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2020-NGSBio-Read-Alignment-Exercises.tex
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2020-NGSBio-Read-Alignment-Exercises.tex
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\documentclass[11pt]{article}
\usepackage{sectsty}
\allsectionsfont{\color{blue}\fontfamily{lmss}\selectfont}
\usepackage{fontspec}
\setmainfont{XCharter}
\usepackage{listings}
\lstset{
basicstyle=\small\ttfamily,
tabsize=8,
columns=flexible,
breaklines=true,
frame=tb,
rulecolor=\color[rgb]{0.8,0.8,0.7},
backgroundcolor=\color[rgb]{1,1,0.91},
postbreak=\raisebox{0ex}[0ex][0ex]{\ensuremath{\color{red}\hookrightarrow\space}}
}
\usepackage{fontawesome}
\usepackage{mdframed}
\newmdenv[
backgroundcolor=gray,
fontcolor=white,
nobreak=true,
]{terminalinput}
\usepackage{parskip}
\usepackage[breakable]{tcolorbox}
\usepackage{parskip} % Stop auto-indenting (to mimic markdown behaviour)
\usepackage{iftex}
\ifPDFTeX
\usepackage[T1]{fontenc}
\usepackage{mathpazo}
\else
\usepackage{fontspec}
\fi
% Basic figure setup, for now with no caption control since it's done
% automatically by Pandoc (which extracts  syntax from Markdown).
\usepackage{graphicx}
% Maintain compatibility with old templates. Remove in nbconvert 6.0
\let\Oldincludegraphics\includegraphics
% Ensure that by default, figures have no caption (until we provide a
% proper Figure object with a Caption API and a way to capture that
% in the conversion process - todo).
\usepackage{caption}
\DeclareCaptionFormat{nocaption}{}
\captionsetup{format=nocaption,aboveskip=0pt,belowskip=0pt}
\usepackage[Export]{adjustbox} % Used to constrain images to a maximum size
\adjustboxset{max size={0.9\linewidth}{0.9\paperheight}}
\usepackage{float}
\floatplacement{figure}{H} % forces figures to be placed at the correct location
\usepackage{xcolor} % Allow colors to be defined
\usepackage{enumerate} % Needed for markdown enumerations to work
\usepackage{geometry} % Used to adjust the document margins
\usepackage{amsmath} % Equations
\usepackage{amssymb} % Equations
\usepackage{textcomp} % defines textquotesingle
% Hack from http://tex.stackexchange.com/a/47451/13684:
\AtBeginDocument{%
\def\PYZsq{\textquotesingle}% Upright quotes in Pygmentized code
}
\usepackage{upquote} % Upright quotes for verbatim code
\usepackage{eurosym} % defines \euro
\usepackage[mathletters]{ucs} % Extended unicode (utf-8) support
\usepackage{fancyvrb} % verbatim replacement that allows latex
\usepackage{grffile} % extends the file name processing of package graphics
% to support a larger range
\makeatletter % fix for grffile with XeLaTeX
\def\Gread@@xetex#1{%
\IfFileExists{"\Gin@base".bb}%
{\Gread@eps{\Gin@base.bb}}%
{\Gread@@xetex@aux#1}%
}
\makeatother
% The hyperref package gives us a pdf with properly built
% internal navigation ('pdf bookmarks' for the table of contents,
% internal cross-reference links, web links for URLs, etc.)
\usepackage{hyperref}
% The default LaTeX title has an obnoxious amount of whitespace. By default,
% titling removes some of it. It also provides customization options.
\usepackage{titling}
\usepackage{longtable} % longtable support required by pandoc >1.10
\usepackage{booktabs} % table support for pandoc > 1.12.2
\usepackage[inline]{enumitem} % IRkernel/repr support (it uses the enumerate* environment)
\usepackage[normalem]{ulem} % ulem is needed to support strikethroughs (\sout)
% normalem makes italics be italics, not underlines
\usepackage{mathrsfs}
% Colors for the hyperref package
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% ANSI colors
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% commands and environments needed by pandoc snippets
% extracted from the output of `pandoc -s`
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% Add ',fontsize=\small' for more characters per line
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% Additional commands for more recent versions of Pandoc
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\newcommand{\WarningTok}[1]{\textcolor[rgb]{0.38,0.63,0.69}{\textbf{\textit{{#1}}}}}
% Define a nice break command that doesn't care if a line doesn't already
% exist.
