For Research Use Only. Not for use in diagnostic procedures. © Copyright 2019 by Pacific Biosciences of California, Inc. All rights reserved. PN 101-742-500 Version 01 (February 2019)

SMRTbell Express Template Prep Kit 2.0:

Large Insert Genomic Library PreparationSequel System v6.0 / SMRT Link v6.0 / Sequel Chemistry v3.0

February.25.2019

SMRTbell Express Template Prep Kit 2.0

Overview: Large Insert Genomic Library

Preparation

1. Summary of Key Features & Benefits, New Reagents & Consumables and

Kit Configuration

2. Supported Large Insert gDNA Library Types, Sample Input Requirements

3. Large Insert Library Sample Preparation & Sequencing Workflow Details

4. Large Insert Library Example Performance Data (Sequel System 6.0 /

Chemistry 3.0)

5. Technical Documentation and Software Download Resources

6. Appendix: General Recommendations for High-Molecular Weight gDNA

QC, Shearing and Handling for SMRTbell Library Construction

SMRTBELL EXPRESS TEMPLATE PREP KIT 2.0 KEY FEATURES

- Improved formulation to accommodate

all gDNA library insert sizes from 10 kb to

>50 kb size-selected libraries*

-Streamlined, consolidated protocols for

improved ease of use for library

construction in ~4 hours

-Addition-only workflow eliminates DNA

loss during AMPure cleanups and enables

lower input requirements while

minimizing handling-induced DNA damage

-Updated multiplexing solutions for

microbial genomes with Express 2.0 Kit

SMRTbell Express Template Prep workflow is compatible with all genomic

DNA SMRTbell library insert sizes

• Single-tube, addition-only workflow

• ~4 hours workflow time†

• One Kit supports up to 18 large-insert

libraries, 48 microbial genomes & 96

amplicon template preparations*

† Not including DNA QC and library size-selection

* Iso-Seq method and multiplexed amplicon template compatibility COMING SOON (Q2/Q3

2019) to accommodate ALL library insert sizes (500 bp to >50 kb)

PACIFIC BIOSCIENCES® CONFIDENTIAL

SUMMARY COMPARISON OF SMRTBELL TEMPLATE PREP KIT (TPK)

PRODUCTS

SMRTbell TPK 1.0 SMRTbell Express TPK SMRTbell Express TPK

2.0

Available Now Discontinued (Available while supplies last)

New Product

For all insert sizes For >15 kb size-selected gDNA

libraries

For all library insert sizes (~500 bp

to >50 kb)

(Support for Iso-Seq method and

amplicon template preparations

≥500 bp COMING SOON!)

Supports 10 reactions Supports 16 reactions Supports 18 gDNA libraries & 48

microbial genomes

(Support for 96 amplicon template

preparations COMING SOON!)

~1.5 Day workflow (not including

DNA QC and library size-

selection); ~3.5 h hands-on time

~3.5 h workflow (not including DNA

QC and library size-selection)

~4 h workflow (not including DNA

QC and library size-selection)

Requires manual sample transfers

between tubes

Streamlined, single-tube addition-

only protocol

Streamlined, single-tube addition-

only protocol

Several AMPure purification steps One AMPure purification step at

end of library construction

One AMPure purification step at

end of library construction

SMRTBELL EXPRESS TPK 2.0 WORKFLOWS OFFER STREAMLINED

AND CONSOLIDATED PROTOCOLS FOR ALL APPLICATIONS

TPK 1.0 (Current) Express TPK 2.0

19 Protocols 5 Protocols

Express TPK 2.0 Supported Applications:

- All large-insert gDNA libraries

- De novo genome assembly

- Low-fold coverage structural variant detection

- Microbial Multiplexing gDNA libraries

Express TPK 2.0 Forthcoming Protocols:

- Iso-Seq method

- Amplicons with Barcoded Overhang Adapters

- Amplicons with F/R Universal Primers

NEW CONSUMABLES PRODUCTS TO SUPPORT SMRTBELL

EXPRESS TPK 2.0 WORKFLOWS

Available Now (Q1 2019):

Coming Soon:

