Whole genome sequencing

A more complete view of the genome to fuel research and discovery

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Whole genome sequencing

Whole genome sequencing (WGS) with PacBio HiFi sequencing delivers a more complete and accurate genome with long, single-molecule reads and 90% of bases at ≥Q30.

Contiguous and phased genome assemblies

The unique combination of length and accuracy enables HiFi reads to resolve difficult repeats, segmental duplications, and centromeres to produce more contiguous and phased assemblies.

Precision and recall for variant calling

HiFi reads also provide precisionFDA-winning accuracy for all variant types, especially in the most challenging parts of the human genome.

Methylation calling

HiFi sequencing also provides simultaneous 5mC detection in CpG contexts without any additional library preparation. This feature enables the resolution of methylation profiles with phased haplotyping and the ability to interrogate methylation abnormalities from a single sequencing experiment.

Whole genome sequencing — how PacBio compares

PacBio HiFi Illumina Oxford Nanopore
Average read length1
15–20 kb
2 x 150 bp 10–100 kb
Average read accuracy1
99.95% (Q33) 99.92% (Q31) 99.26% (Q21)
Unbiased Reduced at low and high [GC] Reduced in low-complexity runs
Variant calling: SNVs
Variant calling: indels
Variant calling: SVs
Genome assembly: contiguity
Genome assembly: accuracy
Epigenetics: 5mC

1. PacBio HiFi: HG003 18 kb library, Sequel II system chemistry 2.0, precisionFDA Truth Challenge V2 (, Illumina: HG002 2×150 bp NovaSeq library, precisionFDA Truth Challenge V2 (, ONT: Q20+ chemistry (R10.4, Kit 12), Oct 2021 GM24385 Q20+ Simplex Dataset Release (
2. HiFi+ONT: Nurk 2021, HiFi+Illumina: Logsdon 2020, ONT: Tan 2022

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Human whole genome sequencing

Completely characterize human genomes to better understand the complexity of health and disease.

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Plant + animal whole genome sequencing

Plant + animal whole genome sequencing

Quickly and affordably produce contiguous, complete, and correct de novo assemblies of even the most complex genomes.

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Microbial whole genome sequencing

Achieve closed chromosomes and plasmids from even the most repeat-dense and GC-rich genomes.

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PacBio Compatible products

Maximize throughput by automating library prep with PacBio Compatible automation partners

PacBio Compatible   Qualified automation protocols   Microlab Prep Scripts

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PacBio qualified automation protocols for WGS library prep and polymerase binding

PacBio has partnered with NGS automation vendors to provide scalable automated library prep methods to meet your lab’s throughput needs. From single samples on the Integra Miro Canvas, to 96 samples on the Hamilton NGS STAR or STAR V systems. Each platform protocol has been qualified through the PacBio Compatible program to ensure quality and great sequencing performance. 

Automated protocols:  

Guide and overview – Automated HiFi prep 96 and HiFi ABC for the Hamilton NGS STAR MOA system 

Guide and overview – Automated HiFi plex prep 96 for the Hamilton NGS STAR MOA system 


Hamilton Integra Revvity Tecan
Pipette tip DNA shearing for the WGS protocol
HiFi sequencing 15 to 20 kb

Microlab Prep

WGS library prep protocol
SMRTbell prep kit 3.0 Miro Canvas SciClone NGSx DreamPrep Compact
HiFi prep kit 96
HiFi plex prep kit 96
Anneal, binding, and clean (ABC) protocol for polymerase binding
Seque II binding kit 3.2 DreamPrep Compact
Revio polymerase kit DreamPrep Compact
Revio polymerase kit 96

New automation protocols will be added as they become available via our PacBio Compatible partners. Please use our automation workflow selector tool to determine which of our PacBio Compatible automation solutions are best for your application.


In PrecisionFDA Challenge, PacBio Hifi reads outperform both short reads and noisy long reads

PacBio HiFi reads delivered the highest precision and recall in all categories: genome-wide, specifically in difficult-to-map regions, and in the major histocompatibility complex.

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Whole genome sequencing datasets

Application Dataset Download literature Technology Sequencing system
Variant detection, assembly, epigenetics Homo sapiens — GIAB trio HG002-4 Application note HiFi long read Revio system
Tumor/normal COLO829 melanoma Application note HiFi long read Revio system
Tumor/normal HCC1395 Application note HiFi long read Revio system
Whole genome sequencing Various plant & animals – mouse, ladybug, oak, mistletoe, and maize Plant + animal biology HiFi long read Revio system
Assembly Food safety & infectious microbes – 96 plex Microbial WGS HiFi long read Sequel IIe system

More datasets

Whole genome sequencing applications

Variant detection

From SNVs to structural rearrangements, highly accurate single-molecule long reads allow you to confidently catalog variants of all kinds.

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De novo sequencing

With HiFi reads up to 25 kb, you can efficiently and affordably generate de novo assemblies of even the most complex genomes.

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Genome structure

HiFi data delivers insights into genome structure that short-read technologies often miss. Generate long reads, even through highly repetitive regions of the genome.

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HiFi sequencing equips you to capture epigenetic modifications during a normal sequencing run — no special chemistry or preparation required.

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PacBio library prep workflow for long-read WGS

Library preparation for WGS is a simple 5-step process that can be completed in a single day.  

Step 1: HMW DNA is mechanical sheared to average size between 15 to 20 kb, with fragments ranging from 5 to 35 kb.  

Steps 2 -4: Sheared DNA from step 1 goes through a round of DNA repair and A-tailing to prepare the fragments for SMRTbell adapter ligation. After ligation, any un-ligated, semi-ligated, or damaged DNA will be removed in a nuclease treatment. All that remains should be DNA with the hairpin SMRTbell adapters on both ends.  