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% Math Jax compatibility definitions
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% Document parameters
% Document title
\title{index}
% Pygments definitions
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\def\PY@tok#1{\csname PY@tok@#1\endcsname}
\def\PY@toks#1+{\ifx\relax#1\empty\else%
\PY@tok{#1}\expandafter\PY@toks\fi}
\def\PY@do#1{\PY@bc{\PY@tc{\PY@ul{%
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\def\PY#1#2{\PY@reset\PY@toks#1+\relax+\PY@do{#2}}
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% Prevent overflowing lines due to hard-to-break entities
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% Setup hyperref package
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colorlinks=true,
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% Slightly bigger margins than the latex defaults
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\begin{document}
\hypertarget{read-alignment}{%
\section{Read Alignment}\label{read-alignment}}
\hypertarget{introduction}{%
\subsection{Introduction}\label{introduction}}
Sequence alignment in NGS is the process of determining the most likely
source of the observed DNA sequencing read within the reference genome
sequence.
\begin{figure}
\centering
\includegraphics{images/alignment.png}
\caption{NGS Read Alignment}
\end{figure}
\hypertarget{why-align}{%
\subsubsection{Why align?}\label{why-align}}
There are typical inferences you can make from an alignment of NGS data
against a reference genome:
\begin{itemize}
\tightlist
\item
Variation from the reference -- could have functional consequence.
\item
Transcript abundance: Instead of a microarray, you could use alignment
to genome to quantify expression: more sensitive
\item
Ab-initio transcript discovery: you can see a pileup from RNA seq data
showing evidence for an exon which was previously missed or an exon
which is being skipped in a transcript.
\end{itemize}
\begin{figure}
\centering
\includegraphics{images/alignment-uses.png}
\caption{Uses of Read Alignment}
\end{figure}
\hypertarget{learning-outcomes}{%
\subsection{Learning outcomes}\label{learning-outcomes}}
On completion of the tutorial, you can expect to be able to:
\begin{itemize}
\tightlist
\item
Perform read alignment using standard tools (BWA-MEM)
\item
Perform the following task and understand their effect on analysis
results
\begin{itemize}
\tightlist
\item
Mark Duplicates
\end{itemize}
\item
Visualise read alignments using IGV (Integrated Visualisation Tool)
\end{itemize}
If there is time you will learn how to:
\begin{itemize}
\tightlist
\item
Merge the results from multiple alignments and understand when it is
appropriate to perform a merge
\end{itemize}
\hypertarget{tutorial-sections}{%
\subsection{Tutorial sections}\label{tutorial-sections}}
This tutorial comprises the following sections:\\
1. \href{alignment.ipynb}{Performing read alignment}\\
2. \href{visualisation.ipynb}{Alignment visualisation}
There is also an additional (optional) section: 3.
\href{workflows.ipynb}{Alignment workflows}
\hypertarget{authors}{%
\subsection{Authors}\label{authors}}
This tutorial was written by
\href{https://github.com/jacquikeane}{Jacqui Keane} based on material
from \href{https://github.com/tk2}{Thomas Keane},
\href{https://github.com/vviyer}{Vivek Iyer} and
\href{https://github.com/vo1}{Victoria Offord}.
\hypertarget{running-the-commands-from-this-tutorial}{%
\subsection{Running the commands from this
tutorial}\label{running-the-commands-from-this-tutorial}}
You can follow this tutorial by typing all the commands you see into a
terminal window. This is similar to the ``Command Prompt'' window on MS
Windows systems, which allows the user to type DOS commands to manage
files.
To get started, open a new terminal on your computer and type the
command below:
\begin{tcolorbox}[breakable, size=fbox, boxrule=1pt, pad at break*=1mm,colback=cellbackground, colframe=cellborder]
\prompt{In}{incolor}{ }{\boxspacing}
\begin{Verbatim}[commandchars=\\\{\}]
\PY{n+nb}{cd} /home/manager/course\PYZus{}data/read\PYZus{}alignment
\end{Verbatim}
\end{tcolorbox}
Now you can follow the instructions in the tutorial from here.
