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Revision as of 11:07, 2 February 2017
, 11:07, 2 February 2017→Download the example data
'''Note:''' the latest version of this practical is available at: [[SeqShop: Sequence Mapping and Assembly Practical]]
* The ones here is the original one from the June workshop (updated to be run from elsewhere)
== Introduction ==
== Introduction ==
See the [[introductory slides]] for an intro to this tutorial.
See the [[Media:SeqShop - GotCloud Align.pdf|introductory slides]] for an intro to this tutorial.
== Goals of This Session ==
== Goals of This Session ==
* What we want to learn
* What we want to learn
** How to evaluate the quality of sequence data
** How to evaluate the quality of sequence data
** How to visualize sequence data to examine the reads aligned to particular genomic positions
** How to visualize sequence data to examine the reads aligned to particular genomic positions
== Setup in person at the SeqShop Workshop ==
''This section is specifically for the SeqShop Workshop computers.''
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''If you are not running during the SeqShop Workshop, please skip this section.''
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{{SeqShopLogin}}
{{SeqShopLogin}}
== Setup your run environment==
=== Setup your run environment===
This will setup some environment variables to point you to
This will setup some environment variables to point you to
* GotCloud program
* [[GotCloud]] program
* Tutorial input files
* Tutorial input files
* Setup an output directory
* Setup an output directory
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</div>
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== Setup when running on your own outside of the SeqShop Workshop ==
''This section is specifically for running on your own outside of the SeqShop Workshop.''
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''If you are running during the SeqShop Workshop, please skip this section.''
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=== Download & Build GotCloud ===
If you do not already have GotCloud:
* cd to where you want GotCloud installed (you can change this to any directory you want)
mkdir -p ~/seqshop
cd ~/seqshop/
* download, decompress, and build the version of gotcloud that was tested with this tutorial:
wget https://github.com/statgen/gotcloud/archive/gotcloud.workshop.tar.gz
tar xvf gotcloud.workshop.tar.gz
mv gotcloud-gotcloud.workshop gotcloud
cd gotcloud/src
make
cd ../..
Remember the path to gotcloud/ that is what you will need to set your GC variable to.
=== Download the example data ===
Download and untar file containing the example data used in the practicals:
wget http://csg.sph.umich.edu//mktrost/seqshopExample.tar.gz
tar xvf seqshopExample.tar.gz
== Examining GotCloud Align Input Files ==
You will see the names of all the files included in the example data scrolling on the screen as they are unpacked from the tar file.
{{SeqShopRemoteEnv}}
== Examining [[GotCloud]] Align Input Files ==
=== Examining Raw Sequence Reads : FASTQs ===
=== Examining Raw Sequence Reads : FASTQs ===
FASTQ : standard file format provided to you by those who did the sequencing.
FASTQ : standard file format provided to you by those who did the sequencing.
* Subset of FASTQs - should map to chromosome 22 36000000-37000000
* Subset of FASTQs - should map to chromosome 22 36000000-37000000
ls ${IN}/fastq/
ls ${SS}/fastq/
There are 24 fastq files: combination of single-end & paired-end.
There are 24 fastq files: combination of single-end & paired-end.
Look at a couple of FASTQs:
Look at a couple of FASTQs:
less -S ${IN}/fastq/HG00551.SRR190851_1.fastq
less -S ${SS}/fastq/HG00551.SRR190851_1.fastq
<code>less</code> is a Linux command that allows you to look at a file.
<code>less</code> is a Linux command that allows you to look at a file.
*<code>-S</code> option prevents line wrap.
*<code>-S</code> option prevents line wrap
* Use the arrow (up/down/left/right) keys to scroll through the file.
* Use the arrow (up/down/left/right) keys to scroll through the file
* Use <code>zless</code> if the file is compressed (it isn't).
