nriddiford / svparser

Explore and filter structural variant calls from Lumpy and Delly VCF files

svParser

Filter, genotype, annotate and combine VCF files from structural variant callers LUMPY, DELLY and novobreak. Run without options or with --help or -h to print usage statement.

This tool is under constant development. Please feel free to contact me, or raise an issue if you encounter any problems.

Table of Contents

• Installation
• Summarise variants
• Genotyping variants
• Filtering variants
• Test filters against true positives
• Inspecting variants
• Browse variants
• Print variants
• Combine variants from multiple sources

Installation

git clone https://github.com/nriddiford/svParser.git

Install cpanm for easy installation of requirements:

wget -O- http://cpanmin.us | perl - -l ~/perl5 App::cpanminus local::lib

To avoid issues with root access, I recommended using a local Perl library, which can be setup as follows (following this):

eval `perl -I ~/perl5/lib/perl5 -Mlocal::lib`

Add the following lines to your bash_profile (or.profile/.bashrc/):

eval `perl -I ~/perl5/lib/perl5 -Mlocal::lib`
export MANPATH=$HOME/perl5/man:$MANPATH

Once you have cpanm installed, then installing the dependencies should be as simple as:

cd svParser
bash install_deps.sh

Summarise variants

A good place to start is with a summary of variants called in VCF file:

Read vcf file from lumpy (or delly/novobreak) and see summary of variants called:

perl script/svParse.pl -v data/Droso_R7.lumpy.vcf It's reccomended to explicity tag files on the caller that has produced them using the --method flag:
perl script/svParse.pl -v data/Droso_R7.lumpy.vcf -m l

Genotyping variants

The defualt behaviour of svParser is to consider all variants present in a VCF file, and classify these according to genotype. Genotype will be assigned as follows:

• germline_recurrent - variants with high quality read support in both a tumour and normal sample and at least one other sample in a Panel of Normals (PON)
• germline_private - variants with high quality read support in both a tumour and normal sample but NOT in a any samples in a PON
• somatic_normal - variants with high quality read support in normal sample, but not in tumour sample
• somatic_tumour - variants with high quality read support in tumour sample, but not in normal sample

Genotypes can also be filtered in using the genotype filters outlined in the filter section

IMPORTANT:

• Variants called by LUMPY must also be genotyped by SVTyper
• Genotyping requires that the samples in the VCF are ordered as follows: TUMOUR NORMAL PON.1 ... PON.N

Filtering variants

The real power of svParser comes from its ability to easily filter variant calls on anumber of different criteria, and quickly assess how this affects your callset. It is highly recommended to play around with differnt combinations of filters that suit your needs. Filter flags can be used with any of the other options aid in fine tuning

Filters available:

su   -   number of tumour reads supporting variant. Expects integer e.g. `-f su=5`
dp   -   minimum depth for both tumour normal at variant site. Expects integer e.g. `-f dp=10`
rdr  -   supporting reads/tumour depth - a value of 1 would mean all reads support variant. Expects integer/float e.g. `-f rdr=0.2`
sq   -   phred-scaled variant likelihood. Expects integer e.g. `-f sq=10`
chr  -   filter out chromosomes not in `chroms.txt` (if not provided, defaults to chromosomes 2L 2R 3L 3R 4 X Y). Expects binary e.g. `-f chr=1`
st   -   only keep somatic TUMOUR variants. Expects binary e.g. `-f st=1`
sn   -   only keep somatic NORMAL variants. Expects binary e.g. `-f sn=1`
gp   -   only keep germline PRIVATE variants. Expects binary e.g. `-f gp=1`
gr   -   only keep germline RECURRENT variants. Expects binary e.g. `-f gr=1`
a    -   apply default combination of filters. Equivalent to:

perl script/svParse.pl \
-v data/Droso_R7.lumpy.vcf \
-f su=4 \     # min 4 reads supporting event in tumour
-f dp=10 \    # min read depth of 10 in both tumour/normal
-f rdr=0.1 \  # min 10% of reads at breakpoint supporting variant
-f sq=10 \    # min Log10 likelihood of 10
-f chr=1      # filter out calls on chromosomes not in 'chroms.txt'

• In addition, users can provide a bed file containing regions to exclude by using -e [path/to/exclude.bed]

Filter-check-filter strategy to hone filters

Filters can be used in combination with other features of svParser to experiment with filters that remove obvious false positives while retaining true positives. E.g:

See summary of variants that have >= 4 reads supporting call in tumour (-f su=4)

perl script/svParse.pl -v data/Droso_R7.lumpy.vcf -m l -f su=4

See summary of variants that have >= 4 reads supporting call in tumour and have both breakpoints on chromosomes contained in chroms.txt

perl script/svParse.pl -v data/Droso_R7.lumpy.vcf -m l -f su=4 -f chr=1

Test filters on true positive variant calls

If you have a set of true positive calls that you want to protect from filtering, you can provide a set of sample-specfic true positives:

sample  caller   type   chrom1   bp1      chrom2    bp2
R7      lumpy    DEL    X        456393   X         4588700

perl script/svParse.pl -v data/Droso_R7.lumpy.vcf -m l -f su=4 -f chr=1 -t tests/

This allows you to see how adjusting filters affects your true positives.

Inspect specific variant call

Sometimes it's useful to see the information behind a specific variant call. svParser makes it easy to dig out a variant using it's id (taken from the VCF column ID) If the variant is affected by supplied filters this will be reported in the summary

Investigate a specific variant (by ID) using the --id flag:

perl script/svParse.pl -v data/Droso_R7.lumpy.vcf -m l -i 1

Browse variants

Browse variants using the --dump flag. This cycles through each line of the VCF file, printing an easy-to-read breakdown for each variant. Press any key to move to the next variant, or q to quit

perl script/svParse.pl -v data/Droso_R7.lumpy.vcf -d

Browse all variants with su>=5 on X chromosome (-c X -f su=5):

perl script/svParse.pl -v data/Droso_R7.lumpy.vcf -m l -f su=5 -d -c X

Browse all variants within a specific window on X chromosome (-c X:3000000-3500000):

perl script/svParse.pl -v data/Droso_R7.lumpy.vcf -m l -d -c X:3000000-3500000

Browse all variants that passed read depth filter (-f dp=20) filter within a specific window on X chromosome:

perl script/svParse.pl -v data/Droso_R7.lumpy.vcf -m l -f dp=20 -d -c X:3000000-3500000

Print variants

Write a new VCF file (filtered/input.filtered.vcf) and a summary txt file containing useful info (filtered/summary/input.filtered.summary.txt).

Print (-p) all variants that passed all defualt filters (-f a):

perl script/svParse.pl -v data/Droso_R7.lumpy.vcf -f a -p

Combining calls from multiple sources

Another useful feature of svParser is its ability to combine calls made by multiple callers into a unified call set per sample

Run on all samples

Generate filtered vcf and summary file for calls made by delly, lumpy and novoBreak:

for file in data/lumpy/*.vcf
do
  perl script/svParse.pl -v $file -f a -m l -p
done

for file in data/delly/*.vcf
do
  perl script/svParse.pl -v $file -f a -m d -p
done

for file in data/novobreak/*.vcf
do
  perl script/svParse.pl -v $file -f a -m n -p
done

Merge calls together in VCF using merge_vcf

Run merge_vcf on all filtered files. (Run from filtered directory):

cd filtered
perl ../script/merge_vcf.pl

Merge summary output foreach sample

Run svMerger.pl for all files in sample group. E.g. input1.delly.filtered.summary.txt input1.lumpy.filtered.summary.txt input1.novobreak.filtered.summary.txt

cd filtered/summary
perl ../../script/svMerger.pl -f input1.*

Cluster sv calls that are close together (+/- 50 bp):

for f in *_merged_SVs.txt
do
  perl ../../script/svClusters.pl $f -d 50
  rm $f
done

Annotate calls from a .gtf file

This takes a user-provided .gtf file and annotates variants with what feature/gene they affect:

features=/path/to/annotations.gtf

for clustered_file in *clustered_SVs.txt
do
  perl ../../script/sv2gene.pl -f $features -i $clustered_file
  rm $clustered_file
done

Cleaning results

The recommended next step from here is to inspect calls that remain. Any obvious false positives can be marked as false positives by entering F in the T/F column for each _annotated.txt file.

These can then be removed by using:

for annofile in *_annotated_SVs.txt
do
  python ../../script/clean.py -f $annofile
done

Plotting results

See svBreaks for plotting functions. This takes as input the annotated SV calls per sample

svparser's Issues

all_bps.txt does not contain `bp_` col

Currently running without reannotation step will cause break in svBreaks with missing bp_ col - need to add this to sv2gene output.

