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Application note

Consolidated analysis
with the PacBio WGS
Variant Pipeline
Introduction
The increased throughput of PacBio® HiFi sequencers
improves the cost and operational efficiency for humanwhole genome sequencing. This resulting growth indata yield has enabled breakthrough discoveries inhuman and population genomics yet underlines theneed for the available analysis tools to pace with data
generation.

This Application note introduces the PacBio WGS
Variant Pipeline that consolidates several state-of-theart analysis tools into a single user-friendly workflow.
This piece also serves as a best practice guide forstreamlining the analysis of HiFi data to generatecompelling biological insight.

© 2023 PacBio. All rights reserved. Research use only. Not for use in diagnostic procedures.
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The WGS Variant Pipeline is powered by the Workflow
Description Language (WDL), a programming language
for genome analysis workflows created by the Broad
Institute and maintained by the OpenWDL community.
WDL defines the structure of a data processing
workflow with a human-readable and writable syntax.
Built from this widely adopted language, the WGS
Variant Pipeline also offers the flexibility to use at thecommand line or through platforms provided by PacBio
Compatible partners so that users with different levelsof bioinformatics experience may access the pipeline.

small and structural variants. Finally, annotation tools
are included for small and structural variants (Table 1).
Analysis

Tool

Description

Alignment

pbmm2

PacBio compatible minimap2
wrapper for alignment toreference

DeepVariant

SNV and small indel caller

HiFiCNVCNV caller

pbsv

Structural variant caller

Paraphase

Variant caller for genes withinsegmental duplications

TRGT

Tandem repeat genotyper

HiPhaseRead phasing for smallvariants from DeepVariant andstructural variants from pbsv

pb-CpG-tools

Summarize site probabilities
for 5mCpG methylation

GLnexus

Joint calling for small variants

pbsv

Joint calling for SVs

WGS Variant Pipeline
ALIGNMENT

Variantcalling

pbmm2
DeepVariant
pbsv

VARIANTCALLING

TRGT
Methylation
probability

Paraphase
HiFiCNV

Cohortanalysis

HiPhaseMETHYLATIONPROBABILITYCOHORTANALYSIS
ANNOTATION

pb-CpG-tools

slivarAnnotation

GLnexussvpackslivar

Developed byPacBio

svpackThird-party tools

Figure 1. Overview of the WGS Variant Pipeline for secondary
and tertiary sequencing analysis.

The WGS Variant Pipeline consolidates secondary andtertiary analysis tools spanning from alignment toannotation (Figure 1) developed by PacBio and thirdparty groups.
This pipeline packages tools for HiFi alignment,
comprehensive variant callers for single nucleotide
variants (SNVs), small insertions and deletions, copy
number variants (CNVs), and structural variants (SVs),
tandem repeat genotyping, typing of genes withinsegmental duplications, phasing of small variants andlarge insertions and deletions, and summarization of 5methylcytosine-pG (5mCpG) methylation probabilities.
For multi-sample cohorts, joint calling is included forTable 1. Analysis tools available in the WGS Variant Pipeline

Workflow setup and execution
For users with all levels of bioinformatics experience,
this workflow can be executed through compatible
software platforms, including those provided byDNAnexus, FormBio, Terra, and DNAstack. Theseplatforms have been vetted by PacBio and allow users
to run bundled workflows in a managed system. This
option provides additional support for compliance andsecurity without the use of internal resources needed
for compute requirements.
For bioinformaticians who can negotiate command line
tool installations and hardware configuration, thepipeline can also be installed from scratch onto one’s
preferred compute system. The pipeline setup requires
configuration of execution engines and input according
to the selected backend environment (Table 2).

© 2023 PacBio. All rights reserved. Research use only. Not for use in diagnostic procedures.
102-326-588 REV01 OCT2023Annotation tool for small
variants
Annotation tool for structuralvariants

Page 3Choose abackend

Configure aworkflow
executionengine•

Pre-configuration templates are available
for the following environments: HPCS, AWS,Azure, and GCP.