\hypertarget{lets-get-started}{%
\subsection{Let's get started!}\label{lets-get-started}}
This tutorial assumes that you have samtools, bwa, Picard tools and IGV
installed on your computer. These are already installed on the VM you
are using. To check that these are installed, you can run the following
commands:
\begin{tcolorbox}[breakable, size=fbox, boxrule=1pt, pad at break*=1mm,colback=cellbackground, colframe=cellborder]
\prompt{In}{incolor}{ }{\boxspacing}
\begin{Verbatim}[commandchars=\\\{\}]
samtools \PYZhy{}\PYZhy{}help
\end{Verbatim}
\end{tcolorbox}
\begin{tcolorbox}[breakable, size=fbox, boxrule=1pt, pad at break*=1mm,colback=cellbackground, colframe=cellborder]
\prompt{In}{incolor}{ }{\boxspacing}
\begin{Verbatim}[commandchars=\\\{\}]
bwa
\end{Verbatim}
\end{tcolorbox}
\begin{tcolorbox}[breakable, size=fbox, boxrule=1pt, pad at break*=1mm,colback=cellbackground, colframe=cellborder]
\prompt{In}{incolor}{ }{\boxspacing}
\begin{Verbatim}[commandchars=\\\{\}]
picard \PYZhy{}h
\end{Verbatim}
\end{tcolorbox}
\begin{tcolorbox}[breakable, size=fbox, boxrule=1pt, pad at break*=1mm,colback=cellbackground, colframe=cellborder]
\prompt{In}{incolor}{ }{\boxspacing}
\begin{Verbatim}[commandchars=\\\{\}]
igv
\end{Verbatim}
\end{tcolorbox}
This should return the help message for samtools, bwa and Picard tools
respectively. The final command should launch the genome viewer IGV. You
can close the IGV software, we will use it later in this tutorial to
visualise alignments.
To get started with the tutorial, head to the first section:
\href{alignment.ipynb}{Performing read alignment}
% Add a bibliography block to the postdoc
\newpage
\hypertarget{performing-read-alignment}{%
\section{Performing Read Alignment}\label{performing-read-alignment}}
Here we will use the BWA aligner to align a smll set of Illumina
sequencing data to the \textit{Mus Musculus} reference genome. We will
align genomic sequence (from Whole-Genome Sequencing) from a mouse
embryo which has been mutagenised while the one-cell stage using
CRISPR-Cas9 and a gRNA targeting an exon of the Tyr gene. The successful
mutation of the gene will delete one or both alleles. A bi-allelic null
Tyr mouse will be albino, but otherwise healthy.
First, check you are in the correct directory.
\begin{tcolorbox}[breakable, size=fbox, boxrule=1pt, pad at break*=1mm,colback=cellbackground, colframe=cellborder]
\prompt{In}{incolor}{ }{\boxspacing}
\begin{Verbatim}[commandchars=\\\{\}]
\PY{n+nb}{pwd}
\end{Verbatim}
\end{tcolorbox}
It should display something like:
\texttt{/home/manager/course\_data/read\_alignment}
\hypertarget{viewing-the-reference-genome}{%
\subsection{Viewing the reference
genome}\label{viewing-the-reference-genome}}
Go to the \texttt{ref} directory that contains the fasta files of the
reference genomes:
\begin{tcolorbox}[breakable, size=fbox, boxrule=1pt, pad at break*=1mm,colback=cellbackground, colframe=cellborder]
\prompt{In}{incolor}{ }{\boxspacing}
\begin{Verbatim}[commandchars=\\\{\}]
\PY{n+nb}{cd} \PYZti{}/course\PYZus{}data/read\PYZus{}alignment/data/ref
\end{Verbatim}
\end{tcolorbox}
Fasta files (.fa) are used to store raw sequencing information before
aligning data. A single chromosome from the mouse genome is contained in
the file GRCm38.68.dna.toplevel.chr7.fa.gz
View the file with zless (we use zless instead of less because the file
is compressed):
\begin{tcolorbox}[breakable, size=fbox, boxrule=1pt, pad at break*=1mm,colback=cellbackground, colframe=cellborder]
\prompt{In}{incolor}{ }{\boxspacing}
\begin{Verbatim}[commandchars=\\\{\}]
zless GRCm38.68.dna.toplevel.chr7.fa.gz
\end{Verbatim}
\end{tcolorbox}
\textbf{Q1: What is the length of chromosome 7 of the mouse genome?