Use of uninitialized value

Hi,
I am trying to run the perl script svParse.pl on a structural variant vcf (normal-tumor) generated with Lumpy and genotyped with svTyper.
I want to apply the filters that you suggested to filter in somatic variants as follows:

perl $PERLDIR/svParse.pl \
-v $MYDIR/LumpyDir/$SAMPLE/NBD99-smoove.genotyped.vcf -f su=4 \     # min 4 reads supporting event in tumour
-f dp=10 \    # min read depth of 10 in both tumour/normal
-f rdr=0.1 \  # min 10% of reads at breakpoint supporting variant
-f sq=10 \    # min Log10 likelihood of 10
-f chr=0      # filter out calls on chromosomes not in 'chroms.txt' // this is actually not set 

What I unluckily got is thousands of lines of error messages with this pattern:

Use of uninitialized value $t_SR in numeric eq (==) at /srv/ngs/analysis/dalteriog/Tools/svParser/script/../bin/svParser.pm line 191, <$_[...]> line 11737.
(this is one of the last, but it happens for every field).

here I print the first 5 lines (along with the header) of my vcf
cat $MYDIR/LumpyDir/$SAMPLE/NBD99-smoove.genotyped.vcf | head -146

##fileformat=VCFv4.2
##FILTER=<ID=PASS,Description="All filters passed">
##fileDate=20220519
##reference=/srv/ngs/analysis/aievola/references/Homo_sapiens/GATK/GRCh37/Sequence/WholeGenomeFasta/hs37d5.fa
##INFO=<ID=SVTYPE,Number=1,Type=String,Description="Type of structural variant">
##INFO=<ID=SVLEN,Number=.,Type=Integer,Description="Difference in length between REF and ALT alleles">
##INFO=<ID=END,Number=1,Type=Integer,Description="End position of the variant described in this record">
##INFO=<ID=STRANDS,Number=.,Type=String,Description="Strand orientation of the adjacency in BEDPE format (DEL:+-, DUP:-+, INV:++/--)">
##INFO=<ID=IMPRECISE,Number=0,Type=Flag,Description="Imprecise structural variation">
##INFO=<ID=CIPOS,Number=2,Type=Integer,Description="Confidence interval around POS for imprecise variants">
##INFO=<ID=CIEND,Number=2,Type=Integer,Description="Confidence interval around END for imprecise variants">
##INFO=<ID=CIPOS95,Number=2,Type=Integer,Description="Confidence interval (95%) around POS for imprecise variants">
##INFO=<ID=CIEND95,Number=2,Type=Integer,Description="Confidence interval (95%) around END for imprecise variants">
##INFO=<ID=MATEID,Number=.,Type=String,Description="ID of mate breakends">
##INFO=<ID=EVENT,Number=1,Type=String,Description="ID of event associated to breakend">
##INFO=<ID=SECONDARY,Number=0,Type=Flag,Description="Secondary breakend in a multi-line variants">
##INFO=<ID=SU,Number=.,Type=Integer,Description="Number of pieces of evidence supporting the variant across all samples">
##INFO=<ID=PE,Number=.,Type=Integer,Description="Number of paired-end reads supporting the variant across all samples">
##INFO=<ID=SR,Number=.,Type=Integer,Description="Number of split reads supporting the variant across all samples">
##INFO=<ID=BD,Number=.,Type=Integer,Description="Amount of BED evidence supporting the variant across all samples">
##INFO=<ID=EV,Number=.,Type=String,Description="Type of LUMPY evidence contributing to the variant call">
##INFO=<ID=PRPOS,Number=.,Type=String,Description="LUMPY probability curve of the POS breakend">
##INFO=<ID=PREND,Number=.,Type=String,Description="LUMPY probability curve of the END breakend">
##ALT=<ID=DEL,Description="Deletion">
##ALT=<ID=DUP,Description="Duplication">
##ALT=<ID=INV,Description="Inversion">
##ALT=<ID=DUP:TANDEM,Description="Tandem duplication">