- 96 Barcoded Overhang Adapter Plate

- 96 Barcoded Universal F/R Primers v2

Part Number Description Comments

100-938-900SMRTbell Express Template Prep Kit

2.0

• Core Kit required for all SMRTbell Express

TPK 2.0 workflows

101-633-500 Elution Buffer, 50 ml Bottle

• Stand-alone product to support additional

AMPure PB purification reactions for

multiplexed applications

101-628-400 Barcoded Overhang Adapter Kit-8A

• 8 Barcoded Overhang Adapters

• Supports 6 reactions per barcode

• Compatible with SMRTbell Express 2.0

101-628-500 Barcoded Overhang Adapter Kit-8B

• 8 Barcoded Overhang Adapters

• Support 6 reactions per barcode

• Compatible with SMRTbell Express 2.0

SMRTBELL EXPRESS TEMPLATE PREP KIT 2.0 CONFIGURATION

- Part Number: 100-938-900

- Each kit supports preparation of up to 18 large-insert gDNA libraries, 48 microbial gDNA libraries, and

96 amplicon template libraries

- Tubes are arranged in workflow order

- Diversity of tube cap colors to more easily distinguish components

- Overhang Adapter v3 contains a ‘T’ overhang

- Sequencing Primer v4 is only compatible with Diffusion loading

SMRTbell Express Template Prep Kit 2.0Tube # Description

1 TUBE, DNA Prep Buffer

2 TUBE, NAD

3 TUBE, Enzyme Dilution Buffer

4 TUBE, Nuclease-free Water

5 TUBE, DNA Prep Additive

6 TUBE, DNA Prep Enzyme

7 TUBE, DNA Damage Repair Mix v2

8 TUBE, End Prep Mix

9 TUBE, Overhang Adapter v3

10 TUBE, Ligation Mix

11 TUBE, Ligation Additive

12 TUBE, Ligation Enhancer

13 TUBE, Elution Buffer

13 TUBE, Elution Buffer

13 TUBE, Elution Buffer

14 TUBE, 10X Primer Buffer v2

15 TUBE, Sequencing Primer v4

BARCODED OVERHANG ADAPTER KIT 8A/8B CONFIGURATION

Barcoded Overhang Adapter Kit - 8A

Tube Image # Description1 TUBE, Bar Over Adapt - bc1001

2 TUBE, Bar Over Adapt - bc1002

3 TUBE, Bar Over Adapt - bc1003

4 TUBE, Bar Over Adapt - bc1008

5 TUBE, Bar Over Adapt - bc1009

6 TUBE, Bar Over Adapt - bc1010

7 TUBE, Bar Over Adapt - bc1011

8 TUBE, Bar Over Adapt - bc1012

Barcoded Overhang Adapter Kit - 8B

Tube Image # Description1 TUBE, Bar Over Adapt - bc1015

2 TUBE, Bar Over Adapt - bc1016

3 TUBE, Bar Over Adapt - bc1017

4 TUBE, Bar Over Adapt - bc1018

5 TUBE, Bar Over Adapt - bc1019

6 TUBE, Bar Over Adapt - bc1020

7 TUBE, Bar Over Adapt - bc1021

8 TUBE, Bar Over Adapt - bc1022

Each Barcoded Overhang Adapter Kit Supports 6 Reactions per Barcode

Barcoded Overhang Adapter Kit – 8A

Barcoded Overhang Adapter Kit – 8B

SMRTBELL EXPRESS TEMPLATE PREP KIT 2.0 USES A/T LIGATION

INSTEAD OF BLUNT END LIGATION FOR (BARCODED AND NON-

BARCODED) ADAPTER LIGATION REACTIONS

Why?

1. Reduces chimera formation

2. Reduces adapter dimerization (requires fewer AMPure cleanup steps)

SMRTBELL EXPRESS TEMPLATE PREP KIT 2.0 REAGENT

HANDLING RECOMMENDATIONS

- Several tubes in the kits are sensitive to temperature and vortexing

- PacBio highly recommends:

- Never leaving tubes at room temperature

- Working on ice at all times when preparing master mixes

- Finger tapping followed by a quick-spin prior to use

Supported Large Insert gDNA Library Types

and Sample Input Requirements

SUPPORTED LARGE-INSERT GENOMIC DNA LIBRARY TYPES

AND SAMPLE INPUT REQUIREMENTS

- SMRTbell Express Template Prep Kit 2.0 is designed for diffusion loading of large-

insert gDNA libraries on the Sequel System for de novo genome assembly and structural

variation detection applications

Recommended Shearing Methods and Input DNA Amounts for Large

Insert gDNA Library Construction (10 kb to >50 kb)

