## Key Learning Outcomes

After completing this practical the trainee should be able to:

• Perform a simple NGS data alignment task, with Bowtie2, against one interested reference data

• Interpret and manipulate the mapping output using SAMtools

• Visualise the alignment via a standard genome browser, e.g. IGV browser

## Resources You’ll be Using

### Tools Used

BEDTools:

UCSC tools:

IGV genome browser:

SAM Specification:
http://samtools.sourceforge.net/SAM1.pdf

Explain SAM Flags:

### Sources of Data

http://www.ebi.ac.uk/arrayexpress/experiments/E-GEOD-11431

## Author Information

Contributor(s): Xi (Sean) Li sean.li@csiro.au

## Introduction

The goal of this hands-on session is to perform an unspliced alignment for a small subset of raw reads. We will align raw sequencing data to the mouse genome using Bowtie2 and then we will manipulate the SAM output in order to visualize the alignment on the IGV browser.

## Prepare the Environment

We will use one data set in this practical, which can be found in the ChIP-seq directory on your desktop.

Open the Terminal.

First, go to the right folder, where the data are stored.

cd /home/trainee/chipseq


The .fastq file that we will align is called Oct4.fastq. This file is based on Oct4 ChIP-seq data published by Chen et al. (2008). For the sake of time, we will align these reads to a single mouse chromosome.

## Alignment

You already know that there are a number of competing tools for short read alignment, each with its own set of strengths, weaknesses, and caveats. Here we will try Bowtie2, a widely used ultrafast, memory efficient short read aligner.

Bowtie2 has a number of parameters in order to perform the alignment. To view them all type

bowtie2 --help


Bowtie2 uses indexed genome for the alignment in order to keep its memory footprint small. Because of time constraints we will build the index only for one chromosome of the mouse genome. For this we need the chromosome sequence in FASTA format. This is stored in a file named mm10, under the subdirectory bowtie_index.

STOP

DO NOT run this command. This has already been run for you.

** bowtie2-build bowtie_index/mm10.fa bowtie_index/mm10 **

This command will output 6 files that constitute the index. These files that have the prefix mm10 are stored in the bowtie_index subdirectory. To view if they files have been successfully created type:

ls -l bowtie_index


Now that the genome is indexed we can move on to the actual alignment. The first argument for bowtie2 is the basename of the index for the genome to be searched; in our case this is mm10. We also want to make sure that the output is in SAM format using the -S parameter. The last argument is the name of the FASTQ file.

Align the Oct4 reads using Bowtie2:

bowtie2 -x bowtie_index/mm10 -q Oct4.fastq > Oct4.sam


The above command outputs the alignment in SAM format and stores them in the file Oct4.sam.

In general before you run Bowtie2, you have to know what quality encoding your FASTQ files are in. The available FASTQ encodings for bowtie are:

–phred33-quals : Input qualities are Phred+33 (default).

–phred64-quals : Input qualities are Phred+64 (same as –solexa1.3-quals).

–solexa-quals : Input qualities are from GA Pipeline ver. < 1.3.

–solexa1.3-quals : Input qualities are from GA Pipeline ver. >= 1.3.

–integer-quals : Qualities are given as space-separated integers (not ASCII).

The FASTQ files we are working with are Sanger encoded (Phred+33), which is the default for Bowtie2.

Bowtie2 will take 2-3 minutes to align the file. This is fast compared to other aligners which sacrifice some speed to obtain higher sensitivity.

Look at the top 10 lines of the SAM file using head (record lines are wrapped). Then try the second command, note use arrow navigation and to exit type ’q’.

head  Oct4.sam
less -S Oct4.sam


Question

Can you distinguish between the header of the SAM format and the actual alignments?

The header line starts with the letter ‘@’, i.e.:

@HD VN:1.0 SO:unsorted
@SQ SN:chr1 LN:195471971
@PG ID:Bowtie2 PN:bowtie2 VN:2.2.4 CL:“/tools/bowtie2/bowtie2-default/bowtie2-align-s –wrapper basic-0 -x bowtie_index/mm10 -q Oct4.fastq”

SRR002012.45 0 etc
SRR002012.48 16 chr1 etc

Question

What kind of information does the header provide?

• @HD: Header line; VN: Format version; SO: the sort order of alignments.

• @SQ: Reference sequence information; SN: reference sequence name; LN: reference sequence length.