(Hint: Look at the fasta header for chromosome 7)}
\begin{tcolorbox}[breakable, size=fbox, boxrule=1pt, pad at break*=1mm,colback=cellbackground, colframe=cellborder]
\prompt{In}{incolor}{ }{\boxspacing}
\begin{Verbatim}[commandchars=\\\{\}]
\end{Verbatim}
\end{tcolorbox}
Similar to a BAM file, to allow fast retrieval of data, an index file is
often required. You should check for the presencen of fasta indexes for
the genome in the `ref' directory:
\texttt{GRCm38.68.dna.toplevel.chr7.fa.gz.amb\ ...\ GRCm38.68.dna.toplevel.chr7.fa.gz.sa}
These are created by BWA: suffixtrees, bwt transform etc etc.
If these index files don't exist, then you can run the indexing with the
command
\texttt{bwa\ index\ GRCm38.68.dna.toplevel.chr7.fa.gz}
Beware -- this indexing process can take 3-5 minutes so please only run
it if the index files do not exist!
\hypertarget{align-the-data-with-bwa}{%
\subsection{Align the data with bwa}\label{align-the-data-with-bwa}}
Go to the
\texttt{\textasciitilde{}/course\_data/read\_alignment/data/Exercise1/fastq/}
directory - you can use this command:
\begin{tcolorbox}[breakable, size=fbox, boxrule=1pt, pad at break*=1mm,colback=cellbackground, colframe=cellborder]
\prompt{In}{incolor}{ }{\boxspacing}
\begin{Verbatim}[commandchars=\\\{\}]
\PY{n+nb}{cd} ../Exercise1/fastq
\end{Verbatim}
\end{tcolorbox}
Use the \texttt{bwa\ mem} command to align the fastq files to the mouse
reference genome. By default bwa outputs SAM format directly to the
standard output (in this case your terminal window), therefore you will
have to redirect the result into a SAM file.
\begin{tcolorbox}[breakable, size=fbox, boxrule=1pt, pad at break*=1mm,colback=cellbackground, colframe=cellborder]
\prompt{In}{incolor}{ }{\boxspacing}
\begin{Verbatim}[commandchars=\\\{\}]
bwa mem ../../ref/GRCm38.68.dna.toplevel.chr7.fa.gz md5638a\PYZus{}7\PYZus{}87000000\PYZus{}R1.fastq.gz md5638a\PYZus{}7\PYZus{}87000000\PYZus{}R2.fastq.gz \PYZgt{} md5638.sam
\end{Verbatim}
\end{tcolorbox}
This may take a few minutes, please be patient.
\hypertarget{convert-a-sam-file-to-a-bam-file}{%
\subsection{Convert a SAM file to a BAM
file}\label{convert-a-sam-file-to-a-bam-file}}
Now use samtools to convert the SAM file \texttt{md5638.sam} created in
the previous step into a BAM file called \texttt{md5638.bam}.
\begin{tcolorbox}[breakable, size=fbox, boxrule=1pt, pad at break*=1mm,colback=cellbackground, colframe=cellborder]
\prompt{In}{incolor}{ }{\boxspacing}
\begin{Verbatim}[commandchars=\\\{\}]
samtools view \PYZhy{}O BAM \PYZhy{}o md5638.bam md5638.sam
\end{Verbatim}
\end{tcolorbox}
\textbf{Q2: How much space is saved by using a BAM file instead of a SAM
file?}
\begin{tcolorbox}[breakable, size=fbox, boxrule=1pt, pad at break*=1mm,colback=cellbackground, colframe=cellborder]
\prompt{In}{incolor}{ }{\boxspacing}
\begin{Verbatim}[commandchars=\\\{\}]
\end{Verbatim}
\end{tcolorbox}
\hypertarget{sort-and-index-the-bam-file}{%
\subsection{Sort and index the BAM
file}\label{sort-and-index-the-bam-file}}
The BAM files produced by BWA are sorted by read name (same order as the
original fastq files). However, most viewing and variant calling
software require the BAM files to be sorted by reference coordinate
position and indexed for rapid retrieval. Therefore, use `samtools sort'
to produce a new BAM file called \texttt{md5638.sorted.bam} that is
sorted by position.
