For Research Use Only. Not for use in diagnostic procedures. © Copyright 2019 by Pacific Biosciences of California, Inc. All rights reserved. PN 101-784-500 Version 01 (April 2019)

SMRTbell Library Preparation for High

Fidelity (HiFi/CCS) Long Read SequencingSequel System II v7.0.0 / SMRT Link v7.0.0 / Sequel II Chemistry v1.0

April.23.2019

SMRTbell Library Preparation for High

Fidelity (HiFi /CCS) Long Read Sequencing

1. HiFi Long Read Sequencing Features & Applications Overview

2. HiFi Library Sample Preparation & Sequencing Workflow Details

3. HiFi Long Read Sequencing Performance Example Data (Sequel II System)

4. Variant Detection Application Best Practices

5. Technical Documentation & Software Download Resources

HiFi Long Read Sequencing Features &

Applications Overview

HiFi Sequencing Workflow Provides High

Accuracy and Long Read Lengths

HIGHLY ACCURATE LONG READS: A NEW PARADIGM IN DNA

SEQUENCING

Advantages of High-Fidelity

(HiFi / CCS) Long Reads

- Generate higher quality genome

assemblies using a single technology

- Call all variant types – from single

nucleotides to structural variants

- Phase allele-specific haplotypes

- Identify full-length isoform transcripts

– no assembly required

HIFI READS: FROM DNA TO HIGH-QUALITY DATA

Start with high-quality

double stranded DNA

Ligate SMRTbell

Adapters and Size

Select

Anneal Primers

and Bind DNA

PolymeraseDNA is now circularized and ready

for SMRT Sequencing

Circularized DNA is sequenced in

repeated passes

The polymerase reads are trimmed

of adapters to yield subreads

Consensus is called from subreads

to generate highly accurate long

readsHiFi Read

HIFI READS: FROM DNA TO HIGH-QUALITY DATA (CONT.)

99% accuracy with 4 passes

Sequel System (1M)

Sequel II System (8M)

Read length (kb)

0 50 100 150 200 250 300

Yie

ld p

er

un

it r

ea

d le

ng

th (

kb

)

0

25

50

100

150

175

75

125

HIFI READ YIELD AND ACCURACY PERFORMANCE ON SEQUEL

SYSTEMS

Passes

5 10 15 200

30

0

10

20

40

50

Ac

cu

rac

y (P

hre

d)

30

0

10

20

40

50

85 15 200 10

Passes

Sequel (1M)

Sequel II (8M)

Subread Yield 318 Gb

CCS Yield 16 Gb

CCS Accuracy 99.8%

Data shown above from a 12 kb size-selected human library using the SMRTbell Template Prep Kit 1.0 on a Sequel II

System (v1.0 Chemistry, Sequel II System Software v7.0, 30-hour movie). Read lengths, reads/data per SMRT Cell 8M

and other sequencing performance results vary based on sample quality/type and insert size.

- For Researchers who want to detect variants comprehensively

in a whole human genome, the Sequel II System provides high

precision and recall for single-nucleotide variants, indels,

structural variants, and copy-number variants, including in

repetitive regions of the genome.

- HiFi reads from the Sequel II System combine the benefits of

short and long reads: high accuracy and long read length.

- With the Sequel II System, comprehensive detection of variants

in a whole human genome requires 3-4 SMRT Cells 8M.

- Variant calling with HiFi reads uses standard software like

Google DeepVariant and GATK.