•Cromwell is supported on HPC, AWS, Azure,and GCP.
Miniwdl is supported on HPC and AWS.

••

Clone therepo andconfigure
input

•

•

•
Run analysis•

The workflow can be run on Azure, AWS, GCP, andHPC. The choice of backend is largely determined bydata location. The open-source workflow enginesCromwell and miniwdl are supported for execution.
Following engine configuration, the repo is cloned tothe chosen environment and input template files areconfigured. Backend-specific documentation andtemplates are available at the WGS Variant Pipeline
GitHub. The workflow can be directly run on themachine from which it was deployed (see Figure 2 forsample run summary).

The PacBio WGS Variant Pipeline is publicly
available on PacBio GitHub. Users can clone
the repo to their correspondingenvironment.
Reference data used in the analysis isavailable for download. A copy is alsoavailable at each supported cloudenvironment.
An input JSON template file is present in theworkflow repo. The template should be
updated for any customization and point to
the input data.

Conclusion
The consolidation of these analysis tools into the WGS
Variant Pipeline streamlines the analysis of HiFi
sequencing data into a single accessible, scalable
workflow. This simplified process enables users tofocus on biological insight and discovery through theinterpretation of human whole genome datasets withstate-of-the-art tools.

The workflow can be directly run on themachine from which the workflow has been
deployed.
Depending on the configuration, workflows
may also be submitted via schedulers orother methods.

Table 2. WGS Variant Pipeline workflow setup.
Sample 1A)

Sample 2
Sample 3
Cohort analysis
Tertiary analysis

024B)

86

10

12

pbmm2

14

16

18

20

22hours

deepvariant

pbsv
HiPhaseTRGT
HiFiCNVPb-CpG-tools
Paraphase

02

4

6

8

10

12

14

16

18hours

3. Run timeforsummary
from HG002/HG003/HG004
The analysis is done on atriolargecommercial
HPC was performed on aFigure 2. RunFigure
time summary
WGS Variant
Pipeline performedTrio.
on HG002/HG003/HG004
data.
This analysis
infrastructure
withoutqueuingtime.
large commercial HPC infrastructure without queuing time. A) Overall summary for the trio analysis. B) Run time for each major
Figure 3a is an overall summary for the trio analysis
component inFigure
a per-sample
analysis.
3b describes
run time for each major component in a per-sample analysis© 2023 PacBio. All rights reserved. Research use only. Not for use in diagnostic procedures.
102-326-588 REV01 OCT2023

Page 4Resources
WGS Variant Pipeline GitHub
Open WDL community
Reference datasets

Partner information
PacBio Compatible program
FormBio
DNAnexus
Terra
DNAstack

Technical resources
CromwellMiniwdl
DeepVariant
GLnexusslivarFor additional information on computational tools andworkflows for HiFi applications visit the PacBio
Computational tools webpage.
Note:

Command line tools are considered indevelopment, can be updated frequently, andare not supported as part of the PacBio
software product suite.

Research use only. Not for use in diagnostic procedures. © 2023 Pacific Biosciences of California, Inc. (“PacBio”). All rights reserved. Informationin this document is subject to change without notice. PacBio assumes no responsibility for any errors or omissions in this document. Certain
notices, terms, conditions and/or use restrictions may pertain to your use of PacBio products and/or third-party products. Refer to the applicable
PacBio terms and conditions of sale and to the applicable license terms at pacb.com/license. Pacific Biosciences, the PacBio logo, PacBio,Circulomics, Omniome, SMRT, SMRTbell, Iso-Seq, Sequel, Nanobind, SBB, Revio, and Onso are trademarks of PacBio.

© 2023 PacBio. All rights reserved. Research use only. Not for use in diagnostic procedures.
102-326-588 REV01 OCT2023