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PACIFIC BIOSCIENCES® CONFIDENTIAL
For Research Use Only. Not for use in diagnostic procedures. © Copyright 2019 by Pacific Biosciences of California, Inc. All rights reserved. PN 101-784-400 Version 03 (July 2019)
Sequel II System v7.0 & SMRT Link v7.0
OverviewSequel II System v7.0 / SMRT Link v7.0 / Sequel II Chemistry v1.0
July 2019
Sequel II System v7.0 & SMRT Link
v7.0 OverviewA. Sequel II System v7.0 Overview
What’s Different with Sequel II Instrument Hardware / ICS / Consumables?
Sample Preparation Workflow Updates
SMRT Link v7.0 Sample Setup, Run Design & Run QC Updates
Example Sequel II System Performance Data
B. SMRT Link v7.0 Overview
Summary Overview of Major Improvements
SMRT Analysis Applications Feature Enhancements
Run Design and Analysis Support for CCS Sequencing Mode
High Throughput System Utilization and Data Traceability
SMRT Link v7.0 General Usability Improvements
C. Applications Best Practices Overview
Whole Genome Sequencing for De Novo Assembly of Large Genomes
Whole Genome Sequencing for Structural Variation Detection
Human Variant Detection
D. Technical Documentation & Software Download Resources
PACIFIC BIOSCIENCES® CONFIDENTIAL
For Research Use Only. Not for use in diagnostic procedures. © Copyright 2019 by Pacific Biosciences of California, Inc. All rights reserved.
Sequel II System v7.0 Overview
Sequel II System v7.0 / Sequel II Chemistry v1.0
July 2019
SEQUEL II SYSTEM – SEQUENCE WITH CONFIDENCE
Increased Throughput Capacity With the Sequel II System
1 million ZMWs
SMRT Cell 1M
8 million ZMWs
SMRT Cell 8M
Sequel System Sequel II System
-~8-fold increase in data yield
-Reduced project time
-Lower project cost
-Equivalent performance
SEQUEL II SYSTEM KEY APPLICATIONS
Human Variant Detection
Whole Genome Sequencing for De novo Assembly
of Large Genomes
Structural Variation Detection
Iso-Seq Whole Transcriptome Analysis
SMRT SEQUENCING WORKFLOW
-Complete template preparation in ~6 hours
-Accepts a variety of sample types and insert sizes
-Size-selection options to enrich for longest inserts
-Multiplexing and barcoding solutions available
-Rapid sequence time (0.5 to 30 hours)
-Serially process up to 8 SMRT Cells
-Walk away time up to four days
-Variety of analysis methods available through SMRT
Analysis and PacBio Analytical Portfolio
-DevNet and Analytical Partners applications
-Advanced data visualization and mining
What’s Different with Sequel II Instrument
Hardware / ICS / Consumables?
WHAT’S DIFFERENT WITH THE SEQUEL II SYSTEM?
- Instrument
-Work Deck
- Chassis
- Optics & Stage
- Computers
- Environmental
-Consumables
- SMRT Cell 8M
- Sequel II Binding Kit
- Sequel II Internal Control
- Sequel II Sequencing Kit (4 RXN)
-SMRT Link
- User selectable support for Sequel and/or
Sequel II
- Support for new High Fidelity (HiFi / CCS)
data type
SEQUEL
SEQUEL II INSTRUMENT HARDWARE: WHAT’S DIFFERENT?
SEQUEL II
Work Deck:
2 Cell Tray slots &
no MagBead Station
Chassis
Optics &
Stage
Environmental
Computers
SEQUEL II SYSTEM INSTRUMENT CONTROL SOFTWARE (ICS)
Sequel II System ICS Features:
-No substantial changes from Sequel
-Selectable run designs from SMRT Link
-Guided work deck loading
-Real-time run feedback and run metrics
-Directed post-run instrument clean-up
- ICS warnings and error messages are
the same for both Sequel and Sequel II
instruments
SEQUEL II SYSTEM INSTRUMENT CONTROL SOFTWARE
No Icons
for Tray
Slots 3 & 4
SEQUEL II SYSTEM CONSUMABLES
SMRTbell Template Preparation (No changes from Sequel)
- Standard SMRTbell Template Prep Kit 1.0; or
- SMRTbell Express Template Prep Kit 2.0
1M 8M
Sequel II Binding Kit
- Sequel II DNA Polymerase 1.0
- Sequel II DNA Internal Control 1.0
SMRT Cell 8M
- Only one type of SMRT Cell available for Sequel II
- SMRT Cell 8M supports movie times up to 30 hours
- SMRT Cell 8M requires a larger on-plate sample
volume (115 µl) compared to SMRT Cell 1M
Sequel II Sequencing Kit
- Only 4-rxn Kit is currently available
Sequel II Sample Preparation Workflow
Updates
SMRTBELL LIBRARY CONSTRUCTION
Existing Sequel library construction
procedures are compatible with Sequel II
-SMRTbell Template Prep Kit 1.0
- HiFi (CCS) Libraries
-Metagenomic Shotgun (CCS) Libraries
-SMRTbell Express Template Prep Kit 2.0
- Large insert gDNA library (≥10 kb)
-Microbial multiplexing (10 kb)
- Iso-Seq Express Method
- 16S Amplicon Sequencing
https://www.pacb.com/support/documentation/
-Sequel II System is Diffusion Loading only (no MagBead Loading option)
-Sequel II SMRTbell library ABC requirements:
- Recommended method for complex cleanup for all insert sizes is AMPure PB
purification (no changes)
- Final on-plate sample volume = 115 µl (robot aspirates 105 µl from sample plate; adds
105 µl of Dilution Buffer; and then dispenses 200 µl into a SMRT Cell 8M) (Sequel on-
plate sample volume is 85 µl)
-Sequel II DNA Internal Control:
- Expect ~3000 internal control reads per SMRT Cell 8M
SMRTBELL LIBRARY ANNEALING, BINDING & CLEANUP
WORKFLOW AND DNA INTERNAL CONTROL (SEQUEL II SYSTEMS)
Add 3.8 µl of serially diluted DNA Internal Control to final on-
plate sample dilution (Sequel internal control volume is 2.8 µl)
RECOMMENDED SAMPLE SETUP AND RUN DESIGN CONDITIONS
FOR SPECIFIC APPLICATIONS (SEQUEL II SYSTEM V7.0 / SEQUEL II
CHEMISTRY V1.0)
Application TPKSample Setup
Method Options
Seq. Mode
Seq. Primer
Annealing (nM)
Binding (nM)
Primer:Template
Poly:Template
Annealing Time (h)
Binding Time (h)
Complex Cleanup Method
Movie Time (h)
Pre-Ext. Time (h)
WGS for De Novo Assembly
Express TPK 2.0
SMRT Link v7.0, Excel
Workbook 6.2CLR V4 1.0 0.5 20:1 30:1 1 1
AMPure PB Beads
15.0NO Pre-
Extension
Variant Detection w/ HiFi Reads (11 -13 kb)
SMRTbell TPK 1.0
SMRT Link v7.0 (with V2 Primer
swap), Excel Workbook 6.2
CCS V2 1.0 0.5 20:1 10:1 1 4AMPure PB Beads
30.0 2.0
16S Amplicon Sequencing (1.6 - 2.5 kb)1,2
Express TPK 2.0
Excel Workbook 6.2
CCS V4 1.0 0.5 20:1 30:1 1 1AMPure PB Beads
10.0 0.4
10 kb Shotgun Metagenomics
SMRTbell TPK 1.0
SMRT Link v7.0 (with V2 Primer
swap), Excel Workbook 6.2
CCS V2 1.0 0.5 20:1 10:1 1 4AMPure PB Beads
30.0 2.0
Iso-Seq Analysis2
Express TPK 2.0
Excel Workbook 6.2
CCS V4 1.0 0.5 20:1 15:1 1 1ProNex Beads
24.0 2.0
Microbial Multiplexing
Express TPK 2.0
Excel Workbook 6.2
CLR V4 1.0 0.5 20:1 30:1 1 1AMPure PB Beads
15.0 2.0
1 Sequel II System QRC (Version 02 June 2019) only includes sample setup recommendations for 16S amplicon sequencing (1.6 – 2.5 kb); other amplicon
sizes are not currently supported.