* Use the <code>space bar</code> to jump down a page
Use <code>'q'</code> to exit out of <code>less</code>
Use <code>'q'</code> to exit out of <code>less</code>
q
q
Look at the paired read:
Look at the paired read:
less -S ${IN}/fastq/HG00551.SRR190851_2.fastq
less -S ${SS}/fastq/HG00551.SRR190851_2.fastq
Remember, use <code>'q'</code> to exit out of <code>less</code>
Remember, use <code>'q'</code> to exit out of <code>less</code>
=== Reference Files ===
=== Reference Files ===
Reference files can be downloaded with GotCloud or from other sources
Reference files can be downloaded with [[GotCloud]] or from other sources
* See [[GotCloud: Genetic Reference and Resource Files]] for more information on downloading/generating reference files
* See [[GotCloud: Genetic Reference and Resource Files]] for more information on downloading/generating reference files
Take a look at the chromosome 22 reference files included for this tutorial:
Take a look at the chromosome 22 reference files included for this tutorial:
ls ${REF}
ls ${SS}/ref22
<ul>
<ul>
<li>View Screenshot</li>
<li>View Screenshot</li>
<div class="mw-collapsible-content">
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[[File:RefDir.png|500px]]
[[File:RefDir.png|700px]]
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Let's read the reference FASTA file (all reference bases for the chromosome):
Let's read the reference FASTA file (all reference bases for the chromosome):
less ${REF}/human.g1k.v37.chr22.fa
less ${SS}/ref22/human.g1k.v37.chr22.fa
Remember, use <code>'q'</code> to exit out of <code>less</code>
Remember, use <code>'q'</code> to exit out of <code>less</code>
<li>The ends of a chromosome are 'N' - unknown bases</li>
<li>The ends of a chromosome are 'N' - unknown bases</li>
<li>Let's look at 5 lines of the file starting at line 300,000</li>
<li>Let's look at 5 lines of the file starting at line 300,000</li>
tail -n+300000 ${REF}/human.g1k.v37.chr22.fa |head -n 5
tail -n+300000 ${SS}/ref22/human.g1k.v37.chr22.fa |head -n 5
[[File:Fasta.png|500px]]
[[File:Fasta.png|500px]]
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</ul>
</ul>
</ul>
</ul>
If you want to access the FASTA file by position, you can use <code>samtools faidx</code> command
${GC}/bin/samtools faidx ${SS}/ref22/human.g1k.v37.chr22.fa 22:36000000 | less
or
${GC}/bin/samtools faidx ${SS}/ref22/human.g1k.v37.chr22.fa 22:36000000-36000100
=== GotCloud FASTQ Index File ===
=== GotCloud FASTQ Index File ===
Let's look a look at the index file I prepared for this tutorial:
Let's look a look at the index file I prepared for this tutorial:
less -S ${IN}/align.index
less -S ${SS}/align.index
Remember, use <code>'q'</code> to exit out of <code>less</code>
Remember, use <code>'q'</code> to exit out of <code>less</code>
<li>Need a reminder of the format?</li>
<li>Need a reminder of the format?</li>
<div class="mw-collapsible-content">
<div class="mw-collapsible-content">
[[File:fqindex.png|650px]]
[[File:fqindex.png|750px]]
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<ul>
<li>Note: in the screenshots, the fields are shifted into clear columns to make it easier to read</li>
<ul>
<li>When you view the file, the fields will not line up in neat columns and it can be hard to read</li>
</ul>
</ul>
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<div class="mw-collapsible mw-collapsed" style="width:500px">
<li>Hard to read the index? Need a hint?</li>
<li>Hard to read the index? Need a hint?</li>
<ul>
<ul>
<li>Use cut to extract just the MERGE_NAME & RGID fields </li>
<li>Use cut to extract just the MERGE_NAME & RGID fields </li>
cut -f 1,4 ${IN}/align.index
cut -f 1,4 ${SS}/align.index
</ul>
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</div>
<div class="mw-collapsible mw-collapsed" style="width:500px">
<div class="mw-collapsible mw-collapsed" style="width:500px">
<li>Answer:</li>
<li>Answer:</li>
<li>HG00553 & HG00640</li>
<li>HG00553 & HG00640</li>
<li>They have multiple unique values in the RGID field</li>
<li>They have multiple unique values in the RGID field</li>
[[File:fqindexRG.png|650px]]
[[File:fqindexRG.png|800px]]
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** Most values should be left as the defaults
** Most values should be left as the defaults
* Specify values in your configuration file as:
* Specify values in your configuration file as:
** <code>KEY = value</code>
* Use $(KEY) to refer to another key's value
* Use $(KEY) to refer to another key's value
* If a KEY is specified twice, the later value is used
* If a KEY is specified twice, the later value is used
Let's look at the configuration file I created for this test:
Let's look at the configuration file I created for this test:
more ${IN}/gotcloud.conf
more ${SS}/gotcloud.conf
Use the <code>space bar</code> to advance if the whole file isn't displayed.