VARIANT DETECTION APPLICATION

POWERED BY HIFI READS

Gain complete views of human genetic diversity

with PacBio high-fidelity long-read sequencing

HiFi Library Sample Preparation &

Sequencing Workflow Details

PROCEDURE & CHECKLIST – PREPARING SMRTBELL LIBRARIES

FOR HIFI LONG READ SEQUENCING ON SEQUEL AND SEQUEL II

SYSTEMS

- Procedure for constructing SMRTbell libraries

suitable for generating high accuracy long reads

on the Sequel and Sequel II Systems

- Protocol document contains:

1. Recommendations for gDNA QC and quantification

2. Recommendations for shearing gDNA to the

desired target mode size using the Megaruptor

System from Diagenode

3. Enzymatic steps for preparation of a HiFi

SMRTbell library

4. Instructions for size-selection of the HiFi SMRTbell

library using the SageELF System from Sage

Science

PROCEDURE & CHECKLIST – PREPARING HIFI LIBRARIES USING

THE STANDARD SMRTBELL TEMPLATE PREPARATION KIT 1.0

List of Required Materials and Equipment for Library Construction and Size

Selection

PACIFIC BIOSCIENCES® CONFIDENTIAL

HIFI SMRTBELL LIBRARY PREPARATION WORKFLOW OVERVIEW

1. gDNA QC & Shearing

- CHEF Mapper, FEMTO Pulse or Pippin Pulse sizing QC

- Qubit DNA concentration measurement QC

- Megaruptor shearing

2. SMRTbell Library Construction

- Standard SMRTbell Template Preparation Kit 1.0

- 2-Day LC workflow includes overnight ligation step

3. Size-select & Purify SMRTbell Library

- 10 – 15 kb size selection using SageELF

- AMPure PB purification of final SMRTbell library

4. Sample A/B/C & Sequence

- Anneal v2 Primer, Bind Polymerase, AMPure PB Complex

Cleanup

- Follow QRC for diffusion loading recommendations

5. Analyze

- Manage data through SMRT Link GUI

- For variant calling analysis, use GATK or Google Deep Variant

HIFI LIBRARY PREPARATION WORKFLOW IS MODIFIED FROM

STANDARD LIBRARY CONSTRUCTION PROCEDURES USING

SMRTBELL TEMPLATE PREP KIT 1.0

Library Construction Step High Fidelity Procedure Standard Procedure

Shear gDNA and concentrationMegaruptor

Target = 15 kb

Megaruptor or g-TUBE,

Target up to 75 kb

Remove ssDNA ends Exo VII Exo VII

DNA damage repair DNA Damage Repair Mix DNA Damage Repair Mix

Blunt-end repair ER Mix ER Mix

AMPure Purification Yes Yes

Ligation Blunt Adapter, overnight Blunt Adapter, overnight

AMPure Purification Yes Yes

Size-selectionSageELF, Target = 9-15 kb

(Collect 3 fractions)

BluePippin size selection (≥10-40 kb

lower cutoff)

Annealing, Binding and Cleanupv2 primer; 10:1 = Pol:Template; 4 hr

binding; AMPure clean up

v4 primer; 30:1= Pol:Template; 1 hr

binding; AMPure clean up

ExoVII Pre-Tx

0.45X AMPure

Adapter Ligation

ExoIII / ExoVII

Elf Fractionation

0.45X AMPure

ER

DDR

Day 1

Day 2

Day 30.45X AMPure

SMRTbell Template Prep Kit 1.0

15 m

45 m

O/N

60 m

5 h

45 m

15m

60 m

45 m

3 m

10 m

5 m

3 m

0.5 h

10 m

3 m

3 m

10 m

Step H-O W-A

Day 1

Day 2

Day 3H-O: Hands-on Time

W-A: Walk-away Time

HIFI SMRTBELL LIBRARY PREPARATION WORKFLOW TIME

HIFI LIBRARY INPUT GENOMIC DNA SAMPLE REQUIREMENTS

Library Construction Step Requirements

Recommended input gDNA size for shearing >40 kb

Recommended input gDNA amount for shearing 15 µg

Required sheared gDNA amount for Exo VII pre-treatment step 10 µg*

* Maximum input amount for Exo VII pre-treatment step is 10 µg. For >10 µg, scale the reaction volumes.

