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We have installed SVdetect for you under $BI/bin so it . It should be in your $PATH. Installation is a bit difficult,   so you can please skip this part.!

Install SVDetect scripts

Navigate to the SVDetect project page

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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 structural_variation
cd structural_variation

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Map data using BWA

You should submit the bwa aln and bwa sampe commands as jobs to the queue, one after the other.

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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 options.

idev -m 60 -q development -a CCBB

bowtie2-build NC_012967.1.fasta 61FTVAAXX_2_1.fastq
bwa aln -f 61FTVAAXX_2_2.sai NC_012967.1.fasta 61FTVAAXX_2_2.fastq
bwa sampe -n 0 -N 100 -f 61FTVAAXX.sam
bowtie2 -p 12 -X 5000 --rf -x NC_012967.1 -1.fasta 61FTVAAXX_2_1.sai 61FTVAAXX_2_2.saifastq -2 61FTVAAXX_2_12.fastq -S 61FTVAAXX_2_2.fastq
.sam

Possibly unfamiliar options:

• -

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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.
  

If you use bowtie (version 1) to do your mapping, you won't predict any read SVs. Why?

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.

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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=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.

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.

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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
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INTRA   NORMAL_SENSE    -       chrNC_012967    599566-601002601025   -       chrNC_012967    662994663036-664998664898   430 1577    100%    -       -       1       -       -
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INTRA   NORMAL_SENSE    -       chrNC_012967    3-2025  -       chrNC_012967    4627019-4628998 989288     100%    -       -       1       -       -
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INTRA   REVERSE_SENSE   -       chrNC_012967    16999-1890319033     -       chrNC_012967    27759792775082-27778382777014 873274     100%    -       -       1       -       -

Any idea what sorts of mutations produced these three structural variants?

Very, Very Advanced Exercise

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