Use the <code>space bar</code> to advance if the whole file isn't displayed.
; If your input and references are at different paths than specified, what would you change?
; If your references are in a different path than what is specified, what would you change?
<ul>
<ul>
<div class="mw-collapsible mw-collapsed" style="width:200px">
<div class="mw-collapsible mw-collapsed" style="width:300px">
<li>Answer:</li>
<li>Answer:</li>
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<div class="mw-collapsible-content">
<ul>
<ul>
<li>You would change <code>IN_DIR</code> & <code>REF_DIR</code> to the new paths</li>
<li>You would change <code>REF_DIR</code> to the new path</li>
[[File:gcConf.png|500px]]
[[File:gcConf.png|800px]]
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</ul>
</ul>
== Run GotCloud Align ==
== Run [[GotCloud]] Align ==
[[File:AlignDiagram.png|500px]]
[[File:AlignDiagram.png|500px]]
Now that we have all of our input files, we need just a simple command to run them
Now that we have all of our input files, we need just a simple command to run them
${GC}/gotcloud align --conf ${IN}/gotcloud.conf --numcs 2
${GC}/gotcloud align --conf ${SS}/gotcloud.conf --numcs 2 --base_prefix ${SS} --outdir ${OUT}
* <code>${GC}/gotcloud</code> runs GotCloud
* <code>align</code> tells GotCloud you want to run the alignment pipeline.
* <code>--conf</code> tells GotCloud the name of the configuration file to use.
** The configuration for this test was downloaded with the seqshop input files.
* <code>--numcs</code> means to run 2 samples at a time.
* <code>--numcs</code> means to run 2 samples at a time.
** How many you can run concurrently depends on your system
** How many you can run concurrently depends on your system.
* <code>--base_prefix</code> tells GotCloud the prefix to append to relative paths.
** The Configuration file cannot read environment variables, so we need to tell GotCloud the path to the input files, ${SS}
** Alternatively, gotcloud.conf could be updated to specify the full paths
* <code>--out_dir</code> tells GotCloud where to write the output.
** This could be specified in gotcloud.conf, but to allow you to use the ${OUT} to change the output location, it is specified on the command-line
[[File:gcalignStart.png|850px]]
[[File:gcalignStart.png|850px]]
If you cancelled GotCloud part way through, just rerun your GotCloud command and it will pick up where it left off.
If you cancelled GotCloud part way through, just rerun your GotCloud command and it will pick up where it left off.
Inside GotCloud align, not only sequence alignment but also pre-processing of sequence data, including deduplication and base quality recalibration are performed along with quality assessment, as illustrated below.
