PacBio structural variant (SV) calling and analysis tools
pbsv is a suite of tools to call and analyze structural variants
in diploid genomes from PacBio single molecule real-time sequencing (SMRT) reads.
The tools power the Structural Variant Calling analysis workflow in
PacBio's SMRT Link GUI.
pbsv calls insertions, deletions, inversions, duplications, and translocations.
Both single-sample calling and joint (multi-sample) calling are provided.
pbsv is most
- insertions 20 bp to 10 kb
- deletions 20 bp to 100 kb
- inversions 200 bp to 10 kb
- duplications 20 bp to 10 kb
- translocations between different chromosomes or further than 100kb apart on a single chromosome
Latest version can be installed via bioconda package
Please refer to our official pbbioconda page for information on Installation, Support, License, Copyright, and Disclaimer.
Version 2.3.0: Full changelog here
pbsv workflow is:
- Align PacBio reads to a reference genome, per movie. (
- Discover signatures of structural variation. (
- Call structural variants and assign genotypes, all samples. (
1. Align PacBio reads to a reference genome
The recommended aligner is
pbmm2 that can be installed via
conda install pbmm2.
For each movie (
.ccs.fq) align records to a
reference genome (
Subreads BAM input:
pbmm2 align ref.fa movie1.subreads.bam ref.movie1.bam --sort --median-filter --sample sample1
CCS BAM input:
pbmm2 align ref.fa movie1.ccs.bam ref.movie1.bam --sort --preset CCS --sample sample1
CCS FASTQ input:
pbmm2 align ref.fa movie1.Q20.fastq ref.movie1.bam --sort --preset CCS --sample sample1 --rg '@RG\tID:movie1'
The sample name, stored in the
SM tag of the read groups, associates
aligned reads with a particular sample. It is required for downstream
2. Discover signatures of structural variation
For each aligned BAM or set of aligned BAMs, identify signatures
of structural variation. This reduces all aligned reads to those that are relevant
to calling structural variants. The signatures are stored in a
pbsv discover ref.movie1.bam ref.sample1.svsig.gz pbsv discover ref.movie2.bam ref.sample2.svsig.gz
It is highly recommended to provide one tandem repeat annotation
of your reference to
pbsv discover via
--tandem-repeats. This increases
sensitivity and recall. Feel free to use the following for human SV calling:
Sample names are transferred from the
RG headers to the
3. Call structural variants and assign genotypes
Call structural variants from structural variant signatures, jointly for all
samples of interest. One or more
.svsig.gz files are accepted, including multiple
.svsig.gz for a single sample and/or
svsig.gz for multiple samples.
If the input is CCS reads, please add
--ccs to the following call:
pbsv call ref.fa ref.sample1.svsig.gz ref.sample2.svsig.gz ref.var.vcf
Variant calls for all samples are output in a single
Parallel processing per chromosome
For large genomes with high sequencing coverage, it is recommended to process chromosomes separately. After aligning each movie:
.svsig.gz files per chromosome
for i in $(samtools view -H hg38.movie1.bam | grep '^@SQ' | cut -f2 | cut -d':' -f2); do pbsv discover --region $i hg38.movie1.bam hg38.sample1.$i.svsig.gz done
# -j is number of threads pbsv call -j 8 hg38.fa hg38.sample1.*.svsig.gz hg38.sample1.vcf
Algorithm Overview and Advanced Parameters
Cluster options used during
SV Signature Cluster Options: --cluster-max-length-perc-diff Do not cluster signatures with difference in length > P%.  --cluster-max-ref-pos-diff Do not cluster signatures > N bp apart in reference. 
Number of flanks used for consensus generation:
Consensus Options: -x,--max-consensus-coverage Limit to N reads for variant consensus. 
Split deletions: Deletions that are not fully aligned using the
D cigar are recovered up to a
size of 100kb. Deletions greater than 100kb are currently called as translocations.
Insertion calling workflow is identical to the above described deletion workflow, except for one additional criteria, the inserted sequence similarity check during clustering:
SV Signature Cluster Options: --cluster-min-basepair-perc-id Do not cluster signatures with basepair identity < P%. 
Split insertions: Insertions can be recovered from split split reads, if the
reference overlap of those split reads is less than
and if the query distance is larger than 500 bp.
The explicit upper limit on the insertion size can be adjusted in
but be aware that predicting larger insertions will consume more memory!
--max-ins-length Ignore insertions with length > N bp. ["10K"]
An inversion signature is detected if a single read is split into three
alignments with different orientations / strands, either
Gaps in the reference, as depicted as
A, are not constrained.
The maximum permitted reference overlap
B, between consecutive alignments, is
-k,--max-skip-split Ignore alignment pairs separated by > N bp of a read or reference. ["100"]
Clustering is performed on the inverted segment and uses the same criteria as deletion clustering.
The VCF call marks the most likely position and size of the inverted segment, as shown in this IGV screenshot:
Translocations are identified using breakends of individual split reads with
a query skip of less than
The minimum reads that support a BND (total over all samples), can be defined
All four breakend combinations are supported:
From split reads
Duplications can be identified from the following split-read signatures:
whereas a duplication has to include on fully spanning read to be flagged as PASS; otherwise, it is filtered with NotFullySpanned.