1. CHEF Mapper XA System (Bio-Rad)

2. FEMTO Pulse System (Agilent)

3. Pippin Pulse System (Sage Science)

https://www.pacb.com/wp-content/uploads/Procedure-Checklist-Using-the-

Sage-Science-Pippin-Pulse-Electrophoresis-Power-Supply-System.pdf

https://www.pacb.com/wp-content/uploads/Procedure-Checklist-Using-the-BIO-

RAD-CHEF-Mapper-XA-Pulsed-Field-Electrophoresis-System.pdf

https://www.aati-us.com/instruments/femto-pulse/

➢ Up to 10 Mb

➢ >16 Hour Run Time

➢ Up to 165 kb

➢ 1 Hour Run Time

➢ Up to 80 kb

➢ 16 Hour Run Time

Recommended methods for determining gDNA size distribution:

Lane 1: 8-48 kb Ladder (Bio-Rad)

Lane 2: 5 kb Ladder (Bio-Rad)

Lane 3: HMW gDNA

Lane 4: Degraded gDNA

Lane 1: High MW gDNA

Lane 2: Degraded gDNA

Lane 3: 165 kb Ladder

Evaluation of gDNA quality using A) Bio-Rad CHEF Mapper and

B) Advanced Analytical FEMTO Pulse. Lanes A3 and B1 show an

example of a high quality, high molecular weight gDNA sample,

where most of the DNA migrates as a prominent band at the top of

the gel image. Lanes A4 and B3 show an example of a partially

degraded gDNA sample where most of the DNA migrates below ~40

kb and is thus not suitable for constructing a HiFi SMRTbell library.

A B

RECOMMENDATIONS FOR EVALUATING GENOMIC DNA QUALITY

Recommended Megaruptor Shearing Conditions

Hydropore type: Long

Megaruptor software setting: 15 kb

DNA sample concentration: 50 ng/µl

DNA sample Volume: Up to 300 µl

MEGARUPTOR TOOL IS RECOMMENDED FOR SHEARING DNA

FOR HIFI SMRTBELL LIBRARY CONSTRUCTION

Megaruptor generates a tight DNA shearing distribution profile that results

in good recoveries during SMRTbell library size selection

- For HiFi library preparation, PacBio recommends shearing gDNA to a mode size of

approximately 12 kb

- For high quality gDNA, typical yields of sheared and concentrated DNA are >60%

- Because 10 μg of sheared gDNA is needed for the subsequent enzymatic steps, PacBio

recommends starting the shearing procedure with 15 μg of input gDNA

- Because the response of individual gDNA samples to recommended shearing parameters

may differ, small scale test shears are highly recommended (e.g., 50 μL at 50 ng/μL)

- Under- or over-shearing gDNA will result in low yields of final, size fractionated library

MEGARUPTOR DNA SHEARING EXAMPLE

- FEMTO Pulse analysis of human gDNA sheared to a 12-kb mode size using the ‘15 kb’

shear size setting in the Megaruptor software.

PERFORMING SHEARING OPTIMIZATIONS IS IMPORTANT

.

Sample 1

12,296 bp

Sample 2

10,288 bp

Sample 3

13,492 bp

- Above Figure shows examples of 3 different gDNA samples sheared using the same protocol.

- Under- or over-shearing impacts yield of fractions recovered from the library size selection step, hence

impacting the number of SMRT Cells that can be achieved per library prep

Performing test shears is recommended since different genomic DNA

samples may shear differently

SageELF TOOL IS THE GOLD STANDARD FOR PERFORMING HIFI

LIBRARY SIZE SELECTION

Fraction 4: 12977 bp

Fraction 5: 10993 bp

Fraction 6: 9540 bp

- 12 size fractions are collected per SageELF gel cassette (Run Time is ~4.5 hours)

- SMRTbell libraries with insert sizes of ~9 kb, ~11 kb and ~13 kb are suitable for HiFi (CCS) sequencing

- SageELF Cassette Definition File and Run Protocol Setup

- “0.75% 1-18kb v2”; ‘Size-based’ separation mode; Target value = 3400 (move the bar slider to

select Well #12)

- Recommended SMRTbell library input amount per lane: 3-5 µg

- Typical recovery yields (for the total sum of 3 collected fractions of interest) are ~15-30% per lane – but

will be highly dependent on the size distribution of the starting sheared DNA

Righthand Figure shows a FEMTO

Pulse analysis of recovered human

HG002 SMRTbell library fractions after

size selection using a SageELF system.