2 For running 16S amplicon and Iso-Seq samples on the Sequel II System, please contact PacBio Technical Support or your local Field Applications Scientist
for specific details about additional sample setup and SMRT Link Run Design requirements.
RECOMMENDED SAMPLE SETUP AND RUN DESIGN CONDITIONS
FOR SPECIFIC APPLICATIONS (SEQUEL II SYSTEM V7.0 / SEQUEL II
CHEMISTRY V1.0) (CONT.)
Continuous Long Reads (CLR) Sequencing Mode
- CLR data correspond to reads with a subread length approx. equivalent to the polymerase read length
indicating that the sequence is generated from a single continuous template from start to finish
- CLR Sequencing Mode is useful for applications such as WGS de novo assembly and structural variation
detection
Circular Consensus Sequencing (CCS) Sequencing Mode
- CCS data correspond to reads generated from the consensus sequence resulting from alignment between
subreads taken from a single ZMW
- CCS Sequencing Mode is useful for applications such as variant detection and Iso-Seq studies
SEQUEL II LOADING AND PRE-EXTENSION RECOMMENDATIONS
- Refer to Diffusion Loading and Pre-
Extension Recommendations for the
Sequel II System Quick Reference Card
for sample loading recommendations
https://www.pacb.com/support/documentation/
SMRT Link v7.0 Sample Setup, Run
Design & Run QC Updates
SMRT LINK V7.0 SAMPLE SETUP TOGGLE FEATURE
- Toggle to specify either Sequel or Sequel II
- Filters relevant parameter fields in Sample Setup and Run Design
NEW AND UPDATED SMRT LINK V7.0 SAMPLE SETUP FIELDS
FOR SEQUEL II SYSTEM
- Chip Type (8mChip for Sequel II)
- Sequencing Mode (CLR or CCS)
- Selection of CLR vs. CCS Sequencing Mode in
Sample Setup toggles relevant parameters to
support recommended conditions for annealing
and binding samples for either CLR or CCS
sequencing applications, respectively
Continuous Long Reads (CLR)
- CLR data correspond to reads with a subread length
approx. equivalent to the polymerase read length
indicating that the sequence is generated from a single
continuous template from start to finish.
Circular Consensus Sequencing (CCS)
- CCS data correspond to reads generated from the
consensus sequence resulting from alignment between
subreads taken from a single ZMW.
NEW AND UPDATED SMRT LINK V7.0 SAMPLE SETUP FIELDS
FOR SEQUEL II SYSTEM (CONT.)
- Iso-Seq Experiment field is active only if CCS
sequencing mode is selected
- Iso-Seq Version
- Iso-Seq v1
- Iso-Seq Express
- Use Cleanup Step
- Replaces the previous “Cleanup” and “AMPure
Cleanup” fields
- Advanced Options
- Use Predictive Loading (Beta)
- Minimum Pipetting Volume (µl)
- Lock
- Locks the calculation to prevent further changes.
This is required before samples can be imported
into the Run Design module, and also sends a
copy of the instructions to the server for use in
Data Set reports.
- Locking ensures that calculations are always
synchronized with their run time state if a report is
generated at a later date.
NEW AND UPDATED SAMPLE SETUP SELECTION OPTIONS
Sample Setup Field Sequel Sequel II
Chip Type 1mChip 8mChip
Loading Diffusion / MagBead N/A (Sequel II is Diffusion ONLY)
Sequencing Mode N/A CLR / CCS
Sequencing Primer Sequencing Primer v3 / v4 Sequencing Primer v3 / v4
Binding Kit Sequel Binding Kit 2.0
Sequel II Binding Kit 1.0Sequel Binding Kit 2.1
Sequel Binding Kit 3.0
Internal Control Sequel DNA Internal Control 2.0
Sequel II DNA Internal Control 1.0Sequel DNA Internal Control 2.1
Sequel DNA Internal Control 3.0
Actions Copy / Remove Copy / Remove / Lock
- No MagBead Loading option in Sequel II
NEW SMRT LINK V7.0 SAMPLE SETUP FEATURES: ADVANCED
OPTIONS
Use Predictive Loading (Beta)
- This is a new feature under development. Predictive Loading uses active monitoring of the
ZMW loading process to predict a favorable loading end point. AMPure PB-based complex
cleanup requires a different buffer (not currently available) if this feature is used.
NEW SMRT LINK V7.0 SAMPLE SETUP FEATURES: ADVANCED
OPTIONS (CONT.)
Minimum Pipetting Volume
- This allows you to set a lower limit on pipetting volumes to use in certain protocol steps,
such as sample annealing and binding.
- PacBio recommends setting this to 1 µl, though in some cases, for example if sample
availability is very limited, it may be appropriate to set a value below 1 µl.
- Some protocol steps include fixed values of 1 µl that will not be affected by this setting.
