HiFi Sequencing and Software v10.1 Release Technical Overview for Sequel II System and Sequel IIe System Users

HiFi Sequencing and Software v10.1 Release:

Technical Overview for Sequel II System & Sequel IIe System Users

Sequel II and IIe Systems ICS v10.1 / SMRT Link v10.1

HiFi Sequencing and Software v10.1 Release:

Technical Overview for Sequel II & IIe Systems Users

A. Sequel II and IIe Systems v10.1 Release Overview

Summary Overview of Key Features & Improvements

Sequel II and IIe System Instrument Control Software v10.1 Updates

Sequel II and IIe System Consumables and Sample Preparation Workflow Updates

SMRT Link Sample Setup, Run Design & Run QC Updates

Example HiFi Library Sequencing Performance Data

B. SMRT Link v10.1 Release Overview

Summary Overview of Key Features & Improvements

CCS Analysis Application Features & Reports

SMRT Link v10.1 Cloud

SMRT Link Applications Updates

SMRT Link General Usability Improvements

SMRT Link Fixed & Known Issues

C. Sequel II and IIe Systems Applications Support Resources

HIFI SEQUENCING AND SOFTWARE V10.1 RELEASE:

KEY FEATURES & IMPROVEMENTS

▪ New consumables enable improved

HiFi data quality

▪ Updated HiFi sample prep protocol for

WGS applications enables reduced

DNA input requirements and

higher sample throughput / yr

▪ Updated Sequel II and IIe System

Instrument Control Software v10.1 enables

on-instrument sequencing

workflow improvements

▪ Updated SMRT Link v10.1 software

features new analysis applications

and improves Sample Setup & Run

Design ease of use

New Consumables

- SMRTbell Enzyme Clean Up Kit 2.0 (101-932-600) (NEW)

- Sequencing Primer v5 (102-067-400) (NEW)

- Polymerase Binding Kit 2.2 (101-894-200) (NEW)

Updated HiFi Sample Prep Protocol for De Novo Assembly

and Variant Detection

- Enables reduced minimum input gDNA (≥5 µg) for running multiple

SMRT Cells

- Supports high-throughput sample processing and automation

Sequel II and IIe System Instrument Control Software v10.1

- Updated on-instrument robotic workflow for improved fluidic handling

- Supports identification of new barcoded overhang adapters

- Supports new Adaptive Loading (AL) feature

SMRT Link v10.1

- Support for new consumables

- Application-specific Sample Setup and Run Design*

- New Adaptive Loading feature in Run Design for WGS applications

- New SARS-CoV-2 analysis application for COVID-19 surveillance

- Usability and user experience improvements

HIFI SEQUENCING AND SOFTWARE V10.1 RELEASE:

KEY FEATURES & IMPROVEMENTS

▪ New consumables enable improved

HiFi data quality

▪ Updated HiFi sample prep protocol for

WGS applications enables reduced

DNA input requirements and

higher sample throughput / yr

▪ Updated Sequel II and IIe System

Instrument Control Software v10.1 enables

on-instrument sequencing

workflow improvements

▪ Updated SMRT Link v10.1 software

features new analysis applications

and improves Sample Setup & Run

Design ease of use

* Feature first introduced with SMRT Link v10.0 limited release

Sequel II and IIe Systems v10.1 Release Overview

Subhead should be no longer than 1 line

SEQUEL II AND IIe SYSTEMS V10.1 RELEASE:

SUMMARY OVERVIEW OF KEY FEATURES & IMPROVEMENTS

▪ New consumables enable improved

HiFi data quality

▪ Updated HiFi sample prep protocol for

WGS applications enables reduced

DNA input requirements and

higher sample throughput / yr

▪ Updated Sequel II and IIe System

Instrument Control Software v10.1 enables

on-instrument sequencing

workflow improvements

Updated HiFi Sample Prep Protocol for De Novo Assembly

and Variant Detection

- Enables reduced minimum input gDNA (≥5 µg) for running multiple

SMRT Cells

- Supports high-throughput sample processing and automation

Sequel II and IIe System Instrument Control Software v10.1

- Updated on-instrument robotic workflow for improved fluidic handling

- Identification of new barcoded overhang adapters

- Supports new Adaptive Loading (AL) feature

New Consumables

- SMRTbell Enzyme Clean Up Kit 2.0 (101-932-600) (NEW)

- Sequencing Primer v5 (102-067-400) (NEW)

- Polymerase Binding Kit 2.2 (101-894-200) (NEW)

Sequel II and IIe System Instrument Control

Software v10.1 Updates

SEQUEL II AND IIE SYSTEM INSTRUMENT CONTROL SOFTWARE

V10.1 UPDATES

Updated Sequel II and IIe System ICS v10.1 enables several sequencing

workflow improvements

- Updated on-instrument robotic workflow enables

improved fluidic handling during reagent/sample

aspiration & dispense steps to minimize the impact of

drying in low-humidity environments and results in

more uniform loading across the SMRT Cell 8M

surface area

- Enables new Adaptive Loading (AL) feature to

monitor kinetics of immobilization of polymerase

complexes to ZWWs leading to reduced loading

variability and reduced risk of sample overloading

conditions

- Enables support for new barcoded overhang adapters

leading to more streamlined analysis of multiplexed

samples. (Note: New redesigned barcoded overhang

adapter sequences will become available in a future

protocol release.)

SEQUEL II AND IIE SYSTEM INSTRUMENT CONTROL SOFTWARE

V10.1 UPDATES (CONT.)

Updated Sequel II and IIe System ICS v10.1 enables several sequencing

workflow improvements

- Enables improved environmental systems control

leading to increased instrument reliability

▪ Note: Reagent chiller no longer cools the work deck

when the instrument is idle and no sequencing kit is

detected by the NFC reader

▪ NFC reader will scan for the presence of unused reagents

in the sequencing plate upon instrument power up, upon

door closure, and upon end of a sequencing run

▪ A sample plate should not be loaded on the work deck

without a sequencing plate present since there is no NFC

tag on sample plates.

Improved environmental

systems control helps

extend the service life

of the reagent chiller.

Sequel II and IIe System Consumables and

Sample Preparation Workflow Updates

NEW SEQUEL II AND IIE SYSTEM REAGENT KIT PRODUCT

DESCRIPTIONS

SMRTbell Enzyme Clean Up Kit 2.0 (101-932-600)

- Improved formulation enables more efficient removal of damaged/incomplete

SMRTbell template constructs from final library samples

- For use with HiFi WGS de novo assembly and variant detection applications

Sequencing Primer v5 (102-067-400)

- Sequencing Primer v5 is recommended for use with Sequel II Binding Kit 2.2

and enables more processive sequencing of SMRTbell DNA templates leading

to improved HiFi data quality

Sequel II Binding Kit 2.2 and DNA Internal Control 1.0 (102-089-000)

- Faster Sequel II Polymerase 2.2 resulting in more subread passes and

improved HiFi data quality

- No change to spike-in DNA Internal Control

Reagent Kit or Component Part Number Quantity

No. of Reactions

Supported

SMRTbell Enzyme Clean Up Kit 2.0 (NEW) 101-932-600 18

SMRTbell Enzyme Clean Up Mix 1 Tube

SMRTbell Enzyme Clean Up Buffer 1 Tube

Sequencing Primer v5 (NEW) 102-067-400 10

Sequencing Primer v5 1 Tube

Sequel II Binding Kit 2.2 and Internal Control 1.0 (NEW) 102-089-000 24

Sequel II DNA Polymerase 2.2 1 Tube

Adaptive loading Buffer 6 Tubes

Sequel Binding Buffer 2 Tubes

Sequel dNTP 1 Tube

Sequel Complex Dilution Buffer 2 Tubes

Nuclease-Free Water 1 Tube

DNA Internal Control 1.0 1 Tube

NEW SEQUEL II AND IIE SYSTEM CONSUMABLES PRODUCTS

AND REAGENT KIT CONTENTS

Reagent Kit or Component Part Number Quantity

No. of Reactions

Supported

HiFi Express Template Prep Kit 2.0 Bundle (UPDATED) 102-088-900 18

SMRTbell Express Template Prep Kit 2.0 1 Each

SMRTbell Enzyme Clean Up Kit 2.0 (NEW) 1 Each

Sequencing Primer v5 (NEW) 1 Tube

Sequel II HiFi Bundle-18 2.0 (UPDATED) 102-104-700 18

SMRTbell Express Template Prep Kit 2.0 1 Each

SMRTbell Enzyme Clean Up Kit 2.0 (NEW) 1 Each

Sequencing Primer v5 (NEW) 1 Each

Sequel II Binding Kit 2.2 and Internal Control 1.0 (NEW) 1 Each

AMPure PB, 5 mLs 1 Tube

NEW SEQUEL II AND IIE SYSTEM CONSUMABLES PRODUCTS

AND REAGENT KIT CONTENTS

UPDATED HIFI LIBRARY PREPARATION PROTOCOL FOR WGS

DE NOVO ASSEMBLY & VARIANT DETECTION APPLICATIONS

- Procedure & Checklist – Preparing HiFi SMRTbell Libraries using SMRTbell Express

Template Prep Kit 2.0 (PN 101-853-100) protocol document has been updated and

describes a method for constructing SMRTbell libraries (~15 kb - 20 kb) that are suitable for

generating highly accurate long reads on the Sequel II and IIe Systems for WGS de novo

assembly and variant detection applications

- Updated workflow supports high-throughput processing using reduced input genomic DNA

amounts (5 µg per 3 Gb sample genome size)

- Recommend shearing high-quality gDNA using a Megaruptor 3 System (Diagenode)

- Depending on project requirements, SMRTbell libraries can be size-selected using a

PippinHT System (Sage Science), SageELF System (Sage Science), or BluePippin System

