Overview
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 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:
Example: E. coli genome with structural variation
Here's an E. coli genome re-sequencing sample where a key mutation producing a new structural variant was responsible for a new phenotype.
cds cp -r $BI/gva_course/structural_variation/data sv_tutorial cd 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.
File Name | Description | Sample |
|---|---|---|
| Paired-end Illumina, First of mate-pair, FASTQ format | Re-sequenced E. coli genome |
| Paired-end Illumina, Second of mate-pair, FASTQ format | Re-sequenced E. coli genome |
| Reference Genome in FASTA format | E. coli B strain REL606 |
Map data using bowtie2
First we need to (surprise!) map the data. We'll use bowtie2 for this tutorial, but BWA would also work with the proper options.
Do not run on head node
Many of the commands past this point are computationally intensive. You should run them through an idev shell or by qsub. We recommend idev for the tutorial, but you could also qsub any command that takes more than a few seconds to complete.
idev -m 60 -q development -A "UT-2015-05-18"
bowtie2-build NC_012967.1.fasta NC_012967.1 bowtie2 -p 12 -X 5000 --rf -x NC_012967.1 -1 61FTVAAXX_2_1.fastq -2 61FTVAAXX_2_2.fastq -S 61FTVAAXX.sam
Possibly unfamiliar options:
--rftells bowtie2 that your read pairs are in the "reverse-forward" orientation of a mate-pair library-X 5000tells bowtie2 to not mark read pairs as discordant unless their insert size is greater than 5000 bases.
Run SVDetect
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 question: What is the normal insert size for this library?
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:
/full/path/to/61FTVAAXX.ab.sam and /full/path/to/NC_012967.1.lengths on lines 7 and 8 below.<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=1
</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>!!
1<tab>NC_012967<tab>4629812
You'll want to submit the first two commands to the TACC queue or do them in an idev shell. They take a while. Consult the manual for a full description of what these commands and options are doing while the commands are running.
SVDetect linking -conf svdetect.conf SVDetect filtering -conf svdetect.conf SVDetect links2SV -conf svdetect.conf
Take a look at the resulting file: 61FTVAAXX.ab.sam.links.filtered.sv.txt.
We've highlighted a few lines below:
chr_type SV_type BAL_type chromosome1 start1-end1 average_dist chromosome2 start2-end2 nb_pairs score_strand_filtering score_order_filtering score_insert_size_filtering final_score breakpoint1_start1-end1 breakpoint2_start2-end2 ... INTRA NORMAL_SENSE - chrNC_012967 599566-601025 - chrNC_012967 663036-664898 430 100% - - 1 - - ... INTRA NORMAL_SENSE - chrNC_012967 3-2025 - chrNC_012967 4627019-4628998 288 100% - - 1 - - ... INTRA REVERSE_SENSE - chrNC_012967 16999-19033 - chrNC_012967 2775082-2777014 274 100% - - 1 - -
Very, Very Advanced Exercise
SVDetect has a nice option to output a file that can be read by Circos to produce drawings.