The maximum size can be configures in
--max-dup-length Ignore duplications with length > N bp. ["100K"]
In addition, each insertion is tested against its neighboring reference regions and labeled as a duplication if it matches the reference with 80%. Caution: Activating duplication calling has a negative impact when comparing to GIAB, as GIAB labels everything as insertion or deletion.
Copy number variations
Copy number variations (CNVs) are inferred from breakends. Each breakend pair is tested for significant changes in the read depth signal and for each breakend, the longest non-overlapping CNV is being reported.
The minimum length can be configured in
--min-cnv-length Ignore CNVs with length < N bp. ["1K"]
CNVs that are overlapping with or are encapsulated by a deletion are flagged
Calling and Genotyping
An variant is output if it passes all of the following criteria:
- supported by at least
-A,--call-min-reads-all-samples reads total across samples,
- supported by at least
-B,--call-min-bnd-reads-all-samples reads total across samples for BND variants,
- supported by at least
-O,--call-min-reads-one-sample in a sample,
- supported by at least
-P,--call-min-read-perc-one-sample percent of reads in a sample,
- supported by at least
-S,--call-min-reads-per-strand-all-samples reads per strand total across samples,
- assigned a non-reference genotype in at least one sample;
a sample is assigned a non-reference genotype for a variant if at least
--gt-min-reads reads support the variant.
For CCS input, using the
--ccs mode in
pbsv call, thresholds are relaxed to
-A 1 -B 2 -O 1 -S 0 -P 10.
The VCF filter column is
- NearReferenceGap: variant is near (<
--filter-near-reference-gap [1K]) from a gap (run of >= 50 Ns in the reference assembly)
- Decoy: variant involves a decoy sequence, where the chromosome name contains
- NearContigEnd: variant is near (<
--filter-near-contig-end [1K]) from a contig end
- InsufficientStrandEvidence: variant is not supported by at least (
--call-min-reads-per-strand-all-samples ) reads in forward and reverse orientation
- NotFullySpanned: duplication variant does not have any fully spanning reads
Using the publicly available HG002 15kb CCS dataset,
we are tracking
pbsv performance with respect to the genome in a bottle annotation version 0.6.
Step-by-step reproducibility and benchmarks can be found at github.com/PacificBiosciences/sv-benchmark.
To where do I report bugs and ask questions about the pre-release version of
Please refer to our official pbbioconda page to report bugs and ask questions.
Where can I find an example dataset to try
For Genome in a Bottle sample HG002:
The binary does not work on my linux system!
If you get
Illegal instruction upon execution of
pbsv, then your CPU is not supported.
A modern (post-2008) CPU with support for SSE4.1 instructions is required.
Why do I have to use
--median-filter for CLR data?
I'm not sure how deep your knowledge goes for the PacBio technology. You might know that each ZMW contains a polymerase that sequences one SMRTbell, which consists of your piece of DNA, we call insert, and PacBio's hairpin adapters. This SMRTbell is being processed once the acquisition starts and ends when either the polymerase dies or the acquisition ends. Depending how large your insert and how long the movie time is, it might happen that you read the same molecule multiple times. In this case, a ZMW has multiple subreads (also called CLR), originating from the same insert, from the forward and reverse strands. If we were to align all subreads to the genome and call SVs, it will happen that we call SVs that have support from multiple subreads, but all of them are from the same ZMW. In NGS you would call that PCR duplicates or some would refer to them as technical replicates. One does not want to account evidence more than once per ZMW aka per molecule. The median filter picks one subread per ZMW, to be precise the subread of median length, to have exactly one evidence per molecule. If you don't use it, you will get false positive SV calls and the genotypes will be wrong.
- Public release in SMRT Link 9.0.0
- New CLI UX
- Change svsig compression to bgzip to enable indexing via tabix
- Public release in SMRT Link 8.0.0
--sample, used to override input type
- Public release in SMRT Link 7.0.0
- Add duplications and copy number variations
- Improved sensitivity for larger insertions and deletions
- Simplified parameters and relaxed inversion calling criteria
pbsv discoverspeedup and minimize memory footprint
- Add maximum variant limits for duplications and insertions, the latter improves memory footprint of
- Improve error output if reference and svsig contigs do not match
IMPRECISEto VCF header
- Separate filter with semicolon
- Algorithmic improvements to increase recall and sensitivity across all SV lengths
SACStranded Allel Counts for subread input
pbsv fastaand rely on
MATEDIST, distance between two breakends of a translocation if the two breakends are on the same contig
- Fix VCF POS/REF/ALT and sorting to pass GATK
ValidateVariantsand allow VCF
- Allow multiple sample names and multiple BAM inputs for
- Allow length
- Flag NearContigEnd
- Allow breakend calling from single chromosome
- Allow custom
--annotationsfor known sequence types
- Loop termination fix to properly fix the issue in the 2.0.2 patch
- Decrease memory overhead in
2.0.2: Fix rare
pbsv callabort, because of missing coverage
2.0.0: Drop RC for conda release
2.0.0-RC2: First public release candidate for SMRT Link 6.0.0
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