(Sage Science)

https://www.pacb.com/support/documentation/

APPLICATIONS

WHOLE GENOME SEQUENCING

De Novo Assembly

Variant Detection

HIFI LIBRARY PREPARATION & SEQUENCING WORKFLOW

IS EFFICIENT AND SCALABLE

Updated HiFi sample preparation workflow provides improved SMRTbell library construction yields and

supports high-throughput processing using reduced input genomic DNA amounts

Genomic DNA QC & Shearing

▪ ≥5 µg of input gDNA for a 3 Gb sample genome size

▪ Shear up to 8 samples in parallel to ~15 kb - 20 kb

target fragment size with the Megaruptor 3 System

SMRTbell Library Construction (5 hrs)

▪ Process up to 16 samples in parallel manually or up

to 96 samples in parallel using automation

▪ Manual workflow supports construction of ≥32 HiFi

libraries per week

SMRTbell Library Size Selection (2 hrs)

▪ Process up to 20 samples in parallel with the

PippinHT System (2 hour run time)

▪ Higher recovery efficiency with PippinHT provides

sufficient SMRTbell library material to run up to ~4 or

more SMRT Cells 8M per 5 µg of starting input gDNA

Sequencing Preparation

(Sequel II and IIe Systems)

▪ Anneal Sequencing Primer v5 (1 hr), bind Sequel

II Polymerase 2.2 (1 hr) and perform complex

cleanup using AMPure PB beads

HiFi Data Analysis

▪ For variant detection,

can use DeepVariant for

small variants <20 bp

and SMRT Link

Structural Variant Calling

for larger variants >20 bp

▪ For de novo assembly,

can use SMRT Link

Genome Assembly or

other third-party software

PacBio HiFi

reads achieve

>99.9% accuracy

HiFi Read

STEP HANDS-ON (MIN) WALK-AWAY (HRS)

Remove SS to A-Tailing 15 1.4

Adapter Ligation** 5 1.0

AMPure PB Bead Purification 5 0.5

Nuclease Treatment 5 0.5

AMPure PB Bead Purification 5 0.5

Size

selection (PippinHT System)

10 2.0

AMPure PB Bead Purification 5 0.5

Total 50 6.4

PROCESS UP TO 16 SAMPLES IN PARALLEL

MANUALLY USING 0.2-ML TUBE STRIPS*

PROCESS UP TO 96 SAMPLES IN

PARALLEL USING AUTOMATION*

HIFI LIBRARY CONSTRUCTION WORKFLOW TIMING OVERVIEW

DNA Shearing

DNA Damage Repair

DNA End Repair / A-Tailing

Adapter Ligation

1X AMPure PB Bead Purification

Remove Single-Strand Overhangs

1 X AMPure PB Bead Purification

Nuclease Treatment

Size Selection

1 X AMPure PB Bead Purification

Sequencing Preparation

Enzymatic Reactions ~5 hours

Optional Stop Point

** Adapter Ligation reaction can be performed for 1 hour or left incubating overnight

High-throughput library construction and sequencing preparation in 2 days

Day 1

Day 2

* Reagents can be prepared as Master Mixes

Optional Stop Point

THE MEGARUPTOR 3 SYSTEM IS RECOMMENDED FOR HIFI SMRTBELL

LIBRARY CONSTRUCTION

- Megaruptor 3 System (Diagenode) is generally recommended for shearing*

- Allows up to 8 samples to be sheared in parallel for high-throughput applications

- Achieving the same size distribution across multiple samples provides more consistent sequencing

performance

- To maximize HiFi yield per SMRT Cell, PacBio recommends fragmenting the gDNA to a size distribution mode

between 15 kb – 18 kb for human whole genome sequencing

- Note: Libraries with a size distribution mode larger than 20 kb are not recommended for HiFi sequencing.

- Recommended library insert size distributions to use for different WGS applications are summarized below and in

Table 4 in the procedure.

Megaruptor 3 System

Application Recommended Library Insert Size (Mode)

Human Variant Detection 15 kb - 18 kb

Human De Novo Assembly 15 kb - 18 kb

Plant / Animal De Novo Assembly 15 kb - 20 kb

* Note: The g-TUBE (Covaris) device generates a broader DNA fragment size-distribution compared to the Megaruptor 3 system. As a result, HiFi read quality and overall HiFi data yield

may be reduced due to the residual presence of very large DNA fragments generated by g-TUBEs. For additional guidance, see Technical Overview: HiFi Library Preparation Using

SMRTbell Express TPK 2.0 for De Novo Assembly and Variant Detection (PN 101-855-400) or contact PacBio Technical Support or your local Field Applications Scientist.

To use the Megaruptor 3 System, perform two cycles of DNA shearing in the same hydropore-syringe device

- Eliminates very large DNA fragments that may not generate sequencing data that meet HiFi read quality requirements

- Example recommended Megaruptor 3 System software settings to achieve a DNA fragment size distribution mode of ~15 kb - 18 kb

(recommended for human whole genome sequencing applications):

- IMPORTANT: Genomic DNA must be in QIAGEN Buffer EB or PacBio Elution Buffer (EB) or an equivalent low-salt buffer (i.e.,

10 mM Tris-Cl, pH 8.5 - 9.0) for shearing

- To minimize sample loss,** the recovered volume (~53 𝜇L) of sheared DNA is used to go directly into the first enzymatic reaction in

SMRTbell library construction (i.e., no intermediate AMPure PB bead purification step is performed)

gDNA TARGET

SHEAR SIZE MODE

MEGARUPTOR 3 SYSTEM

SHEARING CYCLE

MEGARUPTOR 3 SYSTEM

SPEED SETTING*

RUN TIME

PER SHEARING CYCLE

15 – 18 kb

Cycle 1 Speed Setting 31 40 min

Cycle 2 Speed Setting 32 40 min

THE MEGARUPTOR 3 SYSTEM IS RECOMMENDED FOR HIFI SMRTBELL

LIBRARY CONSTRUCTION (CONT.)

* Note: The shearing instructions described in this HiFi sample prep procedure are not compatible with the Megaruptor or Megaruptor 2 systems from Diagenode. If using a Megaruptor or

Megaruptor 2 system, shearing optimization is necessary before proceeding with this procedure. For additional guidance, see Technical Overview: HiFi Library Preparation Using

SMRTbell Express TPK 2.0 for De Novo Assembly and Variant Detection (PN 101-855-400) or contact PacBio Technical Support or your local Field Applications Scientist.

** Losses are mostly due to dead volume in the Megaruptor 3 System [i.e., 5 – 7 𝜇L (<500 ng)]

Because the response of individual gDNA samples may differ, optimization of shearing

conditions is recommended to achieve the desired fragment distribution

2-CYCLE SHEARING METHOD USING THE MEGARUPTOR 3 SYSTEM IS

RECOMMENDED FOR HIFI SMRTBELL LIBRARY CONSTRUCTION

Femto Pulse DNA sizing QC analysis overlay of 15 human gDNA

samples sheared with 2 cycles of shearing using a Megaruptor 3

System. The size distribution mode of the samples after 2 cycles of

shearing is ~18 kb.

Femto Pulse DNA sizing QC analyses of the same human gDNA

sample sheared with 1 cycle of shearing versus 2 cycles of

shearing using a Megaruptor 3 System. The fragment size

distribution is tighter after performing 2 cycles of shearing compared to

performing 1 cycle of shearing.

1-Cycle Shear

(Speed 32)

2-Cycle Shear

(Speeds 31, 32)

By performing a 2-cycle shear, the resulting DNA fragment size distribution is tighter and more

consistent across multiple samples

2-Cycle Shear

(Speeds 31, 32)

SIZE-SELECTION WITH THE PippinHT SYSTEM IS RECOMMENDED FOR

HIFI SMRTBELL LIBRARY CONSTRUCTION

- PippinHT System (Sage Science) is recommended for size selection

- PippinHT System enables faster run times and higher throughput compared to the

SageELF and BluePippin Systems

- Can process up to 20 samples per instrument run

- 2-hour run time

- SMRTbell templates <10 kb are removed during PippinHT size selection

- PippinHT Cassette Definition File and Run Protocol Setup

- “6-10kb High Pass Marker 75E”

- Using the “Range” selection mode, enter a desired “Start” value of 10000 and a

“End” value of 50000.

- Be sure to assign a marker lane

- PippinHT System shows efficient post-size selection recovery yields (approx. ≥35%

– 50%), which enables reduction of the input gDNA amount required for HiFi

SMRTbell library construction*

* Note: If using BluePippin or SageELF size-selection, library recovery yields may be lower with this HiFi sample prep procedure.

For additional guidance, see Technical Overview: HiFi Library Preparation Using SMRTbell Express TPK 2.0 for De Novo

Assembly and Variant Detection (PN 101-855-400) or contact PacBio Technical Support or your local Field Applications

Scientist.

PippinHT System

With high-quality samples,

higher recovery efficiency with

PippinHT size selection can

provide sufficient SMRTbell

library material to run up to ~4

or more SMRT Cells 8M per

5 µg of starting input gDNA

SIZE-SELECTION WITH THE PippinHT SYSTEM IS RECOMMENDED FOR

HIFI SMRTBELL LIBRARY CONSTRUCTION (CONT.)

16.3 kb Mode

18.6 kb Mean

16.2 kb Mode

18.0 kb Mean

16.0 kb Mode

18.0 kb Mean

15.0 kb Mode

16.0 kb Mean

Before PippinHT Size Selection After PippinHT Size Selection

PippinHT size selection is fast (2-hr run time) and efficient (can process up to 20 samples per instrument run)

Femto Pulse DNA sizing QC analyses of the several human

SMRTbell library samples after PippinHT size selection using a 10-

kb lower cutoff setting. The size distribution mode of the size-selected

samples is similar (~18 kb).

Femto Pulse DNA sizing QC analyses of the several human

SMRTbell library samples before size selection. The size distribution

mode ranges from ~7 kb to ~18 kb for the different samples.