NEW AND UPDATED SMRT LINK RUN DESIGN SAMPLE
INFORMATION FIELDS FOR SEQUEL II SYSTEM
- Removed MagBead Loading field
- Sequencing Mode
- Continuous Long Reads
- CCS Reads
- Movie Time
- SMRT Cell 8M part supports movie
times up to 30 hours
- Advanced Options
- Moved Pre-Extension Time field out
of Advanced Options
- Use Predictive Loading (Beta)
- PacBio highly recommends using
the default immobilization time (2 h)
unless specified otherwise
- No changes to Barcoded Sample
Options
- Other fields appear if CCS
Sequencing Mode is selected (see
upcoming slides)
PRE EXTENSION TIME OPTIONS FOR THE SEQUEL II SYSTEM
-Recommended pre-extension time is based on library insert length
- Calculation: 2*(Insert length + 50)/3.0 bases sec-1/60 sec min-1 /60
min hr-1
- Default recommendations for 600 bp – 20,000 bp inserts are displayed in SMRT Link
Sample Setup
-Pre-extension time is specified in hours (instead of minutes) for Sequel II System
-Maximum Pre-extension time for Sequel II System is 12 hours
DEFAULT RUN DESIGN LAYOUT FOR CONTINUOUS LONG READ
(CLR) SEQUENCING MODE
- Iso-Seq Experiment field is hidden
- Use Pre-Extension = NO by default
Generates one pass on each
library insert molecule
sequenced
Large Insert Sizes
(Approx. >15 kb)
DEFAULT RUN DESIGN LAYOUT FOR CIRCULAR CONSENSUS
SEQUENCING (CCS) SEQUENCING MODE
- Iso-Seq Experiment field appears
- Default value = ‘NO’
- Use Pre-Extension = YES by default
- Recommended Pre-Extension
Time (h) and Pre-Extension Time
(h) fields appear
- Generate CCS Data field appears
- Default value = ‘YES’
Generates multiple passes on each
library insert molecule sequenced
Small Insert Sizes
(Approx. ≤15 kb)
NEW AND UPDATED RUN DESIGN SELECTION OPTIONS
Run Design Field Sequel Sequel II
Sequencing Mode N/A CLR / CCS
MagBead Loading ON / OFF N/A (Sequel II is Diffusion ONLY)
Template Prep Kit SMRTbell Template Prep Kit 1.0 SMRTbell Template Prep Kit 1.0
SMRTbell Express Temp Prep Kit SMRTbell Express Temp Prep Kit*
SMRTbell Express Temp Prep Kit 2.0 SMRTbell Express Temp Prep Kit 2.0
Binding Kit Sequel Binding Kit 2.1Sequel II Binding Kit 1.0
Sequel Binding Kit 3.0
Sequencing Kit Sequel Sequencing Kit 2.1 (8 rxn)Sequel II Sequencing Kit 1.0 (4 rxn)
Sequel Sequencing Kit 2.1 (4 rxn)
Sequel Sequencing Kit 3.0 (8 rxn)
Sequel Sequencing Kit 3.0 (4 rxn)
DNA Control Complex Sequel DNA Internal Control 2.1Sequel II DNA Internal Control 1.0
Sequel DNA Internal Control 3.0
Movie Time per Cell Up to 1200 mins (20 hours) Up to 1800 mins (30 hours)
* Although the SMRTbell Express Template Prep Kit (1.0) is supported in Sequel II, there is no use case for
this discontinued template preparation kit product
SMRT LINK V7.0 RUN DESIGN ADVANCED OPTIONS
Use Predictive Loading (Beta)
- This is a new feature under development. Predictive Loading uses active monitoring of the
ZMW loading process to predict a favorable loading end point.
- If you select YES, fill in the following fields according to the instructions provided in the specific
Procedure & Checklist protocol document:
- Loading Target (P1 + P2): The fraction of ZMWs that the Predictive Loading routine will aim to load
with at least one sequencing complex. The default target for CCS applications is higher to
accommodate loss of complexes during pre-extension, which is generally recommended for all CCS
applications.
- Maximum Loading Time (hours): This defines the maximum time the system will allow loading to
progress before proceeding to sequencing. (Loading time in Predictive Loading is flexible.)
NEW SMRT LINK V7.0 RUN DESIGN FEATURES: PRE-ANALYSIS AND
AUTO-ANALYSIS
Pre-Analysis and Auto-Analysis are new features in SMRT Link v7.0 that allow a specific
analysis to be automatically run after a sequencing Sequel or Sequel II run has finished
and the data is transferred to the SMRT Link Server.
CCS Analysis Demultiplexing Mapping
Pre-Analysis Auto-Analysis
CCS Reads
Sequencing Mode
Auto-Analysis
Demultiplexing Resequencing
HGAP4
LAA
Sequencing Mode
CLR Reads
Pre-Analysis
SV
MVA
- Pre-Analysis is the process of CCS analysis
and/or demultiplexing of Sequel basecalled
data
- Pre-Analysis occurs before Auto-Analysis,
and is defined when you create a Run Design
and specify one or both of the following:
- Read Type = CCS Reads and Generate CCS
Data = Yes (Default)
- Read Type = CCS Reads and Sample is
Barcoded = Yes.
- Read Type = Continuous Long Reads and
Sample is Barcoded = Yes.
- Clicking on Pre-Analysis ID # in the Run
Design Summary displays information about
the Pre-Analysis, including the application
used.
PRE-ANALYSIS
AUTO-ANALYSIS
- Auto-Analysis can be set up in Run Design or SMRT Analysis after the Run
Design is saved and before the run is loaded on the instrument.
- Auto-Analysis can be run on CCS or CLR data and includes all analysis
applications available for the corresponding data type.
- Example Pre-Analysis and Auto-Analysis use cases:
1. Multiplexed CLR experiment:
- Pre-Analysis – Demultiplexing
- Auto-Analysis – Resequencing or de novo assembly, or LAA etc.
2. Non-multiplexed CLR experiment:
- Pre-Analysis – None
- Auto-Analysis – Resequencing, de novo assembly, SV, or LAA etc.
3. Multiplexed CCS experiment:
- Pre-Analysis – CCS analysis and Demultiplexing
- Auto-Analysis – Mapping, etc.
4. Non-multiplexed CCS experiment:
- Pre-Analysis – CCS analysis
- Auto-Analysis – Mapping, etc.
CREATING AN AUTO-ANALYSIS FROM A SMRT LINK V7.0 RUN
DESIGN
1. Create a new Run Design and save it. The Auto-
Analysis button is enabled only after you save the
Run Design.
2. Click Auto-Analysis. This takes you into SMRT
Analysis, where you create the new analysis that will
be associated with the collection.
3. Name the new analysis.
4. Click the numbered Collections link (Column 2 of the Runs table) associated with the run that you defined
in Step 1. (Note: Runs dis- play here only if they are in the Created state – not if they are already running
or have completed.)
5. Select a collection for analysis.
6. Click Next.
CREATING AN AUTO-ANALYSIS FROM A SMRT LINK V7.0 RUN
DESIGN (CONT.)