Large Insert Library Sample Preparation &

Sequencing Workflow Details

PROCEDURE & CHECKLIST – PREPARING GDNA LIBRARIES USING

THE SMRTBELL EXPRESS TEMPLATE PREPARATION KIT 2.0

- Procedures for constructing large insert gDNA

libraries using the SMRTbell Express Template

Prep Kit 2.0 with the following size ranges:

1. ~10 kb (with and without size selection)

2. >15 kb (size-selected using BluePippin system)

3. >30 kb (size-selected using BluePippin system)

- Streamlined and accelerated workflow for all

large-insert gDNA libraries in ~4 hours (not

including gDNA QC and library size selection)

- Protocol document contains:

1. Recommendations for gDNA QC and quantification

2. Recommendations for shearing gDNA to the

desired insert size

3. Enzymatic steps in preparation of the SMRTbell

library preparation

4. Size-selection of the final SMRTbell library (if

applicable)

PROCEDURE & CHECKLIST – PREPARING GDNA LIBRARIES USING

THE SMRTBELL EXPRESS TEMPLATE PREPARATION KIT 2.0

List of Required Materials and Equipment for Library Construction and Size

Selection

SMRTBELL EXPRESS TEMPLATE PREP KIT 2.0 LARGE-INSERT

GENOMIC DNA LIBRARY PREPARATION WORKFLOW OVERVIEW

1. gDNA QC & Shearing

- CHEF Mapper, Femto Pulse or Pippin Pulse sizing QC

- g-Tube, Megaruptor or Needle Shearing

2. SMRTbell Express Library Construction

- Single-Tube, Addition-Only Reactions

- ~4-h hands-on time workflow

3. Size-select & Purify SMRTbell Library

- >15 kb (~5 h BP) or >30 kb (~10 h BP)

- AMPure PB purification of final library

4. Sample A/B/C & Sequence

- Anneal v4 Primer, Bind Polymerase, AMPure PB

Complex Cleanup

- Follow QRC for diffusion loading recommendations

5. Analyze

- Analyze, visualize, and manage data through GUI or

command-line interface.

STANDARD SMRTBELL TPK 1.0 VS. SMRTBELL EXPRESS TPK 2.0

WORKFLOW TIME COMPARISON

Example times shown are for constructing

a >15 kb size-selected large insert library

starting with sheared and purified gDNA

ExoVII Pre-Tx

0.45X AMPure

Adapter Ligation

ExoIII / ExoVII

>15 kb Size Sel.

0.45X AMPure

DDR

0.45X AMPure

ER

DDR

Day 1

Day 2

Day 3

0.45X AMPure

SMRTbell Template Prep Kit 1.0

20 m

35 m

O/N

65 m

>5 h

35 m

65 m

35 m

15 m

65 m

35 m

Step Time

Remove ssDNA OH

Ligation

ER / A-Tailing

DDR

AMPure

∼ 3.5 h

O/N

>15 kb Size Sel.

SMRTbell Express Template Prep Kit 2.0

35 m

35 m

20 m

65 m

45 m

Step Time

>5 h

AMPure 35 m

SPECIAL HANDLING RECOMMENDATIONS FOR PREPARING

LARGE-INSERT GENOMIC DNA SMRTBELL LIBRARIES

1. For small numbers of samples, use DNA Lo-Bind 1.5 mL microcentrifuge tubes for all

enzymatic and AMPure PB bead purification steps.

2. Multi-channel pipettes and PCR strip tubes may be used to efficiently process large

numbers of samples.

3. Use wide-bore tips for all pipette mixing steps when preparing >15 kb and >30 kb size-

selected libraries.

4. Never vortex tubes containing high molecular weight DNA samples. Mix by gentle pipetting

or by flicking the tube.

5. Always follow best practices for DNA quantitation using a Qubit system:

- Use the dsDNA high sensitivity reagent kit

- Prepare the Qubit working solution by diluting the Qubit reagent 1:200 in Qubit buffer. Prepare 200 μl of

working solution for each standard and sample. Always prepare fresh standards for each assay.

- Set up two 190 μl assay tubes for the standards and one 199 μl assay tube for each sample. Add 10 μl

standard (from kit) and 1 μl sample to the respective assay tubes. Both the standard and sample

DNAs should be at room temperature.

- Vortex all tubes for 2 seconds

- Incubate the tubes for 2 minutes at room temperature prior to measurement

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

➢ Up to 165 kb

➢ Up to 80 kb

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 ~50

kb and is thus not suitable for constructing a >30 kb size-selected

library. Depending on the application, the DNA samples in Lane A4

and B3 (although fragmented) may be constructed to a SMRTbell

library without shearing. If size selection is required, a less

aggressive cutoff may be performed for these samples.