\begin{tcolorbox}[breakable, size=fbox, boxrule=1pt, pad at break*=1mm,colback=cellbackground, colframe=cellborder]
\prompt{In}{incolor}{ }{\boxspacing}
\begin{Verbatim}[commandchars=\\\{\}]
samtools sort \PYZhy{}T temp \PYZhy{}O bam \PYZhy{}o md5638.sorted.bam md5638.bam
\end{Verbatim}
\end{tcolorbox}
Finally index the sorted BAM file using `samtools index' command.
\textbf{Note:} indexing a BAM file is also a good way to check that the
BAM file has not been truncated (e.g.~your disk becomes full when
writing the BAM file). At the end of every BAM file, a special end of
file (EOF) marker is written. The Samtools index command will first
check for this and produce an error message if it is not found.
\begin{tcolorbox}[breakable, size=fbox, boxrule=1pt, pad at break*=1mm,colback=cellbackground, colframe=cellborder]
\prompt{In}{incolor}{ }{\boxspacing}
\begin{Verbatim}[commandchars=\\\{\}]
samtools index md5638.sorted.bam
\end{Verbatim}
\end{tcolorbox}
\hypertarget{unix-pipes-to-combine-the-commands-together}{%
\subsection{Unix pipes to combine the commands
together}\label{unix-pipes-to-combine-the-commands-together}}
To produce the sorted BAM file above we had to carry out several
separate commands and produce intermediate files. The Unix pipe command
allows you to feed the output of one command into the next command.
You can combine all of these commands together using unix pipes, and do
all of this data processing together and avoid writing intermediate
files. To do this type:
\begin{tcolorbox}[breakable, size=fbox, boxrule=1pt, pad at break*=1mm,colback=cellbackground, colframe=cellborder]
\prompt{In}{incolor}{ }{\boxspacing}
\begin{Verbatim}[commandchars=\\\{\}]
bwa mem ../../ref/GRCm38.68.dna.toplevel.chr7.fa.gz md5638a\PYZus{}7\PYZus{}87000000\PYZus{}R1.fastq.gz md5638a\PYZus{}7\PYZus{}87000000\PYZus{}R2.fastq.gz \PY{p}{|} samtools view \PYZhy{}O BAM \PYZhy{} \PY{p}{|} samtools sort \PYZhy{}T temp \PYZhy{}O bam \PYZhy{}o md5638\PYZus{}2.sorted.bam \PYZhy{}
\end{Verbatim}
\end{tcolorbox}
Now index the BAM file:
\begin{tcolorbox}[breakable, size=fbox, boxrule=1pt, pad at break*=1mm,colback=cellbackground, colframe=cellborder]
\prompt{In}{incolor}{ }{\boxspacing}
\begin{Verbatim}[commandchars=\\\{\}]
samtools index md5638\PYZus{}2.sorted.bam
\end{Verbatim}
\end{tcolorbox}
\textbf{Note: When the symbol \texttt{-} is used above, Unix will
automatically replace \texttt{-} with the output produced by the
preceeding command (i.e.~the command before the \texttt{\textbar{}}
symbol).}
\hypertarget{mark-pcr-duplicates}{%
\subsection{Mark PCR Duplicates}\label{mark-pcr-duplicates}}
We will use a program called `MarkDuplicates' that is part of Picard
tools (http://picard.sourceforge.net) to remove PCR duplicates that may
have been introduced during the library construction stage. To find the
options for `MarkDuplicates' -- type:
\begin{tcolorbox}[breakable, size=fbox, boxrule=1pt, pad at break*=1mm,colback=cellbackground, colframe=cellborder]
\prompt{In}{incolor}{ }{\boxspacing}
\begin{Verbatim}[commandchars=\\\{\}]
picard MarkDuplicates
\end{Verbatim}
\end{tcolorbox}
Now run MarkDuplicates using the `I=' option to specify the input BAM
file and the `O=' option to specify the output file
(e.g.~md5638.markdup.bam). You will also need to specify the duplication
metrics output file using `M=' (e.g.~md5638.markdup.metrics).