7. Select a secondary analysis application to use for the analysis.
8. (Optional) Click Advanced Analysis Parameters and specify the values of the parameters you would
like to change. Click OK when finished.
9. Click Create.
SMRT LINK V7.0 RUN QC: PRIMARY ANALYSIS METRICS SUMMARY
TABLE INCLUDES UNIQUE MOLECULAR YIELD (UMY)
CALCULATION
- Total Bases (GB): Calculated by multiplying the number of Productive (P1) ZMWs by the
mean Polymerase Read Length; displayed in Gigabases.
- Unique Molecular Yield (UMY): The sum total length of unique single molecules that were
sequenced. It is calculated as the sum of per-ZMW median subread lengths.
CLR Sample 01
CCS HiFi Sample Rep 1
CCS HiFi Sample Rep 2
11 kb CCS (HiFi) Library (30-h movie; 2-h Pre-Extension Time)
CLR Sample 01
CLR Sample 02
80 kb CLR Library (15-h movie; No Pre-Extension Time)
TOTAL BASES VS. UNIQUE MOLECULAR YIELD EXAMPLES
122.02 116.23
117.29 110.35
Total Bases
(GB)
Unique
Molecular Yield
(GB)
SMRT LINK V7.0 RUN QC: IMPROVED INSERT READ LENGTH
DENSITY PLOT
- Displays a (log scale) density plot of reads,
binned according to their Insert Read Length
and Polymerase (HQ) Read Length
- Large-insert libraries: most reads consist of a
single subread and will fall along the diagonal
- Short-insert libraries: subreads will be shorter
than the Polymerase Read Length, and will
appear as horizontal features
SMRT Link v7.0 Improvements to Insert
Read Length Density Plots:
1. Both plot axes are now labeled in units of
base pairs (bp) instead of log10 units
2. Both plot axes feature grid lines to enable
easier visualization and interpretation of
read length data
SMRT LINK V7.0 RUN QC: NEW DISTRIBUTION OF HQ START
POSITIONS PLOT
- This is a histogram of High Quality Region starting positions.
- This plot gives an indication of the fraction of P1 reads resulting from single singly loaded
ZMWs, and from multiply loaded ZMWs where productive sequencing can start later in the
run as competing polymerases terminate, leaving a single active polymerase.
>30 kb BluePippin-Size Selected CLR Library Example (15 h)
Distribution of HQ Start Positions
HQ Region Start Position
Nu
mb
er
of
Re
ad
s
11 kb HiFi Library Examples (10 h)
Under-Loaded Sample (A01) Over-Loaded Sample (C01)
Example Sequel II System Performance
Data
GENOMIC LIBRARY PERFORMANCE
Data shown above from a 35 kb size-selected E. coli library using the SMRTbell Template Prep Kit on a Sequel II System
(1.0 Chemistry, Sequel II System Software v7.0, 15-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) 166
Total Reads 5,201,973
Half of Bases in Reads >51,863
Longest Read Lengths >175,000
- Collaboration with Scott Geib at USDA
- Spotted Lantern Fly (Lycorma delicatula)
- Genome size ~2.4 Gb
- Size Selected (15 kb) SMRTbell large insert gDNA library
Input DNA from a single individual was converted into a SMRTbell library using the SMRTbell Express
Template Prep Kit 2.0 following the Procedure & Checklist - Preparing gDNA Libraries Using the
SMRTbell® Express Template Preparation Kit 2.0
CC BY-SA 3.0
https://commons.wikimedia.org/w/index.php?curid=279717
Sequencing and Assembly Parameters Sequel II (8M)
Number of SMRT Cells 1
Movie time per SMRT Cell (hrs) 30
Total Yield (Gb) 131. 6
Unique Molecular Yield (Gb) 82.4
Subread Length Mean (bp) 14,724
Assembly Size (Gb) 2.45
Contig N50 (Mb) 1.33
SEQUEL II SYSTEM LONG READ WGS SEQUENCING EXAMPLE:
DE NOVO ASSEMBLY OF A 2.5 GB PLANT HOPPER GENOME
COMPARISON OF DE NOVO ASSEMBLY RESULTS TO PREVIOUS
PLANTHOPPER ASSEMBLIES
Highlights of Lycorma delicatula Sequel II System WGS De Novo Assembly
-Wild-caught specimen instead of an inbred lab strain
- At least 100 times fewer individuals needed, and reaching the theoretical limit (1 insect)
- 1 sequencing library, 1 sequencing technology employed
- Assembled genome size is 2 times larger than any previous assembly
- Assembled genome at least 10 times more contiguous than any previous assembly
Nilaparvata lugens
(2014)
Sogatella furcifera
(2017)
Laodelphax
striatellus (2017)
Lycorma delicatula
(this work)
Number of individuals
(source)
~5,000
(F13 from inbred
line)
~120
(F6 from inbred line)
~100
(F22 from inbred
line)
1
(wild caught)
Number of sequencing
libraries
16
(+fosmid libraries)17 47 1
Assembly size 1.14 Gb 0.72 Gb 0.54 Gb 2.45 Gb
Contig N50 24 kb 71 kb 118 kb 1,330 kb
HIGH CONSENSUS ACCURACY
Consensus accuracy is a function of coverage and chemistry. The data above is based on a bacterial genome run on the
Sequel II System (1.0 Chemistry, Sequel II System Software v7.0). Single-molecule accuracy has similar coverage
requirements.
Achieves >99.999% (Q50)
UNIFORM COVERAGE
Mean coverage per GC window across a human sample. Data generated with a 35 kb human library on a Sequel II
System using 1.0 Chemistry and Sequel II System Software v7.0
No amplification required
Even coverage across GC content
CCS (HIFI) LIBRARY PERFORMANCE
Data shown above from a 11 kb size-selected human library using the SMRTbell Template Prep Kit on a Sequel II System (1.0 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 (1.0 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 PERFORMANCE (CONT.)
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%
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 COMPARISON
FOR 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 (1.0 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.
ISO-SEQ PERFORMANCE
Data shown above from a Universal Human Reference RNA (human) and Lexogen SIRV spike-in controls. The library was
constructed using the SMRTbell Template Prep Kit 1.0 on a Sequel II System (0.9 Chemistry, Sequel II System Software v6.1,
20-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) 255
Total Reads 4,460,955
Full Length Non-chimeric Reads 3,436,022
CCS Passes (Mean) 8
PACIFIC BIOSCIENCES® CONFIDENTIAL
For Research Use Only. Not for use in diagnostic procedures. © Copyright 2019 by Pacific Biosciences of California, Inc. All rights reserved.