- Enables greater reproducibility compared with manual-only

methods

- Can process up to 96 samples at a time

HIFI SAMPLE PREPARATION WORKFLOW IS AMENABLE TO AUTOMATION

HiFi SMRTbell library construction can be automated using the Sciclone Liquid Handling

Workstation (Perkin-Elmer)

https://www.perkinelmer.com/category/sciclone-g3-liquid-handling-workstations

Input Requirements:

- Recommend using >6 𝜇g of input sheared gDNA due to liquid dead

volumes

Automated Workflow Steps:

- Enzymatic reactions

- AMPure PB bead purifications

Output:

- SMRTbell libraries ready for downstream size-selection

COMPARISON OF HIFI SAMPLE PREPARATION WORKFLOW CHANGES FOR

DE NOVO ASSEMBLY AND VARIANT DETECTION APPLICATIONS

SAMPLE PREP

WORKFLOW STEP

OLD HIFI PROCEDURE & CHECKLIST

(VERSION 3, JAN. 2020)

NEW HIFI PROCEDURE & CHECKLIST

(VERSION 4, APR. 2021)

LIBRARY CONSTRUCTION

Input gDNA Amount ~15 µg ≥5 µg

DNA Shearing

Megaruptor 1/2/3 System; or

g-TUBEs

Megaruptor 3 System (2-cycle shearing)

Post Shearing AMPure PB Bead

Purification

Yes No

Post-ligation Heat Kill Yes No

Buffer Exchange prior to Nuclease

cleanup (AMPure PB beads)

No Yes

SMRTbell Enzyme Clean up Kit

Version (Reaction Time)

1.0 (60 min) 2.0 (30 min)

Size-Selection Options*

SageELF System; or

BluePippin System; or

AMPure PB Bead Size Selection

PippinHT System; or

SageELF System; or

BluePippin System

* AMPure PB bead size selection is under development for the new HiFi sample preparation Procedure & Checklist (PN 101-853-100, Version 4) and specific guidance will be

provided in a future protocol update.

SAMPLE PREP

WORKFLOW STEP

OLD HIFI PROCEDURE & CHECKLIST

(VERSION 3, JAN. 2020)

NEW HIFI PROCEDURE & CHECKLIST

(VERSION 4, APR. 2021)

SMRT LINK SAMPLE SETUP

Primer Annealing Sequencing Primer v2 Sequencing Primer v5

Polymerase binding

Sequel II Polymerase 2.0

Binding Time = 4 h

Sequel II Polymerase 2.2

Binding Time = 1 h

Complex Cleanup

Dilute Bound Complex Volume by 3.33-fold and

purify sample using 1.2X AMPure PB beads

If Bound Complex Volume is <100 µL, bring up to

100 µL with Complex Dilution Buffer and purify

sample using 1.2X AMPure PB beads.

SMRT LINK RUN DESIGN SETUP

Pre-Extension Time 2 h (<20 kb) or 4 h (≥20 kb) 0 h (No Pre-extension)

Adaptive Loading (AL) OFF ON

COMPARISON OF HIFI SAMPLE PREPARATION WORKFLOW CHANGES FOR

DE NOVO ASSEMBLY AND VARIANT DETECTION APPLICATIONS (CONT.)

ENABLING PRODUCTION-SCALE THROUGHPUTS FOR HUMAN WHOLE

GENOME SEQUENCING FOR RARE AND INHERITED DISEASE RESEARCH

STRUCTURAL VARIANTS

(SVs)

VARIANT DETECTION

(SNVs, INDELs, SVs)

VARIANT DETECTION

(SNVs, INDELs, SVs)

Number of SMRT Cells 8M/sample 1 2 3

Sequencing time per sample (hrs) 30 60 90

Coverage per human genome sample ~10-fold ~20-fold ~30-fold

Variant Detection

Performance

(% Accuracy, F1)

SNV 99.0% 99.8% 99.9%

Indel 92.6% 96.9% 97.9%

SV 92.6% 95.7% 95.8%

Annual Sample

Throughput

1 Sequel IIe System 256 128 84

6 Sequel IIe Systems 1,536 768 504

12 Sequel IIe Systems 3,072 1,536 1,008

This efficient HiFi sample preparation workflow, developed in collaboration with Children’s Mercy Kansas City, provides a scalable

solution for sequencing 100s to 1000s of whole human genomes per year on the Sequel II and IIe Systems.

SMRT Link Sample Setup, Run Design & Run QC

Updates

SMRT LINK V10.1 SAMPLE SETUP & RUN DESIGN

RECOMMENDATIONS FOR SPECIFIC APPLICATIONS

Generally follow SMRT Link Sample Setup & Run Design instructions using the recommendations

provided in the Quick Reference Card – Loading and Pre-Extension Time Recommendations for the

Sequel II and IIe Systems unless specified otherwise in the relevant Procedure & Checklist

https://www.pacb.com/support/documentation/

In SMRT Link v10.1, most Sample Setup

and Run Design parameter fields are

auto-filled with the recommended settings for

each application type.

UPDATED SAMPLE SETUP WORKFLOW: SPECIFYING APPLICATION TYPE

Sample Setup auto-populates application-specific information for selected fields

- The user starts by first entering the Sample Name and then selecting an Application Type

- Once an application is selected, default values are auto-populated for various fields and highlighted in green

UPDATED SAMPLE SETUP WORKFLOW: SPECIFYING APPLICATION TYPE (CONT.)

Auto-populated Sample Setup fields are highlighted in green color

- The following fields are auto-populated and highlighted in green:

- Sequencing Primer

- Binding Kit

- Note: The following auto-populated fields are located in Advanced Options:

- Target Annealing Concentration

- Target Binding Concentration

- Target Polymerase Concentration (Relative)

- Binding Time

- Cleanup Bead Type

- Cleanup Bead Concentration

- If any auto-populated entry is manually changed to a different value, then the

field will be highlighted in yellow color

UPDATED RUN DESIGN WORKFLOW: SPECIFYING APPLICATION TYPE

Run Design auto-populates application-specific information for selected fields

- In not importing sample information from Sample Setup, the user can start by first selecting an Application Type

- Once an application is selected, default values are auto-populated for various fields and highlighted in green

UPDATED RUN DESIGN WORKFLOW: SPECIFYING APPLICATION TYPE (CONT.)

Auto-populated fields are highlighted in green color

- The following fields are auto-populated and highlighted in green:

- Template Prep Kit

- Binding Kit

- Sequencing Kit

- DNA Control Complex

- Movie Time Per SMRT Cell

- Pre-Extension Time (If applicable)

- If any auto-populated entry is manually changed to a different

value, then the field will be highlighted in yellow color

SAMPLE SETUP RECOMMENDATIONS FOR HIFI DE NOVO ASSEMBLY AND

VARIANT DETECTION APPLICATIONS (SEQUEL II AND IIe SYSTEM V10.1 RELEASE)

https://www.pacb.com/support/documentation/

- Follow SMRT Link Sample Setup instructions using the recommendations

provided in the Quick Reference Card – Loading and Pre-Extension Time

Recommendations for the Sequel II/IIe Systems for sequencing HiFi library

samples for De Novo Assembly or Variant Detection applications

→For SMRT Link v10.1 (or higher): Select ‘WGS – De Novo Assembly / HiFi

Reads’ or ‘WGS – Variant Detection / Variant Calling’ from the Application

field drop-down menu in the SMRT Link Sample Setup and SMRT Link Run

Design user interface

* Recommendations for using Sequel II Binding Kit 2.2 with other application use cases aside from WGS de novo assembly and variant detection will be provided in future protocol

documentation releases.

For HiFi WGS de novo assembly and

variant detection applications, we

recommend enabling Adaptive Loading

and using Sequel II Binding Kit 2.2.*

NEW ADAPTIVE LOADING FEATURE FOR SEQUEL II AND IIe

SYSTEMS

Adaptive Loading reduces sample overloading, allowing users to load higher with

confidence

Adaptive Loading (AL) uses active monitoring of polymerase binding

to the bottom of the ZMW during loading to reduce variability and the

risk of overloading with high-concentration samples

- Adaptive loading technology actively monitors

polymerase complex binding to the bottom of

ZMWs during the sample immobilization step.

- Detection of these active polymerase complexes

allows the system to terminate the immobilization

step when the desired loading target has been

achieved.

→ This approach can help reduce sample overloading

and run-to-run yield variability

OVERVIEW OF SMRT LINK V10.1 SAMPLE SETUP AND RUN DESIGN

WORKFLOW TO ENABLE ADAPTIVE LOADING

Adaptive Loading is automatically enabled by default in SMRT Link v10.1 Sample Setup

and Run Design for sequencing applications using Sequel II Binding Kit 2.2

1. Binding Kit Version Selection

- Specify Sequel II Binding Kit 2.2*

2. Complex Cleanup Step

- Use Adaptive Loading (AL) Buffer in place of

Complex Dilution Buffer (CDB) to elute purified

polymerase-bound sample from AMPure PB

beads

3. Final Loading Dilution Step

- Use AL Buffer in place of CDB to dilute the

purified polymerase-bounds sample to the final

on-plate loading concentration

Advanced Options

- Specify Use Adaptive Loading = YES

- Specify Loading Target (P1 + P2)

- Specify Maximum Loading Time (hours)

Load Sample Plate

- Start sequencing run

* In SMRT Link v10.1 Sample Setup, the Adaptive Loading sample setup procedure is only enabled by selecting Sequel II Binding Kit 2.2. Selection of other Sequel II Binding Kit

versions will not enable the Adaptive Loading sample setup procedure.

Binding Kit Version Selection

- Specify Sequel II Binding Kit 2.2*

Complex Cleanup Step

- Use Adaptive Loading (AL) Buffer in

place of Complex Dilution Buffer (CDB) to

elute purified polymerase-bound sample

from AMPure PB beads

Final Loading Dilution Step

- Use AL Buffer in place of CDB to dilute the

purified polymerase-bounds sample to the

final on-plate loading concentration

SMRT LINK V10.1 SAMPLE SETUP WORKFLOW TO ENABLE

ADAPTIVE LOADING

1. Binding Kit Version Selection

* In SMRT Link v10.1 Sample Setup, the Adaptive Loading sample setup procedure is only enabled by selecting Sequel II Binding Kit 2.2. Selection of other Sequel II Binding Kit

versions will not enable the Adaptive Loading sample setup procedure.