A B

RECOMMENDATIONS FOR EVALUATING GENOMIC DNA QUALITY

GENOMIC DNA SHEARING RECOMMENDATIONS

- For plant or animal whole genome assembly projects, it is highly recommended to start with

high molecular weight gDNA which can be sheared and size-selected to 30 kb or larger

- PacBio recommends performing test shears to determine the best shearing condition for

your samples. The response of individual gDNA samples to recommended shearing

parameters may differ and must be determined empirically and evaluated by PFGE or other

suitable systems

- To ensure sufficient yields of final size-selected libraries, the input gDNA sample must be

sheared so that the average size of the fragmented DNA remains well above the desired

size-selection cut-off

SMRTBELL LIBRARY SIZE SELECTION RECOMMENDATIONS

- When constructing large insert SMRTbell libraries for whole genome sequencing

of complex organisms, it is beneficial to remove small insert SMRTbell templates

by performing size selection with the BluePippin System (which collects fragments

above a size cut-off threshold)

- For the latest BluePippin User Manual and guidance on size-selection protocols,

please contact Sage Science (www.sagescience.com)

- Visit Sage's website (http://www.sagescience.com) to verify that your BluePippin

software is up-to-date. (Current version as of February 2019 is v6.31)

- Use Lane 4 for Size Markers

- Recommended Blue Pippin Run Setup Protocols for size selection:

SAMPLE SETUP RECOMMENDATIONS FOR SMRTBELL EXPRESS

TPK 2.0 LARGE INSERT GENOMIC DNA LIBRARIES

- Primer Annealing: Follow the instructions in SMRT Link Sample Setup and select

Sequencing Primer v4

- Anneal Sequencing Primer v4 at a template concentration of 0.833 nM (20:1

Primer:Template ratio); incubate at 20˚C for 1 hour

- Polymerase binding: Follow the instructions in SMRT Link Sample Setup and

select the appropriate version of Sequel Binding Kit

- Bind Sequel Polymerase at a concentration of 0.500 nM (30:1 Polymerase:Template

ratio); incubate at 30˚C for 1 hour

- Complex Cleanup: Follows the instructions in SMRT Link Sample Setup.

- For additional details, refer to the Procedure & Checklist – AMPure PB Bead

Purification of Polymerase Bound SMRTbell Complexes

SAMPLE LOADING RECOMMENDATIONS FOR SMRTBELL EXPRESS

TPK 2.0 LARGE INSERT GENOMIC DNA LIBRARIES

- Refer to Diffusion Loading and

Pre-Extension

Recommendations for the

Sequel System Quick

Reference Card for sample

loading recommendations

- Diffusion Loading

- 10-hour movies

SMRTbell Express Template Prep Kit 2.0

Large Insert Library Example Performance

Data (Sequel System 6.0 / Chemistry 3.0)

EXAMPLE IN-HOUSE QC ASSESSMENT OF UNSHEARED / SHEARED

GENOMIC DNA AND EXPRESS TPK 2.0 SMRTBELL LIBRARIES (PRE-

AND POST-BLUEPIPPIN SIZE SELECTION)

48.5 kb

97 kb

48.5 kbMode ~ 30 kb

20 kb

Example QC gel image for a >15 kb size-selected

SMRTbell Express 2.0 Library showing a mode of

~30 kb

Example QC gel image for a >30 kb size-selected

SMRTbell Express 2.0 Library showing a DNA

fragment smear size extending up to ~97 kb

SMRTBELL EXPRESS TEMPLATE PREP KIT 2.0 EXAMPLE IN-HOUSE

YIELD PERFORMANCE SUMMARY (SEQUEL SYSTEM 6.0 /

CHEMISTRY 3.0 / 10-H MOVIES)

SMRTbell

Library Type

SMRTbell

Library Yielda

Sequencing

Yield (Gb)b

# of SMRT Cells

Achievablec

10 kb Non-size-

selected LibrariesUp to 75% Up to 450 Gb >40

>15 kb Size-selected

LibrariesUp to 27% Up to 250 Gb >20

>30 kb Size-selected

LibrariesUp to 20% Up to 35 Gb >4

a Yield of purified non-size selected or size-selected SMRTbell library per sheared gDNA input amount

b Yield of mapped bases per input µg of sheared gDNA input amount

c Minimum number of SMRT Cells supported per SMRTbell library synthesis reaction

- Note: Sequencing performance, reads/data per SMRT Cell and other expected results

vary based on sample quality/type and insert size

SMRTBELL EXPRESS TEMPLATE PREP KIT 2.0 EXAMPLE IN-HOUSE

SEQUENCING PERFORMANCE SUMMARY (SEQUEL SYSTEM 6.0 /

CHEMISTRY 3.0 / 10-H MOVIES)