\begin{tcolorbox}[breakable, size=fbox, boxrule=1pt, pad at break*=1mm,colback=cellbackground, colframe=cellborder]
\prompt{In}{incolor}{ }{\boxspacing}
\begin{Verbatim}[commandchars=\\\{\}]
picard MarkDuplicates \PY{n+nv}{I}\PY{o}{=}md5638.sorted.bam \PY{n+nv}{O}\PY{o}{=}md5638.markdup.bam \PY{n+nv}{M}\PY{o}{=}md5638.metrics.txt
\end{Verbatim}
\end{tcolorbox}
\textbf{Q3: From looking at the output metrics file - how many reads
were marked as duplicates? What was the percent duplication?}
\begin{tcolorbox}[breakable, size=fbox, boxrule=1pt, pad at break*=1mm,colback=cellbackground, colframe=cellborder]
\prompt{In}{incolor}{ }{\boxspacing}
\begin{Verbatim}[commandchars=\\\{\}]
\end{Verbatim}
\end{tcolorbox}
Don't forget to generate an index for the new bam file using samtools.
\begin{tcolorbox}[breakable, size=fbox, boxrule=1pt, pad at break*=1mm,colback=cellbackground, colframe=cellborder]
\prompt{In}{incolor}{ }{\boxspacing}
\begin{Verbatim}[commandchars=\\\{\}]
samtools index md5638.markdup.bam
\end{Verbatim}
\end{tcolorbox}
\hypertarget{generate-qc-stats}{%
\subsection{Generate QC Stats}\label{generate-qc-stats}}
Use samtools to collect some statistics and generate QC plots from the
alignment in the BAM file from the previous step. Make sure you save the
output of the stats command to a file
(e.g.~\texttt{md5638.markdup.stats}).
\begin{tcolorbox}[breakable, size=fbox, boxrule=1pt, pad at break*=1mm,colback=cellbackground, colframe=cellborder]
\prompt{In}{incolor}{ }{\boxspacing}
\begin{Verbatim}[commandchars=\\\{\}]
samtools stats md5638.markdup.bam \PYZgt{} md5638.markdup.stats
\end{Verbatim}
\end{tcolorbox}
\begin{tcolorbox}[breakable, size=fbox, boxrule=1pt, pad at break*=1mm,colback=cellbackground, colframe=cellborder]
\prompt{In}{incolor}{ }{\boxspacing}
\begin{Verbatim}[commandchars=\\\{\}]
plot\PYZhy{}bamstats \PYZhy{}p md5638\PYZus{}plot/ md5638.markdup.stats
\end{Verbatim}
\end{tcolorbox}
\hypertarget{exercises}{%
\subsection{Exercises}\label{exercises}}
Now look at the output and answer the following questions:
\textbf{Q4: What is the total number of reads?}
\textbf{Q5: What proportion of the reads were mapped?}
\textbf{Q6: How many reads were paired correctly/properly?}
\textbf{Q7: How many read pairs mapped to a different chromosome?}
\textbf{Q8: What is the insert size mean and standard deviation?}
In your web browser open the file called md5638\_plot.html to view the
QC information and anser the following questions:
\textbf{Q9: How many reads have zero mapping quality?}
\textbf{Q10: Which of the first fragments or second fragments are higher
base quality on average?}
Congratulations you have succesfully aligned some NGS data to a
reference genome! Now continue to the next section of the tutorial:
\href{visualisation.ipynb}{Alignment visualisation}.
% Add a bibliography block to the postdoc
\newpage
\hypertarget{alignment-visualisation}{%
\section{Alignment Visualisation}\label{alignment-visualisation}}
You have now made it to the interesting part!