SMRT LINK V10.1 SAMPLE SETUP WORKFLOW TO ENABLE

ADAPTIVE LOADING (CONT.)

Binding Kit Version Selection

- Specify Sequel II Binding Kit 2.2

Complex Cleanup Step

- Use Adaptive Loading (AL) Buffer in

place of Complex Dilution Buffer (CDB) to

elute purified polymerase-bound sample

from AMPure PB beads

Final Loading Dilution Step

- Use AL Buffer in place of CDB to dilute the

purified polymerase-bounds sample to the

final on-plate loading concentration

2. Complex Cleanup Step

SMRT LINK V10.1 SAMPLE SETUP WORKFLOW TO ENABLE

ADAPTIVE LOADING (CONT.)

3. Final Loading Dilution Step*

Binding Kit Version Selection

- Specify Sequel II Binding Kit 2.2

Complex Cleanup Step

- Use Adaptive Loading (AL) Buffer in

place of Complex Dilution Buffer (CDB) to

elute purified polymerase-bound sample

from AMPure PB beads

Final Loading Dilution Step

- Use AL Buffer in place of CDB to dilute the

purified polymerase-bounds sample to the

final on-plate loading concentration

* In SMRT Link v10.1 Sample Setup, no DTT or Sequel Additive is added during the Final Loading Dilution step for sample complexes prepared with Sequel II Binding Kit 2.2.

SMRT LINK V10.1 RUN DESIGN WORKFLOW TO ENABLE

ADAPTIVE LOADING

Advanced Options

- Specify Use Adaptive Loading = YES

- Specify Loading Target (P1 + P2)

- Specify Maximum Loading Time (hours)

For HiFi WGS de novo assembly and variant detection applications

using Sequel II Binding Kit 2.2, we highly recommend using the

Adaptive Loading default values of 0.75 for the Loading

Target and 2 hours for Maximum Loading Time.

Run Design Advanced Options

ADAPTIVE LOADING REQUIRES SERIALIZATION OF THE

SEQUEL II/IIe SYSTEM INSTRUMENT ROBOTIC WORKFLOW

Adaptive Loading Workflow: SMRT Cell prep occurs in series with data acquisitions

SMRT Cell 1 Prep (1 h)

+ AL Monitoring (0.5 h)

+ Loading (0 – 2 h)

SMRT Cell 1 Prep (1 h)

+ Loading (2 h)

SMRT Cell 1 Data Acquisition

SMRT Cell 2 Prep (1 h)

+ Loading (2 h)

SMRT Cell 3 Prep (1 h)

+ Loading (2 h)

SMRT Cell 2 Data Acquisition

SMRT Cell 4 Prep (1 h)

+ Loading (2 h)

SMRT Cell 3 Data Acquisition

Standard Workflow: SMRT Cell prep occurs in parallel with data acquisitions

SMRT Cell 1 Data Acquisition

SMRT Cell 2 Prep (1 h)

+ AL Monitoring (0.5 h)

+ Loading (0 – 2 h)

SMRT Cell 2 Data Acquisition

- With the Adaptive Loading feature enabled, instrument run times will be longer compared to non-AL runs depending on

the actual duration of the AL monitoring + immobilization (loading) time period (up to ~2.5 hours) per SMRT Cell 8M.

NEW RUN QC VISUALIZATION PLOTS IN SMRT LINK V10.1

The following plots below are generated for any run where CCS processing is enabled on-instrument

(Sequel IIe System) or through CCS Pre-Analysis (Sequel or Sequel II Systems)

Displays a histogram distribution of

HiFi Reads (QV ≥20), other CCS

Reads (three or more passes, but QV

<20), and other reads, by read

length.

Displays a histogram distribution of

HiFi Reads (QV ≥20) and other CCS

Reads by read quality.

Displays a heat map of CCS Read

lengths and predicted accuracies.

The boundary between HiFi Reads

and other CCS Reads is shown as a

dashed line at QV 20.

HiFi Reads

(≥Q20)

Q20

Example HiFi Library Sequencing Performance

Data

EXAMPLE SEQUENCING PERFORMANCE OF A 18-KB HUMAN HIFI LIBRARY

FOR WGS VARIANT DETECTION APPLICATIONS

HiFi Reads 2.0 M

HiFi Base Yield** 34.7 Gb

Mean HiFi Read Length 17,212 bp

Median HiFi Read Quality Q30

Size-Selected HiFi Library QC Insert Read Length Density Plot HiFi Read Length Distribution

* 40 pM on-plate loading concentration

generated P1 = 70% using a 30-hour

movie collection time (Sequel IIe System)

50 kb

10 kb

Mean HiFi

Read Length

= 17.2 kb

IRLD plot shows most HiFi read

lengths are ~10 – 30 kb*

** For this human library data set, typical HiFi base

yields were ~25 Gb – 35 Gb per SMRT Cell 8M.

Input gDNA for Megaruptor 3 Shearing 5 µg

Post-Library Construction Recovery (%)

[Pre-Nuclease Treatment]

3540 ng

(71%)

Post-Library Construction Recovery (%)

[Post-Nuclease Treatment]

1368 ng

(27%)

Post-PippinHT Size Selection Recovery (%)

640 ng

(13%)

~17.5 kb Mode

(Pre-Size Selection)

~18 kb Mode

(Post-Size Selection

with PippinHT System)

EXAMPLE SEQUENCING PERFORMANCE OF A 20-KB PLANT HIFI LIBRARY

FOR WGS DE NOVO ASSEMBLY APPLICATIONS

HiFi Reads 1.3 M

HiFi Base Yield 25.1 Gb

Mean HiFi Read Length 19,696 bp

Median HiFi Read Quality Q28

Size-Selected HiFi Library QC Insert Read Length Density Plot HiFi Read Length Distribution

* 60 pM on-plate loading concentration

generated P1 = 85% [Adaptive Loading

Target (P1 + P2) = 0.85] using a 30-hour

movie collection time (Sequel IIe System)

50 kb

10 kb

Mean HiFi

Read Length

= 19.7 kb

IRLD plot shows most HiFi read

lengths are ~10 – 30 kb*

Input gDNA for Megaruptor 3 Shearing 5 µg

Post-Library Construction Recovery (%)

[Post-Nuclease Treatment]

1900 ng

(38%)

Post-PippinHT Size Selection Recovery (%)

850 ng

(17%)

~20 kb Mode

(Post-Size Selection

with PippinHT System)

SEQUEL II DNA INTERNAL CONTROL PERFORMANCE UPDATE

- Sequel II Binding Kits 2.0, 2.1 and 2.2 include Sequel II DNA Internal Control Complex

pre-bound with Sequel II Polymerase 1.0

- Sequel II Polymerase 2.2 is bound (tethered) slightly higher above the surface of the

ZMW compared to Sequel II Polymerases 2.0 and 2.1

- A higher laser power setting is required to illuminate the ZMW when sequencing samples

bound to Sequel II Polymerase 2.2

→ Sequel II DNA Internal Control Complex 1.0 shows reduced mean read length

performance when used with samples bound to Sequel II Polymerase 2.2

compared to samples bound with Sequel II Polymerase 2.0 or 2.1

Sequel II DNA Internal Control Complex 1.0 is included with Sequel II Binding Kit 2.0 / 2.1 / 2.2

Polymerase Version

Bound to Sample

Estimated DNA Internal Control Complex 1.0

Mean Polymerase Read Length (30-h Movie)

Sequel II Polymerase 2.2 ~30 kb

Sequel II Polymerase 2.0

Sequel II Polymerase 2.1

~50 kb

Comparison of Sequel II Polymerase 2.0-

DNA Template complex (A) vs. Sequel II

Polymerase 2.2-DNA Template complex (B)

immobilized to a ZMW.

The higher laser power setting required for sequencing samples bound to Sequel II

Polymerase 2.2 results in increased photodamage to the Sequel II DNA internal

Control Complex 1.0 and hence shorter control polymerase read lengths

SMRT Link v10.1 Overview

Subhead should be no longer than 1 line

SMRT LINK V10.1 SOFTWARE RELEASE: KEY FEATURES &

IMPROVEMENTS

▪ Supports complete SMRT Link analysis

workflow on Amazon Cloud (SMRT Link

Cloud)

▪ Features several new and improved

analysis applications for HiFi de novo

assembly, SV calling, multiplexed Iso-Seq

analysis and SARS-CoV-2 full-viral genome

sequencing

▪ Reduces HPC requirements to enable

lower-cost data analysis & storage

configurations

▪ Provides a simplified user experience for

run setup and includes usability

improvements to support high-throughput

sequencing

Supports Complete SMRT Link Workflow on Amazon Cloud*

- Flexible data analysis optimized for speed or cost based on user

preferences

- No need for internal HPC

Features New and Improved Analysis Applications

- New Genome Assembly application for HiFi data*

- New SARS-CoV-2 application for COVID-19 surveillance

- Updated Iso-Seq application for improved multiplexed sample analysis

- Updated SV Calling application for improved precision

- Enhanced alignment concordance in mapping applications

- Bioconda: New HiFi Amplicon Analysis application*

- Bioconda: New Single-cell Iso-Seq application*

Provides Simplified User Experience & Usability Improvements

- New application-centric Sample Setup and Run Design*

- New HiFi sequencing metrics and data visualizations in Run QC*

- Reduced HPC requirements to enable lower-cost configurations*

- New Sample Setup import feature to support for high-throughput

production environments

SMRT LINK V10.1 SOFTWARE RELEASE: KEY FEATURES &

IMPROVEMENTS

▪ Supports complete SMRT Link analysis

workflow on Amazon Cloud (SMRT Link

Cloud)