##ALT=<ID=INS,Description="Insertion of novel sequence">
##ALT=<ID=CNV,Description="Copy number variable region">
##FORMAT=<ID=GT,Number=1,Type=String,Description="Genotype">
##FORMAT=<ID=SU,Number=1,Type=Integer,Description="Number of pieces of evidence supporting the variant">
##FORMAT=<ID=PE,Number=1,Type=Integer,Description="Number of paired-end reads supporting the variant">
##FORMAT=<ID=SR,Number=1,Type=Integer,Description="Number of split reads supporting the variant">
##FORMAT=<ID=BD,Number=1,Type=Integer,Description="Amount of BED evidence supporting the variant">
##FORMAT=<ID=GQ,Number=1,Type=Integer,Description="Genotype quality">
##FORMAT=<ID=SQ,Number=1,Type=Float,Description="Phred-scaled probability that this site is variant (non-reference in this sample">
##FORMAT=<ID=GL,Number=G,Type=Float,Description="Genotype Likelihood, log10-scaled likelihoods of the data given the called genotype for each possible genotype generated from the reference and alternate alleles given the sample ploidy">
##FORMAT=<ID=DP,Number=1,Type=Integer,Description="Read depth">
##FORMAT=<ID=RO,Number=1,Type=Integer,Description="Reference allele observation count, with partial observations recorded fractionally">
##FORMAT=<ID=AO,Number=A,Type=Integer,Description="Alternate allele observations, with partial observations recorded fractionally">
##FORMAT=<ID=QR,Number=1,Type=Integer,Description="Sum of quality of reference observations">
##FORMAT=<ID=QA,Number=A,Type=Integer,Description="Sum of quality of alternate observations">
##FORMAT=<ID=RS,Number=1,Type=Integer,Description="Reference allele split-read observation count, with partial observations recorded fractionally">
##FORMAT=<ID=AS,Number=A,Type=Integer,Description="Alternate allele split-read observation count, with partial observations recorded fractionally">
##FORMAT=<ID=ASC,Number=A,Type=Integer,Description="Alternate allele clipped-read observation count, with partial observations recorded fractionally">
##FORMAT=<ID=RP,Number=1,Type=Integer,Description="Reference allele paired-end observation count, with partial observations recorded fractionally">
##FORMAT=<ID=AP,Number=A,Type=Integer,Description="Alternate allele paired-end observation count, with partial observations recorded fractionally">
##FORMAT=<ID=AB,Number=A,Type=Float,Description="Allele balance, fraction of observations from alternate allele, QA/(QR+QA)">
##contig=<ID=1,length=249250621>
##contig=<ID=2,length=243199373>
##contig=<ID=3,length=198022430>
##contig=<ID=4,length=191154276>
##contig=<ID=5,length=180915260>
##contig=<ID=6,length=171115067>
##contig=<ID=7,length=159138663>
##contig=<ID=8,length=146364022>
##contig=<ID=9,length=141213431>
##contig=<ID=10,length=135534747>
##contig=<ID=11,length=135006516>
##contig=<ID=12,length=133851895>
##contig=<ID=13,length=115169878>
##contig=<ID=14,length=107349540>
##contig=<ID=15,length=102531392>
##contig=<ID=16,length=90354753>
##contig=<ID=17,length=81195210>
##contig=<ID=18,length=78077248>
##contig=<ID=19,length=59128983>
##contig=<ID=20,length=63025520>
##contig=<ID=21,length=48129895>
##contig=<ID=22,length=51304566>
##contig=<ID=X,length=155270560>
##contig=<ID=Y,length=59373566>
##contig=<ID=MT,length=16569>
##contig=<ID=GL000207.1,length=4262>
##contig=<ID=GL000226.1,length=15008>
##contig=<ID=GL000229.1,length=19913>
##contig=<ID=GL000231.1,length=27386>
##contig=<ID=GL000210.1,length=27682>
##contig=<ID=GL000239.1,length=33824>