ALWAYS PERFORM SIZING QC ON RECOVERED SMRTBELL

LIBRARY FRACTIONS AFTER SageELF SIZE SELECTION

- To determine which fractions are suitable for generating HiFi reads, QC all recovered SMRTbell

library fractions after SageELF size selection using FEMTO Pulse, Fragment Analyzer, CHEF

Mapper or Pippin Pulse

- FEMTO Pulse sizing QC method is highly recommended since it requires very small amounts of DNA

sample (≤500 pg)

- SMRTbell libraries with insert sizes of ~9 kb, ~11 kb and ~13 kb are suitable for HiFi (CCS) sequencing

FEMTO Pulse analysis of HG002 library fractions after SageELF

size selection

CHEF Mapper analysis of HG002 library

fractions after SageELF size selection

48 kb

10 kb

4 5 6 4 5 6

20 kb

EXAMPLE SMRTBELL LIBRARY YIELDS AFTER SageELF

FRACTIONATION OF FOUR DIFFERENT HUMAN SAMPLES

Sample

Input

Sheared

gDNA (ng)

SMRTbell

Library (ng)

Input into

SageELF

(ng)

SageELF

Fraction:

9 kb (ng)

SageELF

Fraction:

11 kb (ng)

SageELF

Fraction:

13 kb (ng)

Final

SMRTbell

Yield (%)*

1 8,000 3,456 3,456 270 365 424 13.2 %

2 8,000 4,032 4,032 216 296 318 10.3 %

3 8,000 5,888 5,000 546 584 622 21.0 %

4 8,000 4,700 4,700 506 424 278 15.1 %

* Final SMRTbell library yield is calculated from the total sum of 3 collected SageELF fractions per input sheared gDNA amount.

For HiFi library sequencing, the collected SageELF fractions should have a size distribution mode between ~9 – 15 kb

- SMRTbell library yield after SageELF size selection is highly dependent on the final SMRTbell

library size distribution

SAMPLE SETUP RECOMMENDATIONS FOR SEQUENCING HIFI

SMRTBELL LIBRARIES USING THE SEQUEL II SYSTEM

(CHEMISTRY V1.0)

- Sequence each size-selected fraction of interest individually

- For Sequel II Systems: PacBio recommends sequencing fractions with insert size mode 11 kb

- Final purified SMRTbell library amount from each fraction is sufficient for approx. 3-4 Sequel II

SMRT Cell 8M

Parameter Recommendation

HiFi Library Insert Size 9 kb, 11 kb, 13 kb

Sequencing Primer Primer v2, 20:1 (Primer:Template)

Sequel II Polymerase 1.0 10:1 (Polymerase:Template)

Pre-extension Time 2 hours

Movie Collection Time 30 hours

On-plate loading concentration (OPLC) 25-50 pM

Target % P1 50-70%

In SMRT Link v7.0.0 Sample Setup, choose ‘Sequencing Primer v3’ from the dropdown menu but substitute

in Sequencing Primer v2 when setting up HiFi library annealing and binding reactions using the Sequel II

System (Chemistry v1.0). (Note: SMRT Link v7.0.0 does not include an option to specify Primer v2.)