▪ Features several new and improved

analysis applications for HiFi de novo

assembly, SV calling, multiplexed Iso-Seq

analysis and SARS-CoV-2 full-viral genome

sequencing

▪ Reduces HPC requirements to enable

lower-cost data analysis & storage

configurations

▪ Provides a simplified user experience for

run setup and includes usability

improvements to support high-throughput

sequencing

SMRT Link

Cloud

SMRT

Analysis

* Feature first introduced with SMRT Link v10.0 limited release

Head Node

Cores

RAM

64 GB

Local Storage

1 TB SSD/Flash storage

db_datadir

(Local Storage)

250 GB

Compute Nodes

Cores (Total)

Minimum RAM per slot (1 slot = 1 core)

>4 GB

Local Storage

100 GB

Shared Data Storage

Sequencing Data

20 TB

Analysis Data

40 TB

Network

10 GbE strongly recommended, 1GbE required

SEQUEL IIe SYSTEM ENABLES REDUCED COMPUTE

REQUIREMENTS FOR SMRT LINK INSTALLATIONS

Storage is calculated for one Sequel IIe System, assuming 100 human genomes per year at

30-fold HiFi coverage, de novo assembly

Connection between the Head Node and Sequel IIe System

Reduced HPC requirements for the

Sequel IIe System enable lower-cost

compute configurations

compared to the Sequel II System

- Approx. 5-fold lower HPC costs: $20K (Sequel IIe

System) vs. $100K (Sequel II System)

- Note: For a single Sequel IIe System deployment, a

Single System Compute configuration is available

– contact PacBio Technical Support or your local

Bioinformatics FAS for details. (Supports ONE

Sequel IIe system ONLY. Not suggested for sites

with multiple instruments.)

Previously 384

(Sequel II System)

Previously >100 TB

(Sequel II System)

CCS Analysis Application Features &

Reports

CCS ANALYSIS ALGORITHM AND DATA OUTPUTS

CCS analysis and output are unified for Sequel IIe System and SMRT Link

- CCS algorithm and output files (metrics, reports) are the same for Sequel IIe System and SMRT Link

On-Instrument CCS

CCS Analysis Application

Sequel IIe System

SMRT Link

HiFi reads achieve

≥99.9% accuracy

CCS ANALYSIS reads.ba m DATA FILE FORMAT

- Sequel IIe System on-instrument CCS (OICCS)* and

SMRT Link CCS Analysis application outputs

a reads.bam file containing one read per

productive ZMW.

- This format is far more compact than subread data.

- There are three classes of reads in this reads.bam

file:

▪ HiFi Reads: CCS reads with QV ≥20

▪ Other CCS Reads: CCS reads with lower quality (<Q20)

▪ Other Reads: Single-pass reads or reads not meeting

minimum CCS requirements

- For OICCS, when the reads.bam file is imported into

SMRT Link, a filtered file containing only HiFi reads

is automatically generated

* Note: Users can optionally specify in SMRT Link Run Design to include polymerase kinetics information (for secondary epigenetics analysis) in the

reads.bam file produced through either on-instrument CCS or SMRT Link – however, BAM file size is 5X larger if kinetics information is included.

HIFI DATA-SPECIFIC FILES ARE AUTOMATICALLY GENERATED IN

SMRT LINK WHEN CCS ANALYSIS IS PERFORMED

HiFi Data File Generation With On-instrument CCS Analysis (Sequel IIe System ICS v10.1+)

- An on-instrument CCS analysis generates a reads.bam file and transfers it to the

network server.

- The reads.bam file contains HiFi Reads and non-HiFi Reads, and should not be

used unfiltered as input for third-party tools that expect ≥QV20 sequencing data .

- SMRT Link automatically launches an Export Reads analysis on the reads.bam to

filter out the HiFi Reads, and generates the following three HiFi data files by default*:

▪ <Movie_Name>.hifi_reads.fastq.gz → FASTQ file with HiFi reads

▪ <Movie_Name>.hifi_reads.fasta.gz → FASTA file with HiFi reads

▪ hifi_reads.bam → BAM file with HiFi reads

- Refer to Sequel IIe System: Location of HiFi Reads Files (PN 102-110-200) to

locate the hifi_reads files generated by SMRT Link when you perform an on-

instrument CCS analysis on the Sequel IIe System.

* If not using SMRT Link for subsequent analysis, please use these three files as input with any third-party analysis tools

HIFI DATA-SPECIFIC FILES ARE AUTOMATICALLY GENERATED IN

SMRT LINK WHEN CCS ANALYSIS IS PERFORMED (CONT.)

HiFi Data File Generation With SMRT Link CCS Analysis Application (SMRT Link v10.1+)

- The Circular Consensus Sequencing (CCS) analysis application in SMRT Link generates one reads.bam file (labeled

‘All Reads (BAM)’ in the File Downloads tab) plus the following three HiFi data-specific files below by default*:

▪ <Movie_Name>.hifi_reads.fastq.gz

→ FASTQ file with HiFi reads

▪ <Movie_Name>.hifi_reads.fasta.gz

→ FASTA file with HiFi reads

▪ hifi_reads.BAM

→ BAM file with HiFi reads

- To download the above files in SMRT Link, go

to the File Downloads tab for the CCS

analysis job

SMRT LINK ANALYSIS APPLICATIONS AND DATA FILTERING

CCS-based analysis applications require a reads.bam file as input and use built-in default

read quality filter settings

- All SMRT Link CCS-based applications* use reads.bam dataset as

input

▪ Built-in default filtering applied prior to analysis execution for each application

▪ All applications except Iso-Seq analysis use default HiFi reads (Q20 or higher)

▪ Iso-Seq application uses reads with Q10 or higher

The following analysis parameters

are deprecated as they no longer

need to be specified by the user:

▪ Minimum Number of Passes

▪ Minimum Predicted Accuracy

* Note: Microbial Assembly, Base Modification Analysis and HGAP4 Assembly remain CLR-based analysis applications

- Custom/non-HiFi data filtering:

▪ Use SMRT Link “Export Reads” application to specify a custom QV value in Advanced Parameters to create

FASTX and/or BAM files containing reads with a specified minimum CCS Predicted Accuracy

▪ Use SMRT Link Data Management to create a Data Set with custom read quality filtering

SMRT LINK DATA MANAGEMENT DATA SET REPORTS

Data Set Overview tab displays summary information for all reads and HiFi reads

SMRT LINK DATA MANAGEMENT DATA SET REPORTS (CONT.)

Raw Data Report tab displays summary metrics for all reads

SMRT LINK DATA MANAGEMENT DATA SET REPORTS (CONT.)

CCS Analysis Report tab includes summary metrics for HiFi Reads (≥Q20) and Other CCS

Reads (<Q20)

SMRT Link v10.1 Cloud

SMRT LINK CLOUD INTEGRATION

A cloud-based end-to-end analysis workflow enabled on Amazon Web Services

- Complete SMRT Link v10.1 functionality is now available on the cloud

- Cloud-agnostic solution – Amazon Web Services (AWS) support is being offered first

- Enabled for all Sequel Systems – Sequel, Sequel II and Sequel IIe Systems

- SMRT Link Cloud Advantages:

- No dependency on having internal compute hardware infrastructure

- Flexible data analysis options to optimize for speed or cost based on user preferences

- Ability to easily share data with collaborators

DATA STREAMING TO THE CLOUD

SMRT Link v10.1 enables data streaming to AWS

- Seamless automated data streaming from the sequencing instrument to AWS

- Once the Sequel / Sequel II / Sequel IIe System, SMRT Link and local network storage are configured,

automated data streaming is enabled through Amazon tools and utilities included with SMRT Link

- Fail-safe data streaming is enabled through use of a local fail-safe network storage server

- The sequencing data for each run is transferred to a local network storage server and then streamed to

AWS

- Network storage server requirements (provided by customer)

▪ Disk space requirements are based on a typical run for Sequel II/IIe Systems

▪ Disk space is managed by the customer – availability, data back up, etc.

* Optionally, a customer-preferred transfer mechanism can be used

DATA STREAMING TO THE CLOUD (CONT.)

SMRT Link provides a solution for streaming data from the network server to AWS

Cloud

(AWS)

Network ServerSequel Instrument Amazon Web Services

Generate Sequencing Data Transfer Data to a Network Server Stream data to AWS*

See SMRT Link Cloud Reference Guide (v10.1) (PN 101-985-900)

SMRT LINK CLOUD WORKFLOW

Versatile post-analysis options

Cloud

(AWS)

SMRT

Sequencing

Data on a

Network Server

SMRT Link

Data Transfer

Network

Server

SMRT Link

SMRT Analysis

Design a

Run

Stream

Sequencing

Data to AWS

Analyze

Sequencing

Data

Analysis

Results

other CCS Reads is shown as a

dashed line at QV 20.

HiFi Reads

(≥Q20)

Q20

SMRT LINK USER INTERFACE AND USABILITY IMPROVEMENTS

Improved Sample Setup Support for High-Throughput Sequencing

- Sample Setup features enhanced support for high-throughput production environments through new

Sample Setup sheet *.CSV import function

Field Name

Required

Description

Sample Name

Yes

Enter alphanumeric characters, spaces, hyphens,

underscores, colons, or periods only.

System Name

Yes

Must be Sequel, Sequel II, or Sequel IIe.

Application

Yes

Enter one of the following values:

• HiFi Reads

• Continuous Long Reads

• Low DNA Input

• Ultra-Low DNA Input

• Microbial Assembly

• Variant Calling

• Structural Variation Calling

• HiFiViral SARS-CoV-2

• Iso-Seq Method

• Full-Length 16S rRNA Sequencing

• Shotgun Metagenomic Profiling or

Assembly

• <3kb Amplicons

• >=3kb Amplicons

• Custom

Available Starting Sample Volume (uL)

Yes

Enter a positive integer. Units are in microliters.

Starting Sample Concentration (ng/uL)

Yes

Enter a positive integer. Units are in nanograms per

microliter.

Insert Size (bp)

Yes

Enter a positive integer. Units are in base pairs.