[[File:Gotcloud_align_detail.png|500px]]
== Examining GotCloud Align Output ==
== Examining GotCloud Align Output ==
[[File:Qplotpdf.png|400px]]
[[File:Qplotpdf.png|400px]]
<li> Look at the PDF I produced when I ran the whole genome:</li>
<li> Look at the PDF I produced when I ran the whole genome:</li>
evince ${IN}/example/HG00551.wg.qplot.pdf&
evince ${SS}/ext/HG00551.wg.qplot.pdf&
</ul>
</ul>
[[File:Qplotpdfwg.png|400px]]
[[File:Qplotpdfwg.png|400px]]
[[File:GcalignOutBAMm.png|600px]]
[[File:GcalignOutBAMm.png|600px]]
Let's examine at the first 5 lines of the BAM file:
Let's examine at the first 5 lines of the BAM file using [http://samtools.sourceforge.net/samtools.shtml#3 samtools view]:
${GC}/bin/samtools view -h ${OUT}/bams/HG00551.recal.bam|head -n 5
${GC}/bin/samtools view -h ${OUT}/bams/HG00551.recal.bam|head -n 5
; What are the chromosome and position of the first record in the BAM file?
; What are the chromosome and position of the first record in the BAM file?
<ul>
<ul>
<div class="mw-collapsible mw-collapsed" style="width:200px">
<div class="mw-collapsible mw-collapsed" style="width:300px">
<li>Need a reminder of the format?</li>
<div class="mw-collapsible-content">
[[File:Bam.png|750px]]
</div>
</div>
<div class="mw-collapsible mw-collapsed" style="width:300px">
<li>Answer</li>
<li>Answer</li>
<div class="mw-collapsible-content">
<div class="mw-collapsible-content">
<li>Chr 22, Pos: 16114122</li>
<li>Chr 22, Pos: 16114122</li>
</ul>
</ul>
[[File:Bam.png|750px]]
[[File:BamRec.png|650px]]
</div>
</div>
</ul>
==== Accessing BAMs by Position ====
BAM's are so big, what if we want to see a position part way through the file?
*[http://samtools.sourceforge.net/samtools.shtml#3 samtools] has an option for that.
Add a region to the view command we used above. Let's find all reads that overlap positions 36907000-36907005:
${GC}/bin/samtools view -h ${OUT}/bams/HG00551.recal.bam 22:36907000-36907005
* Just a few reads.
Let's visualize what reads in that area look like using samtools tview:
${GC}/bin/samtools tview ${OUT}/bams/HG00551.recal.bam ${SS}/ref22/human.g1k.v37.chr22.fa
* Type ‘g’
** Type 22:36907000
* Type ‘n’ to color by nucleotide
* Use the arrow keys to move around and look at the area.
Understanding the syntax:
* '.' : match to the reference on the forward strand
* ',' : match to the reference on the reverse strand
* ACGTN : mismatch to reference on the forward strand
* acgtn : mismatch to reference on the reverse strand
; Do you see anything interesting?
<ul>
<div class="mw-collapsible mw-collapsed" style="width:500px">
<li>Screenshot</li>
<div class="mw-collapsible-content">
<ul>
<li>We will have to remember this region when we run snpcall to see what it says.</li>
</ul>
[[File:tview.png|750px]]
</div>
</div>
</div>
</div>
</ul>
</ul>
Other tview commands:
* Type '?' for a help screen
* Type 'q' to quit tview
Feel free to play around more and browse the BAM files.
==== Other tools for BAMs ====
We have developed a lot of tools that operate on BAM files.
See [[Software#BAM_Util_Tools|Software: BamUtil Tools]] for a list
* Many operations:
** diff : diff 2 BAM files
** stats: per positions statistics
** bam2Fastq : convert a BAM back to a FASTQ (how I created the fastqs for this tutorial)
** Lots of others
* Feel free to try some out
* If you have any questions, let me know, I wrote most of them and am happy to help.
== Logging Off ==
== Logging Off ==
''This section is specifically for the SeqShop Workshop computers.''
<div class="mw-collapsible mw-collapsed" style="width:600px">
''If you are not running during the SeqShop Workshop, please skip this section.''
<div class="mw-collapsible-content">
To logout of seqshop-server, type:
To logout of seqshop-server, type:
exit
exit
When done, log out of the Windows machine.
When done, log out of the Windows machine.
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