SMRT Link v7.0 Overview
SMRT Link v7.0
July 2019
SMRT LINK V7.0 – SUMMARY OVERVIEW OF MAJOR IMPROVEMENTS
Enabled support for the Sequel II System with 8M SMRT Cell
- Scalability, efficiency and robustness improvements
Improved speed and scalability to support analysis applications
- Improved CCS algorithm; pbmm2 replaces blasr → ≥20-fold faster time to results
Enabled support for CCS sequencing mode
- Supports new CCS data type and run design options
Enhanced Structural Variant (SV) analysis application
- Expanded SV types detected → duplications and CNV; better sensitivity for larger indels
Enabled high-throughput system utilization features
- Automated analysis; compute resources utilization options
Improved data traceability
- Extensive PDF reports for data sets
Usability Improvements
- Analysis Setup Wizard; flexible selection of
demultiplexed data
COMPATIBILITY OF SMRT LINK V7.0 WITH SEQUEL AND SEQUEL II
SYSTEMS
-SMRT Link v7.0 supports Sequel and Sequel II Systems
- Customers with both systems can use one SMRT Link v.7.0 instance
-For customers operating a Sequel System only (and not a Sequel II System),
upgrading to SMRT Link v7.0 is optional
-SMRT Link v.7.0 is compatible with Sequel System ICS v6.0 with the following
limitations:
1. Updated CSV file is available for Run Design – compatible with SMRT Link v7.0
2. Automated Analysis feature for Sequel Systems:
- Can do Pre-Analysis only or Auto-Analysis only and not Pre-Analysis followed by Auto-Analysis
3. Predictive Loading (Beta) feature:
- This feature is available for Sequel II Systems only
SEQUEL II SYSTEM COMPUTE ENVIRONMENT REQUIREMENTS
a. For a standard CCS sequencing collection (300 GB sequencing data, 11 kb insert size, 30-hours movie), CCS Analysis takes 27 hours
on this configuration. SMRT Link will work on less powerful compute configurations, however analysis time will be significantly longer.
b. Storage is calculated for one Sequel II System, assuming 100 human genomes per year at 50-fold coverage, de novo assembly.
Head Node
Cores 32
RAM 64 GB
tmp_dir (Local Storage) 1 TB recommended, 500 GB minimum
db_datadir (Local Storage) 250 GB
Compute Nodes
Cores (Total) 384a
Minimum RAM per Slot (1 slot = 1 core) 4 GB/8 GB de novo assembly large complex genomes
tmp_dir (Local Storage) 100 GB
Shared Data Storageb
Sequence Data 40 TB
Analysis Data 70 TB
Network
10 GBE recommended, 1 GBE required
Data Examples
Human Assembly
- Genome size: 3.3 GB; Sequence data 400 GB; Analysis data 700 GB; Analysis time: 72 hours wall. This is a 50-fold coverage
human genome analyzed on the compute configuration listed above.
Rice Assembly
- Genome size: 0.43 GB; Sequence data 55 GB; Analysis data 90 GB; Analysis time: 20 hours wall. This is a 50-fold coverage
rice genome analyzed on the compute configuration listed above.
SEQUEL II SYSTEM COMPUTE ENVIRONMENT REQUIREMENTS
-Requirements are provided in the SMRT Link v7.0 Installation document and
Sequel II System Site Prep Guide
- For a standard CCS sequencing collection (300 Gb sequencing data, 11 kb insert size,
30-hour movie), CCS analysis takes 27 hours on this configuration
- For a de novo assembly of a Human genome with CLR data (50-fold coverage),
analysis time is 72 hours wall time.
-SMRT Link will work on less powerful compute configurations – however, analysis
times will be significantly longer.
-A complete HPC solution is available from BioSoft Integrators
- +1 (619) 793-5095
- https://biosoftintegrators.com/hpc
SMRT LINK V7.0 SETUP CONFIGURATION OPTIONS
SMRT Link v7.0 can be configured to support Sequel II, Sequel and/or
PacBio RS II Systems
-SMRT Link Run Design and Sample Set Up modules support sample preparation
procedures and run designs that are specific to Sequel and Sequel II Systems
DATA TYPES IN SMRT LINK V7.0
SMRT Link Now Supports Three Data Types
1. Sequel Data: Subreads generated from
Sequel or Sequel II instruments.
2. CCS Data: Single-molecule consensus
reads generated from CCS analysis
(HiFi reads). Can be from Sequel or
Sequel II System instruments
3. RS II Data: Subreads generated from
PacBio RS II instruments.
DATA TYPES IN SMRT LINK V7.0 (CONT.)
1. Sequel Data
- Assembly (HGAP4), LAA, CCS, Iso-Seq,
Resequencing, Structural Variant
Calling, Demultiplex Barcodes, Base
Modification Detection, etc.
2. CCS Data
- Mapping, Minor Variants Analysis, etc.
3. RS II Data
- Convert RS II to BAM
SMRT Analysis Applications Are Data Type Dependent
SMRT Analysis Applications Feature
Enhancements
SUMMARY OVERVIEW OF ANALYSIS APPLICATIONS IMPROVEMENTS
Mapping Analysis (for all relevant applications)
- pbmm2 replaces BLASR – enables at least 20 times faster mapping
- Applications: Resequencing, Iso-Seq analysis, Mapping, de novo assembly, SV analysis (SV analysis
uses pbmm2 since SMRT Link v6.0.0)
De novo assembly
- Bug fix: Previously, draft genomes >4 Gb could not proceed to polishing due to a reference
set bug. This has been addressed and they can now be polished in the HGAP4 workflow.