SMRTbell Library

Type

Polymerase

Read length

(Mean)

Polymerase

Read Length

(N50)

Longest

Subread

(Mean)

Longest

Subread

(N50)

10 kb Non-size-

selected Libraries20 to 24 kb 43 to 50 kb 5.5 to 6.5 kb 7.8 to 8.9 kb

>30 kb Size-selected

Libraries18 to 25 kb 31 to 39 kb 17 to 23 kb 39 to 37 kb

- Note: Sequencing performance, reads/data per SMRT Cell and other expected results

vary based on sample quality/type and insert size

SMRTBELL EXPRESS TEMPLATE PREP KIT 2.0 EXAMPLE IN-HOUSE

SEQUENCING ACCURACY PERFORMANCE COMPARISON (SEQUEL

SYSTEM 6.0 / CHEMISTRY 3.0 / 10-H MOVIES)

Express 2.0

Large Insert

Control Cell 1

Express 2.0

Large Insert

Control Cell 2

Express 2.0

Large Insert

Control Cell 3

Express 2.0

Large Insert

Control Cell 4

Express TPK

Large Insert

Control Cell 1

Express TPK

Large Insert

Control Cell 2

Express TPK

Large Insert

Control Cell 3

Technical Documentation and Software

Download Resources

SMRTBELL EXPRESS TPK 2.0 PRODUCT INFO & RESOURCES FOR

LARGE INSERT GENOMIC DNA LIBRARY PREPARATION

-Product Note: An Express Workflow to

Generate SMRTbell Libraries for

Genomic Libraries in as little as 4 Hours

(PN PN103-021919)

Supported Applications Discussed

- De novo genome assemblies

- Low-fold coverage structural variant detection

Key Benefits:

- Reduced gDNA input requirement

- Fast library template preparation

- Minimal handling-induced gDNA damage

Protocol

- Procedure & Checklist - Preparing gDNA Libraries Using

the SMRTbell Express Template Preparation Kit 2.0

Library Generation Time

- Approx. 4 hours (excluding QC and size-selection)

Libraries Supported

- # Rxns: 18 large gDNA libraries

- Insert Sizes: 10 kb to >50 kb

SMRTBELL EXPRESS TPK 2.0 PRODUCT INFO & RESOURCES FOR

LARGE INSERT GENOMIC DNA LIBRARY PREPARATION (CONT.)

-SMRTbell Express Template Prep Kit 2.0 (PN 100-938-900)

-SMRTbell Express Template Prep Kit 2.0 – Product Insert (PN 101-685-400)

-SMRTbell Express Template Prep Kit 2.0 – SDS (PN 101-692-200)

-Procedure & Checklist – Preparing gDNA Libraries Using the SMRTbell Express

Template Preparation Kit 2.0 (PN 101-693-800)

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

SMRTbell Complexes (PN 101-348-800)

-Quick Reference Card – Loading and Pre-extension Recommendations for the

Sequel System (PN 101-461-600)

-Applications Website: Whole Genome Sequencing Application

-Application Brief: Plant and Animal Whole Genome Sequencing Best Practices

(PN BP102-100418)

-Application Brief: Low-coverage, Long-read Whole Genome Sequencing for

Structural Variation (PN BP104-092518)

- Infographic: Gold-Standard Reference Genomes Accelerate Science (PN INF102-

082718)

SMRT LINK 6.0 SOFTWARE DOWNLOAD WEBSITE

https://www.pacb.com/products-and-services/sequel-system/latest-system-release/

General Recommendations for High-

Molecular Weight gDNA QC, Shearing and

Handling for SMRTbell Library Construction

SAMPLE COLLECTION, PREPARATION, AND STORAGE FOR

SMRT SEQUENCING WHOLE GENOME PROJECTS

Technical Note TN100-040518: Preparing

samples for PacBio whole genome sequencing

for de novo assembly – Collection and storage

To obtain the highest quality genomic DNA, it is important to start with

sample types compatible with HMW DNA extraction methods

- This Technical Note provides general guidance on

biological sample collection, preparation, and

storage across a range of commonly encountered

sample types used for SMRT Sequencing whole

genome projects

- Includes sample and storage recommendations for:

- Vertebrates - mammals, birds, fish, amphibians,

reptiles

- Invertebrates - marine, terrestrial

- Arthropods - insects, crustaceans

- Fungi - microorganisms, mushrooms, algae*

- Plants - broad leaf plants, grasses

*Algae is included with fungi due to similar growth and

storage conditions

- Includes additional considerations for planning

HMW DNA isolation

Before gDNA Extraction:

- Microbial gDNA Isolation:

- Avoid culture incubation in complex or rich media

- Harvesting from several replicate cultures rather than a single, high-density culture is preferred

- gDNA isolation from stationary-phase cultures is preferred to help ensure more uniform sequencing read coverage

across the genome

- Extraction of small culture volumes is preferred over large volumes to avoid accumulating high concentrations of

potentially inhibiting secondary components

After gDNA Extraction:

- If gel purification is required, avoid using ethidium/UV based visualization methods. One alternative is

to use SYBR® Safe (Invitrogen) and visualize with blue light

- To help resuspend the DNA, carefully invert the tube several times after adding buffer and/or tap the

tube gently. Avoid vortexing genomic DNA when possible as vortexing can cause shearing of the DNA.

It is also recommended to use wide bore tips in sample handling

- Alternatively, allow the DNA to stand in buffer overnight at 25°C to resuspend

- Overheating can introduce DNA damage. Inactivate DNAase as recommended by the vendor kit. It is

best to avoid heat inactivation when possible

- DNA storage conditions: 4 °C (short-term); -20°C / -80°C (long-term)

GENERAL RECOMMENDATIONS FOR ISOLATING HIGH-MOLECULAR

WEIGHT GENOMIC DNA

EXAMPLE THIRD-PARTY HIGH-MOLECULAR WEIGHT GENOMIC

DNA ISOLATION PROTOCOL AND KIT SOLUTIONS

Bacteria (Gram Negative and Gram Positive)

QIAGEN Genomic-tip 20/100/500/G Kit

- https://www.qiagen.com/de/shop/sample-technologies/dna/genomic-dna/qiagen-genomic-tip-500g/#productdetails

QIAGEN Gentra Puregene Yeast/Bact. Kit

- https://www.qiagen.com/de/shop/sample-technologies/dna/genomic-dna/gentra-puregene-yeastbact-kit/#productdetails

Geneaid™ DNA Isolation (Bacteria) Kit

- http://www.geneaid.com/

Circulomics Nanobind CBB Kit

- https://www.circulomics.com/

Lucigen Masterpure Kit

- https://www.lucigen.com/

Yeast and Fungi

QIAGEN Genomic-tip 20/100/500/G Kit / QIAGEN Gentra Puregene Yeast/Bact. Kit

GeneJET Plant Genomic DNA Purification Kit

- https://www.thermofisher.com/order/catalog/product/K0791

Zymo Research Fungal/Bacterial DNA MidiPrep™ Kit

- https://www.zymoresearch.com/dna/microbial-environmental-dna-isolation-1/bacterial-fungal-dna/zr-fungal-

bacterial-dna-midiprep

Phenol Chloroform Extraction

- Reference: Long-read, whole-genome shotgun sequence data for five model organisms. Scientific Data

1:140045 (2014) DOI: 10.1038/sdata.2014.45 http://www.nature.com/articles/sdata201445

- Reference: Extraction of High Molecular Weight DNA from Fungal Rust Spores for Long Read Sequencing.

Sambasivam Periyannan (ed.),Wheat Rust Diseases: Methods and Protocols, Methods in Molecular Biology, vol.

1659,DOI 10.1007/978-1-4939-7249-4_5 https://www.springer.com/us/book/9781493972487

Plant and Algae Tissue

QIAGEN Genomic-tip 20/100/500/G Kit

Procedure & Checklist – Preparing Genomic DNA for Large Insert SMRTbell Libraries from

Arabidopsis

- http://www.pacb.com/wp-content/uploads/2015/09/Shared-Protocol-Preparing-Arabidopsis-DNA-

for-20-kb-SMRTbell-Libraries.pdf

Unsupported Protocol – Switchgrass (Panicum virgatum) DNA isolation [USDA]

- http://www.pacb.com/wp-content/uploads/2015/09/Switchgrass-DNA-isolation.pdf

Unsupported Protocol – DNA extraction of Chlamydomonas using CTAB [JGI]