• @PG: Read group information; ID: Read group identifier; VN: Program version; CL: the command line that produces the alignment.

Question

To which chromosome are the reads mapped?

Chromosome 1.

## Manipulate SAM output

SAM files are rather big and when dealing with a high volume of NGS data, storage space can become an issue. As we have already seen, we can convert SAM to BAM files (their binary equivalent that are not human readable) that occupy much less space.

Convert SAM to BAM using samtools view and store the output in the file Oct4.bam. You have to instruct samtools view that the input is in SAM format (-S), the output should be in BAM format (-b) and that you want the output to be stored in the file specified by the -o option:

samtools view -bSo Oct4.bam Oct4.sam


Compute summary stats for the Flag values associated with the alignments using:

samtools flagstat Oct4.bam


## Visualize alignments in IGV

IGV is a stand-alone genome browser. Please check their website (http://www.broadinstitute.org/igv/) for all the formats that IGV can display. For our visualization purposes we will use the BAM and bigWig formats.

When uploading a BAM file into the genome browser, the browser will look for the index of the BAM file in the same folder where the BAM files is. The index file should have the same name as the BAM file and the suffix .bai. Finally, to create the index of a BAM file you need to make sure that the file is sorted according to chromosomal coordinates.

Sort alignments according to chromosomal position and store the result in the file with the prefix Oct4.sorted:

samtools sort Oct4.bam -o Oct4.sorted.bam


Index the sorted file.

samtools index Oct4.sorted.bam


The indexing will create a file called Oct4.sorted.bam.bai. Note that you don’t have to specify the name of the index file when running samtools index, it simply appends a .bai suffix to the input BAM file.

Another way to visualize the alignments is to convert the BAM file into a bigWig file. The bigWig format is for display of dense, continuous data and the data will be displayed as a graph. The resulting bigWig files are in an indexed binary format.

The BAM to bigWig conversion takes place in two steps. Firstly, we convert the BAM file into a bedgraph, called Oct4.bedgraph, using the tool genomeCoverageBed from BEDTools. Then we convert the bedgraph into a bigWig binary file called Oct4.bw, using bedGraphToBigWig from the UCSC tools:

genomeCoverageBed -bg -ibam Oct4.sorted.bam -g bowtie_index/mouse.mm10.genome > Oct4.bedgraph
bedGraphToBigWig Oct4.bedgraph bowtie_index/mouse.mm10.genome Oct4.bw


Both of the commands above take as input a file called mouse.mm10.genome that is stored under the subdirectory bowtie_index. These genome files are tab-delimited and describe the size of the chromosomes for the organism of interest. When using the UCSC Genome Browser, Ensembl, or Galaxy, you typically indicate which species/genome build you are working with. The way you do this for BEDTools is to create a “genome” file, which simply lists the names of the chromosomes (or scaffolds, etc.) and their size (in basepairs).

BEDTools includes pre-defined genome files for human and mouse in the genomes subdirectory included in the BEDTools distribution.

Now we will load the data into the IGV browser for visualization. In order to launch IGV double click on the IGV 2.3 icon on your Desktop. Ignore any warnings and when it opens you have to load the genome of interest.

On the top left of your screen choose from the drop down menu Mouse (mm10). If it doesn’t appear in list, click More .., type mm10 in the Filter section, choose the mouse genome and press OK. Then in order to load the desire files go to:

File > Load from File


On the pop up window navigate to Desktop -> chipseq folder and select the file Oct4.sorted.bam.

Repeat these steps in order to load Oct4.bw as well.

Select chr1 from the drop down menu on the top left. Right click on the name of Oct4.bw and choose Maximum under the Windowing Function. Right click again and select Autoscale.

In order to see the aligned reads of the BAM file, you need to zoom in to a specific region. For example, look for gene Lemd1 in the search box.

Question

What is the main difference between the visualization of BAM and bigWig files?

The actual alignment of reads that stack to a particular region can be displayed using the information stored in a BAM format. The bigWig format is for display of dense, continuous data that will be displayed in the Genome Browser as a graph.

Using the + button on the top right, zoom in to see more of the details of the alignments.

Question

What do you think the different colors mean?

In the chipseq folder you will find the file gfp.fastq. Follow the above described analysis, from the bowtie2 alignment step, for this dataset as well. You will need these files for the ChIP-Seq module.