Structural Variant Calling Analysis
- Support for additional variant types – duplications and CNVs
- Improved sensitivity for larger insertions and deletions
Iso-Seq Analysis
- Only one Iso-Seq analysis application in SMRT Link v7.0 (‘Iso-Seq 3’ analysis is now
renamed as ‘Iso-Seq’ analysis)
- Overall faster time-to-results
CCS Analysis
- Redesigned and optimized for longer insert sizes (>3 kb)
- Significantly faster time-to-results – 8-10 times faster
- Example: Time-to-results for 300Gb CCS run with 10-12 kb insert
size, 30-hour movie is 27 hrs. on required HPC
CCS ANALYSIS IMPROVEMENTS
Optimized CCS algorithm for improved support for longer amplicons, enabling
faster time-to-results
- Reduce computational complexity from quadratic to linear
- Windowed draft sequencing generation
- Windowed polishing
- Better multi-threading balance to increase throughput
Example CCS Analysis Application Results in SMRT Link v7.0
STRUCTURAL VARIANT ANALYSIS IMPROVEMENTS
Improved SV analysis application supports calling new variant types and
enables better sensitivity for insertions and deletions
- Added two new variant types
1. Copy Number Variants (CNVs) – For whole genome sequencing data only; must be bound by a
translocation event
2. Duplications
- Improved sensitivity for larger insertions and deletions
- Optimized analysis speed (5 times faster) and memory usage
- Valid VCF file output
CHU Lyon 18A3182 has a copy number loss of hg19.chr3:25,570,417-
25,684,079 (114kb)
PBMM2 REPLACES BLASR – APPLICATIONS WITH MAPPING
Replacement of blasr algorithm with pbmm2 enables significantly faster time-
to-results for analysis applications with mapping
- Mapped yield and concordance metrics: Same or better performance
- Analysis wall time: Significantly faster mapping step (for all analysis applications with mapping)
- de novo assembly: Polishing step no longer limited by genome size
Organism Mapped Yield (bases) Mapped Concordance (%)Wall time (sorting only)
d:hh:mm:ss
blasr pbmm2 blasr pbmm2 blasr pbmm2Speed
up
Rice 26,587,016,552 27,729,575,396 4% 87.5 88.8 1.30% 3:02:41:30 0:01:52:27 97X
Ecoli 17,097,868,456 17,175,528,435 0% 86.9 87.1 0.20% 4:00:31:54 0:00:28:44 167X
Mouse 1,500,612,379 1,542,107,288 2% 85.6 86.1 0.60% 1:05:20:47 0:00:06:37 266X
Arabidopsis 798,461,392 825,051,602 3% 85.2 86.3 1.10% 0:02:47:53 0:00:03:12 52X
HG002 6,399,653,598 6,562,212,083 2% 84 84.8 0.80% 1:03:10:28 0:01:26:15 19X
Run Design and Analysis Support for CCS
Sequencing Mode
SMRT LINK V7.0 SUPPORTS HIGH FIDELITY (HIFI) CCS DATA TYPE
ANALYSIS APPLICATIONS
CCS (HiFi) sequencing workflow provides high accuracy and long read lengths
to enable calling of all variant types – from single nucleotides to structural
variants – using a single technology
- SMRT Analysis algorithmic
improvements for CCS Reads
- Redesigned and optimized for longer
amplicons (>3 kb)
- Analysis applications for CCS Reads
as input
- Mapping, Demultiplexing, SV calling
- SNV Calling and Phasing Tools
- CCS data output are compatible with NGS
SNV tools such as GATK, DeepVariant,
WhatsHap, etc.
DEFAULT RUN DESIGN LAYOUT FOR CIRCULAR CONSENSUS
SEQUENCING (CCS) SEQUENCING MODE
- Generate CCS Data field appears
- Default value = ‘YES’
- Auto-generates high-fidelity CCS read
data type after SMRT Cell data are
imported into SMRT Link
- SMRT Link Data Management module
automatically generates a data set report
with CCS performance statistics
- CCS data can be used as input for
SMRT Link analysis applications
- Note: If the SMRT Link instance
connected to your sequencing
instrument does not communicate
directly with your compute cluster, Set
“Generate CCS Data” = NO when you
are designing your run in Run Design
using the CCS Sequencing mode.
High Throughput System Utilization and
Data Traceability
ENABLEMENT OF HIGH-THROUGHPUT SYSTEM UTILIZATION
Analysis Automation Features
- Defined at Run Set Up or before a run is initiated on the instrument
- Analysis automatically starts when sequencing data are transferred to the SMRT Link server
Analysis Hardware Utilization Options
- Customizable analysis settings based on the available compute power
- SMRT Link Admin configures compute templates on the command line
- SMRT Link User selects template in advanced parameters UI for each analysis application
Data traceability
- From Sample Set Up to Analysis Results – trace data back and forth through the GUI
- Extensive Data Set PDF reports – loading statistics, run set up and QC information, analysis
parameters and results, charts and histograms, list of data output files
NEW SMRT LINK V7.0 AUTOMATED ANALYSIS FEATURES:
PRE ANALYSIS AND AUTO ANALYSIS
Pre Analysis and Auto Analysis are new features in SMRT Link v7.0 that allow a specific
analysis to be automatically run after a sequencing Sequel or Sequel II run has finished
and the data is transferred to the SMRT Link Server.
CCS Analysis Demultiplexing Mapping
Pre-Analysis Auto-Analysis
CCS Reads
Sequencing Mode
Auto-Analysis
Demultiplexing Resequencing
HGAP4
LAA
Sequencing Mode
CLR Reads
Pre-Analysis
SV
MVA
- Pre-Analysis is the process of CCS analysis
and/or demultiplexing of Sequel basecalled
data
- Pre-Analysis occurs before Auto-Analysis,
and is defined when you create a Run Design
and specify one or both of the following:
- Read Type = CCS Reads and Generate CCS
Data = Yes (Default)
- Read Type = CCS Reads and Sample is
Barcoded = Yes.
- Read Type = Continuous Long Reads and
Sample is Barcoded = Yes.
- Clicking on Pre-Analysis ID # in the Run
Design Summary displays information about
the Pre-Analysis, including the application
used.
PRE-ANALYSIS
AUTO-ANALYSIS
- Auto-Analysis can be set up in Run Design or SMRT Analysis after the Run
Design is saved and before the run is loaded on the instrument.
- Auto-Analysis can be run on CCS or CLR data and includes all analysis
applications available for the corresponding data type.
- Example Pre-Analysis and Auto-Analysis use cases:
1. Multiplexed CLR experiment:
- Pre-Analysis – Demultiplexing
- Auto-Analysis – Resequencing or de novo assembly, or LAA etc.
2. Non-multiplexed CLR experiment:
- Pre-Analysis – None
- Auto-Analysis – Resequencing, de novo assembly, SV, or LAA etc.
3. Multiplexed CCS experiment:
- Pre-Analysis – CCS analysis and Demultiplexing
- Auto-Analysis – Mapping, etc.
4. Non-multiplexed CCS experiment:
- Pre-Analysis – CCS analysis
- Auto-Analysis – Mapping, etc.
HOW TO SET UP AN AUTO-ANALYSIS FROM A SMRT LINK V7.0 RUN
DESIGN
1. Create a new Run Design and save it. The Auto-
Analysis button is enabled only after you save the
Run Design.
2. Click Auto-Analysis. This takes you into SMRT
Analysis, where you create the new analysis that will
be associated with the collection.
3. Name the new analysis.
4. Click the numbered Collections link (Column 2 of the Runs table) associated with the run that you
defined in Step 1. (Note: Runs dis- play here only if they are in the Created state - not if they are
already running or have completed.)
5. Select a collection for analysis.
6. Click Next.
HOW TO SET UP AN AUTO-ANALYSIS FROM A SMRT LINK V7.0 RUN
DESIGN (CONT.)
7. Select a secondary analysis application to use for the analysis.
8. (Optional) Click Advanced Analysis Parameters and specify the values of the parameters you would
like to change. Click OK when finished.
