...

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:

...

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.

...

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.

idev  -m 60 -q developmentr CCBB_Bio_Summer_School_2016_Day2 -A "UT-2015-05-18" -N 2 -n 8

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

...

• --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

...

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.

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:

1. What is the normal insert size for this library? 
2. What percent of reads appear to be associated with potential structural variants? 
3. What percent of reads suggest potential novel sequence insertions or contamination with other sources of DNA?
4. 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?

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:

<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>

...

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!

1<tab>NC_012967<tab>4629812

...

  # Use the tab key rather than writing out <tab>!!

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.

...

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:

...

Very, Very Advanced Exercise

...

...

<circos>
organism_id=fish
  <colorcode>
    255,0,0=1,4
    0,255,0=5,10
    0,0,255=11,100000
  </colorcode>
</circos>

.

wget 

https://sourceforge.net/projects/svdetect/files/

latest/download
tar -xvzf SVDetect_

r*.

tar.gz
cd SVDetect_

r*

Move the Perl scripts and make them executable

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:

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$