Step 5: SMRTbell libraries are cleaned up and short fragments depleted using a 3.1X mix of diluted AMPure PB beads.  




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Best practice recommendations for preparing whole genome sequencing libraries

Low throughput: SMRTbell prep kit 3.0 

Mid or high throughput: HiFi prep kit 96 

Human, plant, and animal WGS (for microbial WGS see the HiFi plex prep kit 96 protocol) 

Low throughput Mid or high throughput
Samples prep at a time

≤ 24

≥ 24

DNA quality 70% of DNA mass > 10kb1 70% of DNA mass > 10kb1
DNA input

≥ 2 µg2

≥ 2 µg2

Method Pipette shearing3 Pipette shearing3
Shearing Equipment4 Hamilton Microlab Prep

Hamilton Microlab Prep


Mean insert size 15-20 kb 15-20 kb
Library prep Kit SMRTbell prep kit 3.0 HiFi prep kit 96
Size selection gDNA SRE5 SRE HT5
Library 35% AMPure PB beads 35% AMPure PB beads
ABC Kit Revio polymerase kit Revio polymerase kit 96
Automated protocols available Yes Yes
Methylation: 5mC in CpG Detected Detected
Mean HiFi base yield

≥ 90 µg6

≥ 90 µg

Prep time 6 hours7 13 hours for up to 96 samples8

1This corresponds to a genome quality number (GQN) of 7.0 or higher measured at 10 kb using the Agilent Femto Pulse system.  

2 This is the recommended amount of genomic DNA per Revio SMRT Cell. Lower quality samples may require higher input amounts. Please see protocol details.  

3 This refers to the new method of using automated liquid handlers to mix dilute DNA at high speeds to mechanical shear fragments to an average size between 15 to 20 kb.  

4 Current qualified instruments; please check with your local PacBio FAS for the latest information as we work to expand the number of qualified instruments 

5Short read eliminator is used only on genomic DNA, prior to shearing and library prep.   

6 Average HiFi base yield when using a mean insert size of 15 kb or larger and 24-hour sequencing time.  

7 Manual preparation time when using SRE. Automated time is similar. Time does not include optional QC checkpoints as that will vary by lab equipment used.  

8 Automated prep time for up to 96 samples is 13 hours, including time preparing masters mixes and setting up the runs. Time includes ABC workflow but does not include the time for QC which will vary by user

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New low cost, high-throughput mechanical DNA shearing options for long-read sequencing workflows

Shearing your genomic DNA has never been easier. PacBio has developed new high throughput methods that are both cost-effective and fast to reduce bottlenecks and maximize your HiFi sequencing yields.

Lab Equipment Method Throughput per run Time per run Mode size ranges Consumable cost
Standard Megaruptor 3 Hydropore 8-12 samples 30-40 mins. 7-25 kb $$$
Standard Covaris g-TUBE Centrifugation 1-24 samples 10-30 mins. 6-20 kb $$$
New Hamilton Microlab Prep High-speed pipette tip mixing 8-24 samples 22 mins. for 24 samples

15-22 kb1

New Hamilton NGS STAR/STARlet/STAR V High-speed pipette tip mixing 8-96 samples 8 mins. for 96 samples with MPH

15-22 kb1

New MP Biomedical FastPrep 96 Grinder and lysis system 1-192 samples

1 to 8 mins.2

7-20 kb3

New SPEX MiniG Grinder and lysis system 1-96 samples

1 to 8 mins.2

7-20 kb3

New Tecan DreamPrep NGS and DreamPrep NGS Compact High-speed pipette tip mixing 8-96 samples FCA: 9 min per column MCA: 6 min for 96 samples

15-22 kb4


  1.    Mode shear size will be sample concentration dependent; the high-speed pipette tip mixing cannot shear down to sizes below 15 on average and therefore is only recommended for the WGS application for human, plant, and animal samples.
  2. Time will vary based on the desired shear size distribution. Longer times for smaller
  3. Mode shear size will depend on rpm and time used. Please see application and tech notes for additional guidance.
  4. Costs are estimates based on list prices. Please check with manufacturer for latest pricing or quote. 

Whole genome sequencing in action

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Scaling human whole genome sequencing for rare and inherited disease research

Find out how PacBio HiFi whole genome sequencing is being used in rare disease research.

Read more

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Bacterial genomes from the lakes of Minnesota to NYC hospitals

See how scientists are studying the ecophysiology of Minnesota microbes using long-read…


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Uncovering structural variants associated with rare disease

Discover how Alexander Hoischen’s research group is using long-read sequencing to find…


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Did you know we have a comprehensive library of articles, reports, papers, and videos related to whole genome sequencing?

Explore resources

Whole genome sequencing workflow at a glance

Easy-to-use, high throughput sequencing for the accuracy you need at an affordable cost

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Straightforward library prep

Go from sample to sequencer in a single afternoon

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Complete + accurate sequencing

Obtain accurate, unbiased, and long reads

Directly detect epigenetic modifications

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Universal file types

Analyze data using any of the common genome assembly and analysis tools

Common questions about PacBio whole genome sequencing

HiFi reads provide the most comprehensive view of human genomes with the highest precision and recall for all variant types including variants missed by short-read sequencing: small variants in difficult-to-map regions of the genome and structural variants genome-wide. HiFi reads also phase variants into maternal and paternal haplotypes.

Yes, HiFi sequencing is able to simultaneously determine the identity and methylation status of individual bases in a DNA molecule. The methylation calling relies on kinetic properties (pulse width and interpulse duration) of the sequencing polymerase. A statistical model processes the kinetic information to infer methylation status.

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If you have a question, need to check the status of an order, or are interested in purchasing an instrument, we're here to help.