Sample Name System Name Application

Available Starting

Sample Volume (uL)

Starting Sample

Concentration (ng/uL)

Insert Size (bp)

Sample 1 Demo Sequel IIe HiFi Reads 10 100 18000

SMRT LINK USER INTERFACE AND USABILITY IMPROVEMENTS

We recommend notifying PacBio of your successful SMRT Link v10.1 installation and sending ongoing

SMRT Link analysis usage information to PacBio in order to expedite case troubleshooting and to help us

continually improve our products

SMRT Link Fixed & Known Issues

SMRT LINK V10.1 FIXED ISSUES HIGHLIGHTS

See the latest SMRT Link Release Notes for an updated list of fixed issues

- SV calling: Joint SV calling on demultiplexed Data Sets from the same cell/collection – demultiplexed Data Sets are now

analyzed separately, and the Bio Sample name is used.

- Sample Setup: Columns in the edit/print view can now be drag-and-dropped.

- Copying to the clipboard now works as expected.

- Exporting large analysis directories now works correctly and does not fail.

- Absolute file paths are now included in the subreadset.xml file.

- Login for local SMRT Link WSO2 users is now enabled.

- The outputs analysis directory now includes symbolic links to the BAM files.

- BAM files consolidation for microbial assemblies now works correctly.

- Using the hyphen character "-" in barcode and Bio Sample names no longer causes the Demultiplex Barcodes application

to fail.

- HGAP4 analysis no longer fails if the Genome Size is set to more than 2.0 GB.

- Run Design: When opening a saved Run Design, you will sometimes be asked to save changes when no changes were

made.

- Run Design: The Import from Sample Setup feature does not distinguish between Sample Setup designs created for Sequel II

and for Sequel IIe, instead showing both.

- A cached URL containing the string /welcome at the end of the SMRT Link URL (Example: https://URL/sl/welcome) in the

browser’s history causes an error when accessing SMRT Link.

- Motif detection is designed for microbial genomes and has not been tested on non-microbial genomes; it may run out of

memory on large genomes.

- When copying an analysis using the Demultiplex Barcodes application (using Copy from an Analysis Results page or Copy

From on the New Analysis page), the input of sample names for each barcode is not preserved from the copied analysis. In

the second step of the New Analysis wizard, users must either re-enter the sample names using the Interactive Barcode

Selector and Sample Name Editor, or re- upload a Barcoded Sample File. The Start button is not enabled until users do so.

- When creating a user, ensure that the new user profile has the Username attribute populated with the account/login name.

This is required for the user search in the configuration and project pages to find local users. (See SMRT Link Software

Installation (v10.1) for details.)

- When using Bio Sample Names with a PacBio analysis application, you can enter names that include spaces. Please avoid

using spaces in Bio Sample Names as spaces may lead to third-party compatibility issues.

SMRT LINK V10.1 KNOWN ISSUES HIGHLIGHTS

See the latest SMRT Link Release Notes for an updated list of known issues

Sequel II and IIe Systems Applications Support

Resources

Subhead should be no longer than 1 line

OVERVIEW – SEQUEL SYSTEMS APPLICATION OPTIONS AND

SEQUENCING RECOMMENDATIONS

This document provides high-level application workflow guidance and links to protocols for

preparing samples for sequencing on the Sequel Systems and analysis.

https://www.pacb.com/wp-content/uploads/Overview-Sequel-Systems-Application-Options-and-Sequencing-Recommendations.pdf

Whole Genome

Sequencing

Variant Detection

RNA Sequencing Targeted

Sequencing

Complex

Populations

Epigenetics

WHAT CAN YOU DO WITH ONE SMRT CELL 8M?

With PacBio Single Molecule, Real-Time Sequencing on the Sequel II and IIe Systems you can

characterize whole genomes and transcriptomes with just one SMRT Cell 8M.

*Study design, sample type, and level of multiplexing may affect the number of SMRT Cells 8M required.

†

All prices

are listed in USD and cost may vary by region. Pricing includes library and sequencing reagents run on your Sequel

II System and does not include instrument amortization or other reagents.

Application Brochure: What Can You Do with One SMRT Cell?

SMRT Cell 8M

Whole Genome

Sequencing

Variant Detection

RNA Sequencing Targeted

Sequencing

Complex

Populations

Epigenetics

APPLICATION CONSUMABLE BUNDLES & PURCHASING GUIDE

https://www.pacb.com/wp-content/uploads/Application-Consumable-Bundle-Purchasing-Guide.pdf

Purchasing Guide brochure enables users to easily order required consumables needed to run

a specific type of application on the Sequel II and IIe Systems.

-Customers can use a single part number to order a

consumables bundle containing PacBio-branded reagents

needed for SMRTbell library construction, primer annealing &

polymerase binding

-Exclusions:

- Core PacBio-branded SMRT Sequencing consumables (SMRT

Cells, Sequencing Kits & SMRT Oil), plastics and other 3rd-party

reagents are not included in the application bundles

- For Barcoded Adapter bundles that support >16-plex, PacBio

recommends customers purchase barcoded adapters directly from

a third-party oligo synthesis company.

CORE PACBIO REAGENTS & CONSUMABLES REQUIRED 
FOR SMRTBELL EXPRESS LIBRARY CONSTRUCTION
93
DNA End Repair / A-Tailing
Adapter Ligation
Purify SMRTbell Templates**
Remove Single-Strand Overhangs
DNA Damage Repair
Nuclease Treatment*
SMRTbell Express Template Prep Kit 2.0
SMRTbell Enzyme Cleanup Kit 2.0
* A Nuclease Treatment step is included in some 
protocols to remove non-intact SMRTbell templates
AMPure PB Beads
** Some protocols may specify the use of ProNex Beads 
(instead of AMPure PB Beads) for SMRTbell purification
PacBio Purchasing Guide brochure enables 
users to easily order required consumables needed 
to prepare a SMRTbell library to run a specific type 
of application on the Sequel II/IIe System.***
*** Core PacBio-branded SMRT Sequencing consumables 
(SMRT Cells, Sequencing Kits & SMRT Oil), plastics and 
other 3rd-party reagents are not included in the application 
bundles

SEQUEL IIe SYSTEM QUICK REFERENCE CARD – DIFFUSION

LOADING AND PRE-EXTENSION RECOMMENDATIONS

Follow SMRT Link Sample Setup & Run Design instructions using the recommendations provided in the

Quick Reference Card – Loading and Pre-Extension Time Recommendations for the Sequel II/IIe System

unless specified otherwise in the relevant Procedure & Checklist

In SMRT Link v10.1, most Sample Setup

and Run Design parameter fields are

auto-filled with the recommended settings for

each application type.

https://www.pacb.com/support/documentation/

TECHNICAL DOCUMENTATION & SOFTWARE DOWNLOAD 
RESOURCES
- Sequel II and Sequel IIe Systems Operations Guide (PN 101-774-700)
- Sequel II/IIe System v10.1 Release Notes (PN 102-041-700)
- Sequel IIe System: Location of HiFi Reads Files (PN 102-110-200)
- Quick Reference Card – Loading and Pre-Extension Recommendations for the Sequel II/IIe Systems (PN 101-769-100)
- Pacific Biosciences Glossary of Terms (PN 000-710-267)
Sequel IIe System Documentation
https://www.pacb.com/support/documentation/
- SMRT Link v10.1 Software Download Site:  https://www.pacb.com/support/software-downloads/
- SMRT Link v10.1 Software Installation Instructions (PN 102-036-900)
- SMRT Link v10.1 Release Notes (PN 102-040-000)
- SMRT Link v10.1 User Guide (PN 102-037-000)
- SMRT Link Cloud Reference Guide (v10.1) (PN 102-043-900)
- SMRT Link Web Services API Use Cases (v10.1) (PN 102-040-300)
SMRT Link Documentation
95

TECHNICAL DOCUMENTATION & SOFTWARE DOWNLOAD

RESOURCES (CONT.)

- Overview – Sequel Systems Application Options and Sequencing Recommendations (PN 101-851-300)

- Procedure & Checklist – Using AMPure PB Beads for Size-Selection (PN 101-854-900)

- Whole Genome Sequencing Applications

- De Novo Assembly – HiFi Reads

- Procedure & Checklist – Preparing HiFi SMRTbell Libraries using SMRTbell Express Template Prep Kit 2.0 (PN 101-853-100)

- De Novo Assembly – Low DNA Input

- Procedure & Checklist - Preparing SMRTbell Libraries Using Express Template Prep Kit 2.0 With Low DNA Input (PN 101-730-400)

- De Novo Assembly – Ultra-Low DNA Input

- Procedure & Checklist – Preparing HiFi SMRTbell Libraries from Ultra-Low DNA Input (PN 101-987-800)

- Microbial De Novo Assembly

- Procedure & Checklist – Preparing Multiplexed Microbial Libraries Using SMRTbell Express Template Prep Kit 2.0 (PN 101-696-100)

- Variant Detection

- Procedure & Checklist – Preparing HiFi SMRTbell Libraries using SMRTbell Express Template Prep Kit 2.0 (PN 101-853-100)