- http://www.pacb.com/wp-content/uploads/2015/09/DNA-extraction-chlamy-CTAB-JGI.pdf

QIAGEN User-Developed Prototocol: Isolation of genomic DNA from plants and filamentous fungi

using the QIAGEN Genomic-tip Kit

- https://www.qiagen.com/de/resources/resourcedetail?id=cb2ac658-8d66-43f0-968e-

7bb0ea2c402a&lang=en

Circulomics Nanobind Plant Kit

- https://www.circulomics.com/

Human Tissue

QIAGEN Genomic-tip 20/100/500/G Kit / QIAGEN Gentra Puregene Cell Kit

Unsupported Protocol – Gentra Puregene Cell Kit (Qiagen) DNA Isolation [Univ. Washington]

- http://www.pacb.com/wp-content/uploads/2015/09/Gentra-Puregene-Qiagen-DNA-Isolation.pdf

Reference: Resolving the complexity of the human genome using single-molecule sequencing. Nature 517, 608-611 (29

January 2015) doi:10.1038/nature13907 http://www.nature.com/nature/journal/v517/n7536/full/nature13907.html

Phenol Choloroform Extraction with Phase Lock Gel™ (PLG) [Sick Kids Hospital]

- Please see PacBio 2016 North America UGM Sample Prep Workshop Customer Presentation

“Comparison of g-Tube and Needle Shearing” by Karen Ng (OICR) or contact your local FAS

Insect Tissue

Phenol Chloroform Extraction (Drosophila melanogaster)

- Reference: Long-read, whole-genome shotgun sequence data for five model organisms. Scientific Data

1:140045 (2014) DOI: 10.1038/sdata.2014.45 (Phenol Chloroform Method)

http://www.nature.com/articles/sdata201445

QIAGEN User-Developed Prototocol: Isolation of genomic DNA from mosquitoes or other insects using the

QIAGEN Genomic-tip Kit

- https://www.qiagen.com/ca/resources/resourcedetail?id=b45c3cc3-7f2b-4f4a-aa37-21d814ed3730&lang=en

Fish Tissue

QIAGEN Genomic-tip 20/100/500/G Kit

- Reference: Chromosomal-Level Assembly of the Asian Seabass Genome Using Long Sequence

Reads and Multi-layered Scaffolding. PLOS Genetics 12(4): e1005954. (2016) doi:

10.1371/journal.pgen.1005954

http://journals.plos.org/plosgenetics/article?id=10.1371%2Fjournal.pgen.1005954

QIAGEN User-Developed Prototocol: Purification of archive-quality DNA from 10–20 mg fish tissue

using the Gentra® Puregene® Tissue Kit or Gentra Puregene Mouse Tail Kit

- https://www.qiagen.com/ca/resources/resourcedetail?id=948e5a5e-57c4-482f-a852-

43ec3da97fce&lang=en

Other Application-Specific DNA Extraction Protocols

Contact your Local Field Applications Scientist for details

- Metagenomic DNA Extraction Protocol (Tighe lab, University of Vermont)

- Fecal Microbiome DNA Extraction Protocol (Microbes Environ. Vol. 22, No. 3, 214-222,

2007)

- Stool and tissue DNA Extraction Protocol (Christian Hoffman, Instituto de Ciências

Biológicas, Universidade Federal de Goiás)

- Brain DNA extraction (Taylor Lab, Toronto Hospital for Sick Children)

- Bacterial Genomic DNA Extraction (Ehrlich Lab, Allegheny Singer Research Institute)

High Molecular Weight gDNA Cleanup

Unsupported Protocol – High Salt Phenol Chloroform Cleanup

- http://www.pacb.com/wp-content/uploads/2015/09/Shared-Protocol-Guidelines-for-Using-a-Salt-

Chloroform-Wash-to-Clean-Up-gDNA.pdf

AMPure PB Bead Wash

- Refer to the appropriate Procedure & Checklist protocol document for specific recommendations for

AMPure PB bead purification of different SMRTbell library insert sizes

SMRTbell Library Cleanup

Unsupported Protocol – Purification of Contaminated

SMRTbell™ Library Using Magnetic Bead Capture

- http://www.pacb.com/wp-content/uploads/2015/09/Purifying-Contaminated-SMRTbell-Libraries-