EXAMPLE SMRT LINK V7.0 RUN QC ANALYSIS: 11-KB SageELF SIZE-

SELECTED HUMAN HIFI SMRTBELL LIBRARY (SEQUEL II SYSTEM)

50% of total base yield is found in reads >130 kb

NameMovie Time

(h)

PE Time (hs)

Total Bases (GB)

Unique Mol. Yield (GB)

Read Length ProductivityLocal Base Rate

Polymerase Longest Subread P0 P1 P2

Mean N50 Mean N50

HG005_11kb * 30 2 261.62 38.57 57445 135591 9188 1067742.1%

(3373130)56.8%

(4554204)1.1%(87337)

2.06

HG005_11kb* 30 2 280.78 42.14 56991 132967 9325 1071537.1%

(2974150)61.5%

(4926672)1.4%

(113849)2.05

Insert Read Length Density

Plot indicates many SMRTbell

library inserts are ~10-11 kb

A large proportion of CCS

reads were generated

* The same 11-kb human HiFi SMRTbell library was sequenced on two Sequel II SMRT Cells 8M using 50 pM OPLC

SAMPLE SETUP RECOMMENDATIONS FOR SEQUENCING HIFI

SMRTBELL LIBRARIES USING THE SEQUEL SYSTEM

(CHEMISTRY V3.0)

Parameter Recommendation

HiFi Library Insert Size 9 kb, 11 kb, 13 kb

Sequencing Primer Primer v2, 20:1 (Primer:Template)

Sequel Polymerase 3.0 10:1 (Polymerase:Template)

Pre-extension Time 8 hours

Movie Collection Time 20 hours

On-plate loading concentration (OPLC) 5-7 pM

Target % P1 50-70%

- Sequence each size-selected fraction of interest individually

- For Sequel Systems, PacBio recommends sequencing fractions with insert size mode 13 kb

- Final purified SMRTbell library amount from each fraction is sufficient for approx. 20 Sequel SMRT

Cell 1M

Use the Excel Sample Setup Calculator to set up annealing and binding reactions for sequencing HiFi

SMRTbell libraries using the Sequel System (Chemistry v3.0)

HiFi Long Read Sequencing Performance

Example Data (Sequel II)

HIFI LIBRARY SEQUENCING READ LENGTH

AND YIELD PERFORMANCE

Data shown above from a 11 kb size-selected human library using the SMRTbell Template Prep Kit on a Sequel II System (v1.0 Chemistry,

Sequel II System Software v7.0, 30-hour movie). Read lengths, reads/data per SMRT Cell 8M and other sequencing performance results vary

based on sample quality/type and insert size.

Metric

Number of Raw Bases (Gb) 320

Total Reads 4,053,000

Half of Bases in Reads >166,571

Longest read lengths >300,000

Data shown above from a 11 kb size-selected human library using the SMRTbell Template Prep Kit on a Sequel II System (v1.0 Chemistry,

Sequel II System Software v7.0, 30-hour movie). Read lengths, reads/data per SMRT Cell 8M and other sequencing performance results vary

based on sample quality/type and insert size.

HIFI LIBRARY SEQUENCING ACCURACY PERFORMANCE

Q20 (99%) single-molecule

accuracy

Metric

Insert Size 12 kb

Number of >Q20 Bases 21 Gb

Number of >Q20 Reads 1,855,642

Accuracy (Mean) 99.8%

99.3

95.195.4

98.6

93.0

96.8

90

91

92

93

94

95

96

97

98

99

100

SNVs Small Indels SVs

Precision Recall

Perc

en

tag

e (

%)

HIFI READS ENABLE HIGH PRECISION & RECALL FOR DETECTING

ALL VARIANT TYPES

Variant calls from 15-fold HiFi read coverage of a human genome (HG002) were measured against the Genome in a

Bottle small variant benchmark (v3.3.2) for SNVs and indels and the v0.6 benchmark for SVs.