Sample Library Preparation Documentation

https://www.pacb.com/support/documentation/

TECHNICAL DOCUMENTATION & SOFTWARE DOWNLOAD 
RESOURCES (CONT.)
- RNA Sequencing Applications
- Iso-Seq Method
- Procedure & Checklist – Iso-Seq Express Template Preparation for Sequel and Sequel II Systems (PN 101-763-800)
- Procedure & Checklist – Preparing Single-Cell Iso-Seq Libraries Using SMRTbell Express Template Prep Kit 2.0 (PN 101-892-000)
- Metagenomics Applications
- Full-length 16S Sequencing
- Procedure & Checklist – Amplification of Full-Length 16S Gene with Barcoded Primers for Multiplexed SMRTbell Library Preparation and Sequencing (PN  101-599-700)
- Metagenomics Shotgun Sequencing
- Procedure & Checklist – Preparing 10 kb Library Using SMRTbell Express Template Prep Kit 2.0 for Metagenomics Shotgun Sequencing (PN 101-800-800)
- Targeted Sequencing Applications
- Amplicon Sequencing
- Procedure & Checklist – Preparing SMRTbell Libraries using PacBio Barcoded Overhang Adapters for Multiplexing Amplicons (PN 101-791-700)
- Procedure & Checklist – Preparing SMRTbell Libraries using PacBio Barcoded Universal Primers for Multiplex SMRT Sequencing (PN 101-791-800)
- Procedure & Checklist – Preparing SMRTbell Libraries using PacBio Barcoded M13 Primers for Multiplex SMRT Sequencing (PN 101-921-300)
- No-Amp Targeted Sequencing
- Procedure & Checklist – No-Amp Targeted Sequencing Utilizing the CRISPR-Cas9 System (PN 101-801-500)
Sample Library Preparation Documentation (Cont.)
https://www.pacb.com/support/documentation/
97

TECHNICAL DOCUMENTATION & SOFTWARE DOWNLOAD 
RESOURCES (CONT.)
- Whole Genome Sequencing Applications
- Application Brief: Whole genome sequencing for de novo assembly – Best Practices (PN BP102-121219)
- Application Brief: Variant detection using whole genome sequencing with HiFi reads – Best Practices (PN BP106-092419)
- Application Brief: Microbial whole genome sequencing – Best Practices (PN BP101-013020)
- RNA Sequencing Applications
- Application Brief: Long-read RNA sequencing – Best Practices (PN BP103-062619)
- Application Brief: Single-cell RNA sequencing with HiFi reads - Best Practices (PN BP109-102020)
- Metagenomics Applications
- Application Brief: Metagenomic sequencing with HiFi reads – Best Practices (PN BP108-030220)
- Targeted Sequencing Applications
- Application Brief: Targeted sequencing for amplicons – Best Practices (PN BP105-071919)
- Application Brief: No-Amp targeted sequencing – Best Practices (PN BP107-092319)
Applications Best Practices Guides
https://www.pacb.com/support/documentation/
98

TECHNICAL DOCUMENTATION & SOFTWARE DOWNLOAD 
RESOURCES (CONT.)
- Whole Genome Sequencing Applications
- Technical Overview:  HiFi Library Preparation Using SMRTbell Express Template Prep Kit 2.0 (PN 101-855-400)
- Technical Overview:  Low DNA Input Library Preparation Using SMRTbell Express Template Prep Kit 2.0 (PN 101-781-000)
- Technical Overview:  Ultra-Low DNA Input Library Preparation Using SMRTbell Express Template Prep Kit 2.0 (101-998-000)
- Technical Overview:  Multiplexed Microbial Library Preparation Using SMRTbell Express Template Prep Kit 2.0 (PN 101-742-600)
- RNA Sequencing Applications
- Technical Overview:  Iso-Seq Express Library Preparation Using SMRTbell Express Template Prep Kit 2.0 (PN 101-814-400)
- Technical Overview:  Single-Cell Iso-Seq Library Preparation Using SMRTbell Express TPK 2.0 (PN 101-925-400)
- Metagenomics Applications
- Technical Overview:  Metagenomics Shotgun Library Preparation Using SMRTbell Express Template Prep Kit 2.0 (PN 101-894-900)
- Technical Overview:  Full-Length 16S Library Preparation Using SMRTbell Express Template Prep Kit 2.0 (PN 101-916-900)
- Targeted Sequencing Applications
- Technical Overview:  Multiplexed Amplicon Library Preparation Using SMRTbell Express Template Prep Kit 2.0 (PN  101-814-300)
- Technical Overview:  No-Amp Targeted Sequencing Library Preparation and Data Analysis Technical Overview (PN 101-840-800)
- PacBio HiFiViral Workflow Overview: Multiplexed Amplicon Library Preparation for Full-Viral Genome Sequencing of SARS-CoV-2 (PN 102-084-800)
Applications Technical Training Documentation
https://www.pacb.com/support/documentation/
99

TECHNICAL DOCUMENTATION & SOFTWARE DOWNLOAD

RESOURCES (CONT.)

Data Analysis Documentation

https://www.pacb.com/support/documentation/

- Analysis Procedure – Multiplexed Microbial Assembly with SMRT Link v8.0 and SMRTbell Express Template Prep Kit 2.0

(PN 101-855-300)

- Analysis Procedure – No-Amp Data Preparation and Repeat Analysis (PN 101-801-400)

- Brief Primer and Lexicon for PacBio SMRT Sequencing Webpage (v10.0)

- PacBio Bioinformatics File Formats Documentation Webpage (v10.0)

- SMRT Analysis Barcoding Overview (v9.0) (PN 101-923-200)

- SMRT Tools Reference Guide (v10.1) (PN102-037-300)

- Guide – Step-by-Step Run Performance Evaluation (For Sequel II and Sequel IIe Systems) (PN 101-993-600)

Sequencing Performance Troubleshooting Documentation

100

TECHNICAL DOCUMENTATION & SOFTWARE DOWNLOAD

RESOURCES (CONT.)

Technical Notes

https://www.pacb.com/support/documentation/

- Technical Note: Preparing samples for PacBio whole genome sequencing for de novo assembly – Collection and storage

(PN TN100-040518)

- Technical Note: Preparing DNA for PacBio HiFi sequencing – Extraction and quality control (PN TN101-081420)

101

DNA SAMPLE PREPARATION ONLINE RESOURCE

Literature resource for sample collection and DNA extraction protocol references

www.ExtractDNAforPacBio.com

102

PacBio does not assume responsibilities/guarantees for

these external publications/protocols, but we are happy to

help as best as we can to guide / connect. Please contact

ExtractDNA@pacb.com for more discussions around

your particular species & sequencing project!

SEQUEL II AND IIe SYSTEM BEST PRACTICES OVERVIEW GUIDES

Variant Detection Using Whole Genome Sequencing with HiFi Reads

Whole Genome Sequencing for De novo Assembly

RNA Sequencing / Single-Cell RNA Sequencing

16S / Metagenomics Shotgun Sequencing of Complex Populations

https://www.pacb.com/smrt-science/smrt-resources/pacbio-literature/

No-Amp Targeted Sequencing

103

BEST PRACTICES: WHOLE GENOME SEQUENCING FOR DE NOVO

ASSEMBLY

SMRTbell Template Preparation

- Start with unamplified genomic DNA input (≥5 µg for a ~3-Gb sample genome size) from any sample type (blood, tissue, cell lines)

- Using SMRTbell Express Template Prep Kit 2.0, prepare libraries for HiFi sequencing of up to 16 samples at a time with manual prep, or 96 samples

using an automation-friendly workflow

- Enrich for ~15 kb - 20 kb inserts with size selection.

Sequence on the Sequel II or IIe Systems

- With highly accurate long reads (HiFi reads) from the Sequel II or IIe Systems, you can assemble up to a 2 Gb genome in a single SMRT Cell 8M for

~$1,300* or scale up for larger genomes

- Run up to 200 samples (2 Gb) per year, per Sequel II or IIe System

- Sequence to desired coverage depth based on the complexity of the genome sample:

- Recommend aiming for 10- to 15-fold HiFi read coverage per haplotype for phased de novo assembly

Data Analysis Solutions with the PacBio Analytical Portfolio

- Use SMRT Link Genome Assembly (powered by IPA), or open-source tools including HiCanu or hifiasm to assemble and phase the genome

- Example datasets are available at pacb.com/dataset

* Read lengths, reads/data per SMRT Cell 8M and other sequencing performance results vary based on sample quality/type and insert size.

†

Prices, listed in USD, are approximate and may vary by region. Pricing includes library and sequencing reagents run on a Sequel II or IIe System and does not include instrument

amortization or other reagents.

SMRTbell Template Preparation

- Start with unamplified genomic DNA input (≥5 µg for a ~3-Gb sample genome size) from any sample type (blood, tissue, cell lines)

- Using SMRTbell Express Template Prep Kit 2.0, prepare libraries for HiFi sequencing of up to 16 samples at a time with manual prep, or 96 samples

using an automation-friendly workflow

- Enrich for ~15 kb - 18 kb inserts with size selection. Inserts larger than this range may reduce read and variant calling accuracy

Sequence on the Sequel II or IIe Systems

- With highly accurate long reads (HiFi reads) from the Sequel II or IIe Systems you can comprehensively detect variants in 100s to 1000s of genomes

in a year

- Sequence to desired coverage based on study needs:*

- Aim for ≥15-fold HiFi read coverage of a Human genome for variant detection applications

- Recommend 2 SMRT Cells 8M to achieve ≥15-fold coverage of a human genome for comprehensive variant detection for $2600

†

Data Analysis Solutions with the PacBio Analytical Portfolio

- Detect all variant types – including SNVs, indels, SVs, and CNVs – with the highest precision and recall using SMRT Link Structural Variant Calling

analysis application (powered by pbsv) and Google DeepVariant (PacBio model)

- Use joint calling in pbsv and DeepVariant for multiple samples

- Expand variant calling into previously inaccessible regions of the genome, including repetitive regions and medically relevant genes that are difficult to

map

- Phase small variants into phase blocks using WhatsHap and Confirm variant calls visually with IGV and GenomeRibbon

BEST PRACTICES: VARIANT DETECTION USING WHOLE GENOME

SEQUENCING WITH HIFI READS

* Read lengths, reads/data per SMRT Cell 8M and other sequencing performance results vary based on sample quality/type and insert size.

†

Prices, listed in USD, are approximate and may vary by region. Pricing includes library and sequencing reagents run on a Sequel II or IIe System and does not include instrument

amortization or other reagents.

