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Most approaches for predicting structural variants require you to have paired-end or mate-pair reads. They use the distribution of distances separating these reads to find outliers and also look at pairs with incorrect orientations. Possible As mentioned during several of the presentations, many researchers choose to ignore these types of mutations and combined with the increased difficulty of accurately identifying them, the community is less settled on the "best" way to analyze them. Here we present a tutorial on SVDetect based on the quality of its instructions, and easy of installation despite its use of relatively hefty configuration files.
Other possible tools:
- BreakDancer - hard to install prerequisites on TACC. Requires installing libgd and the notoriously difficult GD Perl module.
- PEMer - hard to install prerequisites on TACC. Requires "ROOT" package.
- SVDetect - good instructions, relatively hefty configuration files.
Good discussion of some of the issues of predicting structural variation:
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cds cp -r $BI/gva_course/structural_variation/data BDIB_sv_tutorial cd BDIB_sv_tutorial |
This is Illumina mate-paired data (having a larger insert size than paired-end data) from genome re-sequencing of an E. coli clone.
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First we need to (surprise!) map the data. We'll use bowtie2 for this tutorial, but BWA would also work with the proper optionsThis will hopefully reinforce the bowtie2 tutorial you just completed, but if you are feeling adventurous you could use BWA as optional reinforcement.
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Many of the commands past this point are computationally intensive. You should run them through an
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bowtie2-build NC_012967.1.fasta NC_012967.1 bowtie2 -p 1248 -X 5000 --rf -x NC_012967.1 -1 61FTVAAXX_2_1.fastq -2 61FTVAAXX_2_2.fastq -S 61FTVAAXX.sam |
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--rf
tells bowtie2 that your read pairs are in the "reverse-forward" orientation of a mate-pair library-X 5000
tells bowtie2 to not mark read pairs as discordant unless their insert size is greater than 5000 bases.
You may notice that these commands complete pretty quickly. Always remember speed is not necessarily representative of how taxing something is for TACC's head node, and always try to be a good TACC citizen and do as much as you can on idev nodes or as job submissions
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bowtie doesn't output discordantly mapped pairs! |
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The first step is to look at all mapped read pairs and whittle down the list only to those that have an unusual insert sizes (distances between the two reads in a pair). You should submit this command to the TACC queue.
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BAM_preprocessingPairs.pl -p 0 61FTVAAXX.sam |
As we discussed in our earlier presentation, SV are often detected by looking for variations in library insert sizes. The stdout of the pearl script will answer the questionquestions:
- What is the normal insert size for this library?
- What percent of reads appear to be associated with potential structural variants?
- What percent of reads suggest potential novel sequence insertions or contamination with other sources of DNA?
- How can you use the above answers to make characterizations about new sequencing data, or use this type of analysis to make implications about your overall sequencing?
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SVDetect demonstrates a common strategy in some programs with complex input where instead of including a lot of options on the command line, it reads in a simple text file that sets all of the required options. Lets look at how to create a configuration file:
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You'll need to substitute your own paths for the output of the pwd command on lines 7 and 8 below in front of "/full/path/to/" , but you will need to leave "61FTVAAXX.ab.sam and /full/path/to/" and "NC_012967.1.lengths on lines 7 and 8 below". |
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<general> input_format=sam sv_type=all mates_orientation=RF read1_length=35 read2_length=35 mates_file=/full/path/to/61FTVAAXX.ab.sam cmap_file=/full/path/to/NC_012967.1.lengths num_threads=148 </general> <detection> split_mate_file=0 window_size=2000 step_length=1000 </detection> <filtering> split_link_file=0 nb_pairs_threshold=3 strand_filtering=1 </filtering> <bed> <colorcode> 255,0,0=1,4 0,255,0=5,10 0,0,255=11,100000 </colorcode> </bed> |
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You also need to create a tab-delimited file of chromosome lengths. Use the tab key rather than writing out <tab>!lengths named NC_012967.1.lengths. YOU CAN NOT COPY PASTE THIS COMMAND!
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1<tab>NC_012967<tab>4629812
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# Use the tab key rather than writing out <tab>!!
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The following commands will take a while and must be completed in order, so no advantages/ability to have them run in the background. Consult the manual for a full description of what these commands and options are doing while the commands are running.
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Take a look at the resulting file: 61FTVAAXX.ab.sam.links.filtered.sv.txt
. Another downside of command line applications is that while you can print files to the screen, the formatting is not always the nicest. On the plus side in 95% of cases, you can directly copy the output from the terminal window to excel and make better sense of what the columns actually are
We've highlighted a few lines below:
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1. This is a tandem head-to-tail duplication of the region from approximately 600000 to 663000. ... Many of the others are due to new insertions of transposable elements. |
Very, Very Advanced Exercise
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<circos>
organism_id=fish
<colorcode>
255,0,0=1,4
0,255,0=5,10
0,0,255=11,100000
</colorcode>
</circos> |
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Optional: Install SVDetectWe have installed SVdetect for you already as installation is a bit difficult . It should be (though still much easier than the alternatives listed in the introduction). You can verify it's location using which SVDetect in your Install SVDetect scriptsNavigate to the SVDetect project page More information: Try to download Download the code yourself onto TACC. Expand | | Here's how... | Here's how..
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wget httphttps://sourceforge.net/projects/svdetect/files/SVDetect/0.70/SVDetect_r0.7m.tar.gz latest/download tar -xvzf SVDetect_r0 r*.7m. tar.gz
cd SVDetect_ r0.7m
r* |
Move the Perl scripts and make them executable
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cp bin/SVDetect $HOME/local/bin chmod 775 scripts/BAM_preprocessingPairs.pl cp scripts/BAM_preprocessingPairs.pl $HOME/local/bin |
Install required Perl modules
SVdetect requires a few Perl modules to be installed. In the default TACC environment, you can use the cpan shell to install most well-behaved Perl modules (with the exception of some complicated ones that require other libraries to be installed or things to compile). Here's how:
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login1$ module load perl login1$ cpan ... cpan[4]> install Config::General ... cpan[4]> install Tie::IxHash ... cpan[4]> install Parallel::ForkManager ... cpan[4]> quit login1$ |