9. Click Create.
- In SMRT Analysis:
- Create New Analysis → select Auto-Analysis – Displays a list with all created runs
available for auto-analysis
- Data Type filters available runs based on selection of either Sequel Data or CCS
Data
HOW TO SET UP AN AUTO-ANALYSIS FROM SMRT ANALYSIS V7.0
HOW TO FIND AUTOMATED ANALYSIS RECORD IDS
- Pre-Analysis and Auto-Analysis IDs are active URL links to SMRT Analysis
This is the Pre-Analysis ID # for all
collections (SMRT Cells) in a Run
These are the ID #s for each Pre-
and Auto-Analysis job for each
SMRT Cell listed in the table
PRE- AND AUTO-ANALYSIS IDS ARE ACTIVE URL LINKS TO SMRT
ANALYSIS
- The Pre-Analyses ID number
displayed in Run Design Run
Information links to a summary
table in SMRT Analysis that lists
all Pre-Analysis jobs for the
corresponding run design
PDF REPORT EXPORT OPTION AVAILABLE FOR EACH DATA SET IN
SMRT LINK V7.0 DATA MANAGEMENT
-PDF Report Includes information for:
- Sample Setup
- Run Design
- Run QC
- Analysis application parameters and results
ADVANCED ANALYSIS PARAMETERS – CUSTOM COMPUTE SETTINGS
SMRT Link v7.0 enables usage of customized compute settings for each
analysis
Customized Compute Settings are configured by the SMRT Link Administrator and saved for
use in the SMRT Link GUI under Advanced Analysis Parameters
- Customized Compute Settings are only available if configured by the SMRT Link Administrator on the
command line using PacBio provided procedure
- Settings are specific for a customer’s compute environment and facilitate better utilization of compute
resources.
- Certain settings may be used based on computational demands of the analysis application – e.g. use
less powerful nodes for analyses with lower compute needs
SMRT Link v7.0 General Usability
Improvements
SMRT LINK V7.0 GENERAL USABILITY
IMPROVEMENTS
Analysis Wizard Feature
- Two-step workflow guiding the user through analysis set up process including visual
indication of the workflow step
Multiplexed SampleData Analysis Improvements
- Enabled selection of multiplexed samples (subsets) from different movie collections
- New ‘List’ feature – displays selected data sets and indicates their number
Miscellaneous Improvements
- Implemented consistent formatting for dates to conform with ISO standards
- Improved annotation of visualization graphs
- Improved visual indications to distinguish between required and optional fields
- Fixed width/wrapping issues with displaying Data Set names
- Improved display of tables in analysis reports
Step 1: Select Data
a. Select Data Type
b. Select Data Sets
➢ List feature icon ( ) indicates how many data sets are selected for analysis
ANALYSIS WIZARD FEATURE
Analysis Wizard is a two-step workflow that guides the user through the
analysis set-up process
Step 2: Select Analysis Application
➢ Specify analysis parameters as needed
ANALYSIS WIZARD FEATURE (CONT.)
MULTIPLEXED SAMPLES: ANALYSIS OF DATA SETS FROM
MULTIPLE DIFFERENT RUNS
The List feature enables selection of subsets from different collections for
analysis
1. Click on the number of
Demultiplexed Subsets of the
collection of interest
- A table with all available
subsets gets displayed.
1
MULTIPLEXED SAMPLES: ANALYSIS OF DATA SETS FROM
MULTIPLE DIFFERENT RUNS (CONT.)
3. Click the Back button above
the List icon to return to the
original list of collections for
analysis.
4. Use the same steps above to
select subsets from the next
collection to include for
analysis.
5. Once you’ve selected all
subsets for analysis, go to the
next step in the Analysis set
up – select Analysis
Application and Start Analysis
2. Select the subsets for analysis
- The List icon ( ) indicates
the number of subsets selected
3
2
5
SMRT LINK V7.0 KNOWN ISSUES
- When an Analysis or Data Sets page includes thumbnail plots, the displayed plots do not expand correctly
when clicked
- Selecting more than 384 Data Sets using the GUI, then clicking the List button, causes an error.
- If you create an analysis using multiple sub-Data Sets, then delete all sub-Data Set analyses, the combined
analysis is not shown as deleted.
- In SMRT Analysis, unchecking the “Failed” filter when the table of analyses is still loading (and says
“Loading...”) causes an error.
- After the initial installation, pressing the Browser refresh button before replying to the “Notify PacBio of
successful installation” dialog causes a "No pb-modal-underlay has been registered!" error.
- SMRT View can fail on Windows clients running older versions of Java. Update to the latest 64-bit Java for
SMRT View support.
- The Active Use time for the Sequencing Kit 3.0, 8-reaction version was incorrectly set to 72 hours. The
correct value is 100 hours. Users may see a warning that this reagent has expired when setting up a run
more than 72 hours after initial use of this item. Please disregard this warning if the elapsed time is less
than 100 hours.
- Auto Analysis cannot be copied to reuse the same analysis parameters.
- In Run Design, the Pre-Extension Time field may not allow entry of a value less than 1.0 hours. A value less
than 1.0 may be achieved through .csv file import if required. Using a value of 1.0 hour when the
recommended time is less than 1.0 should not adversely affect sequencing results.
- The Run Design .csv template file bundled with SMRT Link is an older version, and is not compatible with
this version of the software. A replacement may be obtained by contacting your local FAS representative.
See Release Notes for SMRT Link v7.0.1 for an updated list of known issues
PACIFIC BIOSCIENCES® CONFIDENTIAL
For Research Use Only. Not for use in diagnostic procedures. © Copyright 2019 by Pacific Biosciences of California, Inc. All rights reserved.
