \begin{itemize}
+    \item Easy to make backup procedures.
+  \end{itemize}
+  \bigskip
+
+  Full (online) documentation.
+  \begin{itemize}
+    \item Bypass any of the microservices.
+    \item Bypass everything.
+  \end{itemize}
+\end{pframe}
+
+\subsection{Use case: NIPT}
+\begin{pframe}
+  \begin{lstlisting}[language=none, caption={Summary of run $36$.}]
+    d2e88cea-9d7c-4c1c-b788-b20385d830d8
+      Name: 103033-036
+      Delivered on: 14-06-2017 20:40:53
+      Started on: 14-06-2017 18:58:45
+      Duration: 1:42:08
+      State: successful
+      Samples: 96
+      Remarks:
+  \end{lstlisting}
+
+  Overviews can be made by querying the relevant systems.
+  \bigskip
+  \pause
+
+  Over $21,\!000$ samples per year ($28$\% of the Dutch total).
+\end{pframe}
+
+\subsection{Exome sequencing}
+\begin{pframe}
+  We run four production pipelines at the moment.
+  \bigskip
+
+  Whole exome sequencing:
+  \begin{itemize}
+    \item $20$GB per sample ($50$ million reads).
+    \item $5,\!000$ samples per year (LUMC, EMC).
+    \item $100$ samples per week on average.
+    \item Takes half a year of computation time.
+    \item We deliver in $24$ hours.
+  \end{itemize}
+\end{pframe}
+
+
+% Make the acknowledgements slide.
+\makeAcknowledgementsSlide{
+  \begin{tabular}{ll}
+    Jonathan Vis\\
+    Mark Santcroos\\
+    Martijn Vermaat\\
+    Michel Villerius\\
+    Sander Bollen\\
+    Johan den Dunnen\\
+  \end{tabular}
+}
+
+\end{document}

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