##contig=<ID=GL000235.1,length=34474>
##contig=<ID=GL000201.1,length=36148>
##contig=<ID=GL000247.1,length=36422>
##contig=<ID=GL000245.1,length=36651>
##contig=<ID=GL000197.1,length=37175>
##contig=<ID=GL000203.1,length=37498>
##contig=<ID=GL000246.1,length=38154>
##contig=<ID=GL000249.1,length=38502>
##contig=<ID=GL000196.1,length=38914>
##contig=<ID=GL000248.1,length=39786>
##contig=<ID=GL000244.1,length=39929>
##contig=<ID=GL000238.1,length=39939>
##contig=<ID=GL000202.1,length=40103>
##contig=<ID=GL000234.1,length=40531>
##contig=<ID=GL000232.1,length=40652>
##contig=<ID=GL000206.1,length=41001>
##contig=<ID=GL000240.1,length=41933>
##contig=<ID=GL000236.1,length=41934>
##contig=<ID=GL000241.1,length=42152>
##contig=<ID=GL000243.1,length=43341>
##contig=<ID=GL000242.1,length=43523>
##contig=<ID=GL000230.1,length=43691>
##contig=<ID=GL000237.1,length=45867>
##contig=<ID=GL000233.1,length=45941>
##contig=<ID=GL000204.1,length=81310>
##contig=<ID=GL000198.1,length=90085>
##contig=<ID=GL000208.1,length=92689>
##contig=<ID=GL000191.1,length=106433>
##contig=<ID=GL000227.1,length=128374>
##contig=<ID=GL000228.1,length=129120>
##contig=<ID=GL000214.1,length=137718>
##contig=<ID=GL000221.1,length=155397>
##contig=<ID=GL000209.1,length=159169>
##contig=<ID=GL000218.1,length=161147>
##contig=<ID=GL000220.1,length=161802>
##contig=<ID=GL000213.1,length=164239>
##contig=<ID=GL000211.1,length=166566>
##contig=<ID=GL000199.1,length=169874>
##contig=<ID=GL000217.1,length=172149>
##contig=<ID=GL000216.1,length=172294>
##contig=<ID=GL000215.1,length=172545>
##contig=<ID=GL000205.1,length=174588>
##contig=<ID=GL000219.1,length=179198>
##contig=<ID=GL000224.1,length=179693>
##contig=<ID=GL000223.1,length=180455>
##contig=<ID=GL000195.1,length=182896>
##contig=<ID=GL000212.1,length=186858>
##contig=<ID=GL000222.1,length=186861>
##contig=<ID=GL000200.1,length=187035>
##contig=<ID=GL000193.1,length=189789>
##contig=<ID=GL000194.1,length=191469>
##contig=<ID=GL000225.1,length=211173>
##contig=<ID=GL000192.1,length=547496>
##contig=<ID=NC_007605,length=171823>
##contig=<ID=hs37d5,length=35477943>
##smoove_version=0.2.8
##smoove_count_stats=sample_control_XI003_NB-S_559:28112073,50782761,655708,915464
##smoove_count_stats=sample_tumor_XI003_NB-S_559:45240623,74879219,1439450,1092014
##source=LUMPY
##bcftools_viewVersion=1.15.1+htslib-1.15.1
##bcftools_viewCommand=view -O z -o NBD99-smoove.genotyped.vcf.gz; Date=Thu May 19 18:30:22 2022
#CHROM POS ID REF ALT QUAL FILTER INFO FORMAT sample_control_XI003_NB-S_559 sample_tumor_XI003_NB-S_559
1 10621 1 N 267.12 . SVTYPE=DEL;SVLEN=-132;END=10753;STRANDS=+-:8;CIPOS=-10,9;CIEND=-10,9;CIPOS95=0,0;CIEND95=0,0;SU=8;PE=0;SR=8;PRPOS=9.98739e-33,1.58289e-29,2.50872e-26,3.97605e-23,6.30162e-20,9.98739e-17,1.58289e-13,2.50872e-10,3.97605e-07,0.000630162,0.998739,0.000630162,3.97605e-07,2.50872e-10,1.58289e-13,9.98739e-17,6.30162e-20,3.97605e-23,2.50872e-26,1.58289e-29;PREND=9.98739e-33,1.58289e-29,2.50872e-26,3.97605e-23,6.30162e-20,9.98739e-17,1.58289e-13,2.50872e-10,3.97605e-07,0.000630162,0.998739,0.000630162,3.97605e-07,2.50872e-10,1.58289e-13,9.98739e-17,6.30162e-20,3.97605e-23,2.50872e-26,1.58289e-29 GT:GQ:SQ:GL:DP:RO:AO:QR:QA:RS:AS:ASC:RP:AP:AB 1/1:5:60.25:-6,-1,-0:2:0:2:0:2:0:2:0:0:0:1 1/1:17:206.87:-21,-2,-0:7:0:7:0:7:0:4:2:0:0:1
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Can you tell me where is my mistake?
Thank you in advance,
Giuseppe