Integrative Genome Viewer (IGV) \url{http://www.broadinstitute.org/igv/}
allows you to visualise genomic datasets and is a very useful tool for
looking at the alignment of reads onto a reference genome from BAM
files.
Start IGV by typing:
\begin{tcolorbox}[breakable, size=fbox, boxrule=1pt, pad at break*=1mm,colback=cellbackground, colframe=cellborder]
\prompt{In}{incolor}{ }{\boxspacing}
\begin{Verbatim}[commandchars=\\\{\}]
igv \PY{p}{\PYZam{}}
\end{Verbatim}
\end{tcolorbox}
\hypertarget{igv-main-window}{%
\subsection{IGV main window}\label{igv-main-window}}
When you start IGV, it will open a main window. At the top of this
window you have a toolbar and genome ruler for navigation. The largest
area in the main window is the data viewer where your alignments,
annotations and other data will be displayed. To do this, IGV uses
horizontal rows called tracks. Finally, at the bottom, there is a
sequence viewer which contains the base level information for your
reference genome.
\begin{figure}
\centering
\includegraphics{images/igv-main-window.png}
\caption{IGV - main window}
\end{figure}
\hypertarget{load-the-reference-genome}{%
\subsection{Load the reference genome}\label{load-the-reference-genome}}
IGV provides several genomes which can be selected with the ``Genome
drop-down box'' on the toolbar.
\textbf{Go to ' \textit{Genomes -\textgreater{} Load Genome From
Server\ldots{}} ' and select ``Mouse mm10''. This is a synonym for
GRCm38, which is the current mouse assembly (reference genome)}
\begin{figure}
\centering
\includegraphics{images/load-mouse-ref.png}
\caption{IGV - loading the mouse genome}
\end{figure}
\begin{figure}
\centering
\includegraphics{images/mouse-ref-dialog.png}
\caption{IGV - list of available genomes}
\end{figure}
\hypertarget{igv-toolbar-and-genome-ruler}{%
\subsubsection{IGV toolbar and genome
ruler}\label{igv-toolbar-and-genome-ruler}}
Once the genome has loaded, the chromosomes will be shown on the
\textbf{genome ruler} with their names/numbers above. When a region is
selected, a red box will appear. This represents the visible region of
the genome.
Above the genome ruler is the \textbf{toolbar} which has a variety
controls for navigating the genome:
\begin{itemize}
\tightlist
\item
\textbf{Genome drop-down} - load a genome
\item
\textbf{Chromosome drop-down} - zoom to a chromosome
\item
\textbf{Search} - zoom to a chromosome, locus or gene
\end{itemize}
There are several other buttons which can be used to control the visible
portion of the genome.
\begin{itemize}
\tightlist
\item
\textbf{Whole genome} - zoom back out to whole genome view
\item
\textbf{Previous/next view} - move backward/forward through views
(like the back/forward buttons in a web browser)
\item
\textbf{Refresh} - refresh the display
\item
\textbf{Zoom} - zooms in (+) / out (-) on a chromosome
\end{itemize}
\begin{figure}
\centering
\includegraphics{images/igv-toolbar.png}
\caption{IGV - toolbar and genome ruler}
\end{figure}
\hypertarget{sequence-viewer}{%
\subsubsection{Sequence viewer}\label{sequence-viewer}}
The \textbf{sequence viewer} shows the genome at the single nucleotide
level. You won't be able to see the sequence until you are zomed in.
Let's try it, select the zooom in (+) option in the top right of the
screeen. As you start to zoom in (+), you will see that each nucleotide
is represented by a coloured bar (red=T, yellow=G, blue=C and green=A).
This makes it easier to spot repetitive regions in the genome. Carry on
zooming in (+) and you will see the individual nucleotides.
\begin{figure}
\centering
\includegraphics{images/igv-sequence-viewer.png}
\caption{IGV - sequence viewer}
\end{figure}
\hypertarget{navigation-in-igv}{%
\subsection{Navigation in IGV}\label{navigation-in-igv}}
There are several views in IGV