Sequel II System Applications Best
Practices OverviewSequel II System v7.0 / SMRT Link v7.0 / Sequel II Chemistry v1.0
July 2019
SEQUEL II SYSTEM KEY APPLICATIONS
Human Variant Detection
Whole Genome Sequencing for De novo Assembly
of Large Genomes
Whole Genome Sequencing for Structural Variation
Detection
Iso-Seq Whole Transcriptome Analysis
*Library yield is dependent upon high molecular weight DNA. See Procedure Preparing gDNA Libraries Using the SMRTbell® Express Template Preparation Kit 2.0
**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: Whole Genome Sequencing for De Novo Assembly – Best Practices (April 2019)
BEST PRACTICES: WHOLE GENOME SEQUENCING (WGS) FOR
DE NOVO ASSEMBLY (SEQUEL II SYSTEM)
Template Preparation with SMRTbell Express Template Prep Kit 2.0- Use high-quality, high molecular weight DNA (≥5 µg)
- Prepare long-insert SMRTbell libraries (>30 kb recommended)
- Library yields ~2 SMRT Cell 8M*
- Store library for several months without diminished sequencing performance
- Size select library for optimal yield and read lengths
Sequence on the Sequel II System (CLR Sequencing Mode)- Maximize output and turn-around-time with adjustable sequencing parameters
- 15-hour collection time yields up to 75 Gb per SMRT Cell 8M**
- 30 fold unique molecular coverage per haplotype recommended for assembly
- For a 1 Gb genome, ~1 SMRT Cell 8M* needed for a high-quality genome assembly
Data Analysis Solutions with the PacBio Analytical Portfolio - Generate highly accurate de novo assemblies with megabase-level contiguity
- Push-button assembly for small genomes with HGAP4 using SMRT Analysis
- Phased assembly at the command line with FALCON, FALCON-Unzip, and FALCON-Phase
from PacBio DevNet
- Network of analysis partners for platform or full-service bioinformatics
BEST PRACTICES: WHOLE GENOME SEQUENCING FOR
STRUCTURAL VARIATION DETECTION (SEQUEL II SYSTEM)
Template Preparation with SMRTbell Express Template Prep Kit 2.0- Use recommended high-quality unamplified genomic DNA input (~5 µg) from blood, saliva or cells
- Prepare >15 kb library
- Enrich for longest inserts with size selection
- Library yields ~2 SMRT Cell 8M
- Automated library-prep solutions available
Sequence on the Sequel II System (CLR Sequencing Mode)- Maximize output and turn-around-time with adjustable sequencing parameters
- ~75 Gb per Sequel SMRT Cell 8M* with a 15-hour collection time
- Sequence to desired coverage based on study needs:
- >20-fold: genetic disease studies – identify a variant or gene that causes disease in a cohort of
individuals with a shared phenotype; higher coverage required for de novo SV detection in trios
Data Analysis Solutions with the PacBio Analytical Portfolio - Detect the broadest range of SV types including insertions, deletions, inversions and
translocations
- Discover more SVs with the highest sensitivity – up to 20,000 SVs and 22,000 large indels per
genome
- Identify common SVs across multiple samples with joint calling
- Resolve breakpoints to sequence level
- Limit costly validation efforts with a low false-discovery rate of 5-10%
- Output data in standard file formats (BAM and VCF) for integration with downstream analysis tools
- Confirm SV calls visually with IGV and GenomeRibbon*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: Structural Variant Detection Using Whole Genome Sequencing – Best Practices (April 2019)
*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 (April 2019)
BEST PRACTICES: VARIANT DETECTION USING HIFI READS
(SEQUEL II SYSTEM)
Template Preparation with SMRTbell Template Prep Kit 1.0
- Start with unamplified genomic DNA input (≥15 µg)
- Prepare 10-15 kb library using the procedure: Preparing SMRTbell® Libraries for HiFi Long
Read Sequencing
- Size fractionate using the SageELF System
- Sequence with 11 kb fraction, supports ~4 SMRT Cell 8M
Sequence on the Sequel II System (CCS Sequencing Mode)
- Maximize output and turn-around-time with adjustable sequencing parameters
- Use CCS Sequencing Mode, 2 hour pre-extension, and 30 hour movie collection times
- Generate 15-18 Gb of high quality (≥Q20) long reads per Sequel II SMRT Cell 8M*
- Sequence to desired coverage based on study needs:
- Recommend ~2-3 SMRT Cell 8M to achieve 15-fold human coverage
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 than GATK, particularly for indels
- Phase small variants into phase blocks using WhatsHap
- Output data in standard file formats (BAM and VCF) for seamless integration with
downstream analysis tools
- Confirm variant calls visually with IGV10 and GenomeRibbon
BEST PRACTICES: LONG READ RNA SEQUENCING (ISO-SEQ
ANALYSIS) (SEQUEL AND SEQUEL II SYSTEMS)
* 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: Long-read RNA sequencing – Best Practices (June 2019)
Template Preparation with SMRTbell Express Template Prep Kit 2.0- 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 or Sequel II System (CCS Sequencing Mode)- Maximize output and turn-around-time with adjustable sequencing parameters
- Sequel System: Diffusion loading, 20 hour movies with 4 hours pre-extension is recommended
- Sequel II System: Diffusion loading, 24 hour movies with 2 hours pre-extension is recommended
- Use the Sequel System to generate up to 500,000* full-length, non-concatemer (FLNC) reads per
SMRT Cell 1M
- Use the Sequel II System to generate up to 4 million* FLNC reads per SMRT Cell 8M
- Scale throughput based on project needs – With a single Sequel II System 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
BEST PRACTICES: METAGENOMIC SEQUENCING (SEQUEL II
SYSTEM)
Application Brochure: Explore Metagenomes in High Resolution (June 2019)
BEST PRACTICES: METAGENOMIC SEQUENCING (SEQUEL II
SYSTEM) (CONT.)
Application Brochure: Explore Metagenomes in High Resolution (June 2019)
PACIFIC BIOSCIENCES® CONFIDENTIAL
For Research Use Only. Not for use in diagnostic procedures. © Copyright 2019 by Pacific Biosciences of California, Inc. All rights reserved.
Technical Documentation & Software
Download ResourcesSequel II System v7.0.0 / SMRT Link v7.0.0 / Sequel II Chemistry v1.0
April 2019
PACBIO DOCUMENTATION RESOURCES
- PacBio Documentation page allows you to search for and download the latest guides,
protocols, product information, and more.
https://www.pacb.com/support/documentation/
TECHNICAL DOCUMENTATION & SOFTWARE DOWNLOAD
RESOURCES
- Sequel II System Operations Guide (PN 101-774-700)
- Sequel II System 7.0.0 Release Notes (PN 101-774-400)
- Sequel II System Quick Reference Card - Loading and Pre-Extension Recommendations for the Sequel II
System (PN 101-769-100)
- SMRT Link v7.0.1 Software Download Site: https://www.pacb.com/support/software-downloads/
- SMRT Link v7.0.1 Software Installation Instructions (PN 100-749-900)
- SMRT Link v7.0.1 Release Notes (PN 100-749-600)
- SMRT Link v7.0.0 User Guide (PN 101-039-100)
- SMRT Tools Reference Guide (v7.0.0) (PN 100-939-900)
- Sequel SMRT Link Web Services API Use Cases (v7.0.0) (PN 101-430-800)
- Procedure & Checklist – Preparing SMRTbell Libraries for High Fidelity Long Read Sequencing on Sequel
and Sequel II Systems (PN 101-714-400)
- Pacific Biosciences Glossary of Terms (PN 000-710-267)
- Excel Sample Setup Calculator Workbook: Required for setting up specific types of library samples for
sequencing with the Sequel II System (refer to specific sample setup guidance provided for each
application type in relevant Procedure & Checklists and other documentation)
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
For Research Use Only. Not for use in diagnostic 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
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