VARIANT DETECTION COVERAGE TITRATION

40

50

60

70

80

90

100

0 5 10 15 20 25 30

Perc

enta

ge (

%)

Fold coverage

SNVs with DeepVariant

Precision

Recall

40

50

60

70

80

90

100

0 5 10 15 20 25 30

Pe

rcen

tage

(%

)

Fold coverage

Indels with DeepVariant

Precision

Recall40

50

60

70

80

90

100

0 5 10 15 20 25 30 35

Pe

rcen

tage

(%

)

Fold coverage

Structural Variants

Precision

Recall

15-fold HiFi Coverage

(2-3 SMRT Cells 8M)

provides a good trade-off

between cost and results

Variant Detection Application Best Practices

VARIANT DETECTION BEST PRACTICES (SEQUEL II SYSTEM)

Template Preparation with SMRTbell Template Prep Kit 1.0

- Use recommended high-quality genomic DNA input (≥15 µg)

- Prepare 10-15 kb SMRTbell library using Procedure & Checklist - Preparing SMRTbell

Libraries for HiFi Long Read Sequencing

- Perform SMRTbell library size selection using the SageELF System

- Sequence with 11 kb size-selected library fraction (supports ~4 SMRT Cell 8M)

Sequence on the Sequel II System

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

- Use CCS Sequencing Mode, 2-hour pre-extension time, and 30-hour collection times

- Generate 15-18 Gb of high-quality long reads per Sequel SMRT Cell 8M*

- Sequence to desired CCS coverage based on study needs:

- Recommend ~2-3 SMRT Cell 8M to achieve 15-fold CCS coverage for human studies

Data Analysis Solutions with the PacBio Analytical Portfolio

- Call single-nucleotide variants and small indels with Google DeepVariant or

GATK. DeepVariant provides higher precision and recall, particularly for indels

- Phase variants with WhatsHap

- Call structural variants and copy-number variants with the ‘Structural Variant Calling’

application in SMRT Link (powered by pbsv)

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

among other factors.

Application Brief: Variant detection using whole genome sequencing with HiFi reads – Best Practices

Technical Documentation and Resources

TECHNICAL DOCUMENTATION AND OTHER RESOURCES

- Procedure & Checklist - Preparing SMRTbell Libraries for HiFi Long Read Sequencing on

Sequel and Sequel II Systems (PN 101-714-400)

- Procedure & Checklist – AMPure PB Bead Purification of Polymerase Bound SMRTbell

Complexes (PN 101-348-800)

- SMRT Link v7.0.0 Sample Setup should be used to set up HiFi library annealing and binding

reactions using the Sequel II System (Chemistry v1.0)

- Note: In SMRT Link v7.0.0 Sample Setup, choose ‘Sequencing Primer v3’ from the dropdown menu

but substitute in Sequencing Primer v2

- Excel Sample Setup Calculator should be used to set up HiFi library annealing and binding

reactions using the Sequel System (Chemistry v3.0). [Please contact PacBio or your

Local FAS to request a copy of the Excel Sample Setup Calculator.]

- Application Brief: Variant detection using whole genome sequencing with HiFi reads – Best

Practices (PN: BP106-041919)

-Webinars

- AGBT Presentation (March 1, 2019): HiFi long reads for comprehensive genomic analysis [Mike

Hunkapiller, Pacific Biosciences]

- Publications

- Wenger, A.M., Peluso, P. et al. Highly-accurate long-read sequencing improves variant detection and

assembly of a human genome. BioRxiv Preprint (https://doi.org/10.1101/519025)

For Research Use Only. Not for use in diagnostics procedures. © Copyright 2019 by Pacific Biosciences of California, Inc. All rights reserved. Pacific Biosciences, the Pacific Biosciences logo,

PacBio, SMRT, SMRTbell, Iso-Seq, and Sequel are trademarks of Pacific Biosciences. BluePippin and SageELF are trademarks of Sage Science. NGS-go and NGSengine are trademarks of GenDx.

FEMTO Pulse and Fragment Analyzer are trademarks of Agilent Technologies Inc.

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

www.pacb.com

PACIFIC BIOSCIENCES® CONFIDENTIAL