BEST PRACTICES: RNA SEQUENCING (ISO-SEQ ANALYSIS)

SMRTbell Template Preparation

- Prepare full-length cDNA from 300 ng of total RNA using the NEBNext Single Cell/Low Input cDNA Synthesis & Amplification Module kit

- Use the SMRTbell Express Template Prep Kit 2.0 to prepare libraries in one day

- Multiplex up to 12 samples with barcoding

Sequence on the Sequel II or IIe Systems

- Maximize output and turn-around-time with adjustable sequencing parameters

- Sequel IIe System: 24 hour movies with 2 hours pre-extension is recommended

- Use the Sequel IIe System to generate up to 4 million* full-length, non-concatemer (FLNC) reads per SMRT Cell 8M

- Scale throughput based on project needs – With a single SMRT Cell 8M you can:

- Characterize a whole transcriptome

- Multiplex multiple tissues for genome annotation

Data Analysis Solutions with the PacBio Analytical Portfolio

- Generate highly accurate long reads (HiFi reads), with single-molecule resolution using circular consensus sequencing (CCS) mode

- Use the Iso-Seq analysis in SMRT Link to output high-quality, full-length transcript FASTA sequences, with no assembly required, to characterize

transcripts and splice variants

- Run Iso-Seq analysis with or without a reference genome, and annotate the genome using community tools such as SQANTI2, TAMA, and

LoReAn

* Read lengths, reads/data per SMRT Cell 8M and other sequencing performance results vary based on sample quality/type and insert size.

106

BEST PRACTICES: SINGLE-CELL RNA SEQUENCING (SINGLE-CELL

ISO-SEQ ANALYSIS)

SMRTbell Template Preparation

- Enrich for single-cell cDNA using a single-cell sorting platform that generates full-length cDNA*

- Start library preparation with at least 160 ng of input cDNA (post-single-cell platform PCR reaction) for 1-2 SMRT Cells 8M

- Use the SMRTbell Express Template Prep Kit 2.0 to prepare libraries in one day

Sequence on the Sequel II or IIe Systems

- Use HiFi sequencing on the Sequel II or IIe Systems to generate 3 million full-length transcript reads from one SMRT Cell 8M to obtain ~1,000

unique molecules for 3,000 single cells**

- Use a 24-hour movie collection time with a 2-hour pre-extension time

- For human samples, run up to 240 SMRT Cell 8M/year at a cost of ~$1,300/SMRT Cell 8M, excluding single-cell

- enrichment cost

†

Data Analysis Solutions with the PacBio Analytical Portfolio

- Analyze HiFi reads which allow accurate single-cell barcode and UMI identification

- Use the single-cell Iso-Seq analysis tools on GitHub to output high-quality, full-length transcript FASTA sequences per UMI, with no assembly

required, to characterize transcript variants for each cell

* Number of usable reads, containing the UMI and cell barcode, vary by single-cell platform. Any platform that generates full-length cDNA is compatible with the single-cell RNA sequencing

workflow.

** Read lengths, reads/data per SMRT Cell type and other sequencing performance results vary based on single-cell platform, sample quality/type and insert size.

† Prices, listed in USD, are approximate and may vary by region. Pricing includes library and sequencing reagents run on a Sequel II or IIe System and does not include instrument

amortization or other reagents.

107

BEST PRACTICES: NO-AMP TARGETED SEQUENCING

SMRTbell Template Preparation

- Start with high-quality genomic DNA (~1-20 μg / SMRT Cell)

- Prepare SMRTbell libraries in 2-days with stream-lined protocol

- Block 5’ & 3’ ends to prevent off-target ligation

- Use custom-design guide RNAs to enrich for target regions of interest

- Multiplex up to 48 samples SMRT Cell 8M using barcoded adapters to analyze 5 or more targeted regions per sample

Sequence on the Sequel II or IIe Systems

- Sequence multiplexed targets and/or samples on the Sequel II or IIe Systems using the latest chemistry*

- Run up to 48 samples per SMRT Cell 8M at ~$82-118/sample on the Sequel IIe System

†

Data Analysis Solutions with the PacBio Analytical Portfolio

- Use command line tools to perform de-multiplexing and circular consensus sequencing (CCS) analysis to generate highly accurate long reads

(HiFi reads)

- Output data in FASTQ format for results summary reporting on repeat counts and on-target rates

- Visualize results with IGV and command-line scripts for easy review of repeat count of both alleles, mosaic characterization, identification of

interruption sequences and CRISPR / Cas9 off-targets

* Read lengths, reads/data per SMRT Cell 8M and other sequencing performance results vary based on sample quality/type and insert size.

†

Prices, listed in USD, are approximate and may vary by region. Pricing includes library and sequencing reagents run on a Sequel II or IIe System and does not include instrument

amortization or other reagents.

108

BEST PRACTICES: MICROBIAL WHOLE GENOME SEQUENCING

SMRTbell Template Preparation

- Start with 1.0 µg of high-quality input gDNA per microbial sample and construct a 10 – 15 kb library using SMRTbell Express TPK 2.0

- Reduce costs by multiplexing samples to assemble most bacterial genomes into 5 contigs or fewer, exclusive of plasmids

- Simplify equimolar pooling with Microbial Multiplexing Calculator

- Adjust multiplexing depth to balance cost per genome with genome completeness

- Note: Closure of class III complexity genomes with large repeat regions may require 20–30 kb library preparations & size-selection and may not be

suitable for multiplexing

Sequence on the Sequel II or IIe Systems

- Maximize output and turn-around-time with adjustable sequencing parameters*

- Multiplex up to 48 isolates per SMRT Cell 8M for $70/sample

†

with a 15 hour collection time

- Recommend generating ≥30-fold unique molecular coverage (UMC) per microbial genome

Data Analysis Solutions with the PacBio Analytical Portfolio

- Use SMRT Link for fully automated demultiplexing, assembly, circularization, and polishing of both chromosomes and plasmids to produce gold

standard references

- Achieve high-quality consensus accuracies >99.999%

- Detect and annotate active m6A and m4C Restriction-Modification system motifs with the ‘Base Modification and Motif Analysis’ application in SMRT

Analysis [m4C (≥25-fold coverage per strand) / m6A (≥25-fold coverage per strand)]

* Read lengths, reads/data per SMRT Cell 8M and other sequencing performance results vary based on sample quality/type and insert size.

†

Prices, listed in USD, are approximate and may vary by region. Pricing includes library and sequencing reagents run on a Sequel II or IIe System and does not include instrument

amortization or other reagents.

109

BEST PRACTICES: 16S AMPLICON SEQUENCING

SMRTbell Template Preparation

- Perform library construction with SMRTbell Express TPK 2.0

- PacBio full-length 16S protocol available with recommended barcoded 16S primer sequences; OR use third-party Shoreline Biome kits for DNA

extraction and PCR amplification

- Multiplex up to 192 16S samples per SMRT Cell 8M

Sequence on the Sequel II or IIe Systems

- Produce HiFi reads with circular consensus sequencing (CCS)

- Generate up to 2 Million 16S HiFi (Q30) reads per Sequel SMRT Cell 8M* using a 10-hour movie collection time

- Recommend generating ≥8000 HiFi reads per multiplexed 16S sample for analysis

Data Analysis Solutions with the PacBio Analytical Portfolio

- De-multiplex barcodes within SMRT Link GUI or on the command line

- Output data in standard file formats, (BAM and FASTA/Q) for seamless integration with downstream analysis tools

- Analyze 16S HiFi data using third-party analysis tools like Shoreline Biome SB Analyzer or DADA2

* Read lengths, number of reads, data per SMRT Cell 8M, and other sequencing performance results vary based on sample quality/type and insert size, among other factors

110

BEST PRACTICES: METAGENOMICS SHOTGUN SEQUENCING

SMRTbell Template Preparation

- Start with recommended amount of input gDNA per sample (1.5 μg) and construct a 10 kb library with SMRTbell Express TPK 2.0

- The size distribution of the starting genomic DNA is critical for shearing and PacBio recommends working with samples where the majority of the

input gDNA is greater than 15 kb whenever possible.

Sequence on the Sequel II or IIe Systems

- Produce HiFi (≥Q20) reads with circular consensus sequencing (CCS)

- Generate up to 2.4 Million 16S HiFi (Q20) reads per Sequel SMRT Cell 8M* using a 30-hour movie collection time

- Target coverage recommendations:

- 5-fold coverage (~3000 HiFi reads) of least abundant species for profiling intact genes and operons

- 20-fold coverage (~12,000 HiFi reads) for near-complete genome assemblies

Data Analysis Solutions with the PacBio Analytical Portfolio

- De-multiplex barcodes within SMRT Link GUI or on the command line

- Analyze metagenomic shotgun HiFi reads using third-party analysis tools

- Perform taxonomic classification and functional gene profiling using QIIME and MEGAN

- Perform gene prediction and discovery using FragGeneScan and Prodigal

- Perform metagenomic shotgun assembly directly with HiFi reads using Canu

- Bin contigs and plasmids originating from the same strain by leveraging epigenetic signatures

* Read lengths, number of reads, data per SMRT Cell 8M, and other sequencing performance results vary based on sample quality/type and insert size, among other factors

111

- PacBio Documentation page allows you to search for and download the latest guides, protocols, product information, and more.

https://www.pacb.com/support/documentation/

112

PACBIO DOCUMENTATION RESOURCES

PACBIO TRAINING RESOURCES

- PacBio Training page allows you to search for and download video tutorials and other training resources

http://www.pacb.com/support/training/

113

SMRT SEQUENCING RESOURCES

https://www.pacb.com/smrt-science/smrt-resources/

Scientific Publications PacBio Literature

BLOG

Documentation

Video Gallery

Posters

A Foundation for the Future of Genomic Discovery

114

PacBio, SMRT, SMRTbell, Iso-Seq, and Sequel are trademarks of Pacific Biosciences. Pacific Biosciences does not sell a kit for carrying out the overall No-Amp Targeted Sequencing method.

Use of these No-Amp methods may require rights to third-party owned intellectual property. FEMTO Pulse and Fragment Analyzer are trademarks of Agilent Technologies Inc.

All other trademarks are the sole property of their respective owners.