Using-MagBeads-052013.pdf

GENERAL RECOMMENDATIONS TO HELP CLEANUP HIGH

MOLECULAR WEIGHT GENOMIC DNA AND SMRTBELL LIBRARIES

Starting with high-quality, high molecular weight (HMW) genomic DNA will

result in longer libraries and better de novo assembly performance

METHODS FOR EVALUATION OF GENOMIC DNA QUALITY

Technical Note TN101-040518: Preparing DNA

for PacBio Whole Genome Sequencing for de

novo Assembly: Quality Control and Shearing

- Input genomic DNA must be carefully QC’d to

assess integrity

- PFGE/FIGE or Femto Pulse sizing tool is highly

recommended

- High molecular weight DNA → long read lengths

- Degraded DNA → short read lengths, low library

recovery yields (dependent on BluePippin size

selection parameters)

- DNA purity can be determined by using a NanoDrop

instrument or other spectrophotometer device

- PacBio highly recommends using the Qubit High

Sensitivity fluorometric assay for accurate dsDNA

quantitation

- This Technical Note provides recommendations,

tips and tricks for assessing and preserving the

quality and size of your gDNA sample, shearing

methods, and size selection procedures for

samples intended to be used with whole genome

sequencing for de novo assembly.

1.CHEF Mapper XA System (Bio-Rad)

2.Femto Pulse System (AATI)

3.Pippin Pulse System (Sage Science)

A. DNA Sizing Characterization

http://www.sagescience.com/products/pippin-pulse/

http://www.bio-rad.com/en-us/category/pulsed-field-gel-electrophoresis-systems

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

➢ Up to 10 Mb

➢ Up to 165 kb

➢ Up to 80 kb

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 3

A and 1B are examples of high quality, high molecular weight

genomic DNA. Lanes 4A and 2B are examples of degraded

gDNA and should not be used for production of a >30 kb

library.

A B

- DNA purity can be determined by using a NanoDrop® instrument or other spectrophotometers

- For ultrapure gDNA, A260/280 ratio is typically between ~1.8 - 2.0 and A260/230 ratio is ≥2.0

- If A260/280 and A260/230 readings are out of the range specified above, PacBio recommends

performing an AMPure® purification step followed by re-assessment of quantity and purity of

the gDNA sample

260/280 Ratio

➢A low A260/A280 ratio may indicate the presence of protein, phenol, or other contaminants that absorb

strongly at or near 280 nm. Sometimes it may be caused by a very low concentration of nucleic acid.

➢High 260/280 ratios are not indicative of an issue

260/230 Ratio

➢A low A260/A230 ratio may be the result of:

❑ Carbohydrate carryover (often a problem with plants)

❑ Residual phenol from nucleic acid extraction

❑ Residual guanidine (often used in column-based kits)

❑ Glycogen used for precipitation

➢A high A260/A230 ratio may be the result of:

❑ Making a blank measurement on a dirty pedestal of a Nanodrop instrument

❑ Using an inappropriate solution for the blank measurement

B. DNA Purity Determination

- Accurate quantitation of DNA concentration is critical for PacBio template preparation

procedures

➢Specifically, it is critical to determine the concentration of the double-stranded DNA, since only double-

stranded DNA will be converted into sequencing templates

- PacBio highly recommends using a Qubit® fluorimeter tool for routine DNA quantitation

during SMRTbell library construction

-When assessing gDNA QC, PacBio recommends using both fluorometric and

spectrophotometric methods – for example, using both the Qubit and NanoDrop instruments

➢ If the sample is pure gDNA, free of any RNA contaminants and other small molecules, the two methods

should converge to similar DNA concentration measurement values

C. DNA Quantification

- If the measured NanoDrop concentration is significantly different

(>50%) from the Qubit measurement, PacBio recommends doing an

AMPure purification step (as specified by your chosen library

preparation protocol), followed by a re-measurement with both

methods. Typically, a single AMPure purification step resolves the

discrepancy.

➢ If the concentration measurement discrepancy after one AMPure purification

step is not reduced, we recommend trying another cleanup approach such

as a salt:chloroform wash protocol before a re-measurement with both

methods

METHODS FOR DNA SHEARINGInput gDNA must be sheared carefully so that the average size of fragmented DNA

remains well above the desired size selection cut-off

Recommended methods for shearing genomic DNA to the desired average target insert size:

1. Covaris g-TUBE™ Devices (6 kb – 20 kb)

- https://covaris.com/products/g-tube/

- g-TUBE is a single-use device that shears genomic DNA into selected fragments sizes ranging from 6kb

to 20kb.

- The only equipment needed is a compatible bench-top centrifuge.

2. Needle Shearing with 26 G Needles (>30 kb)

- https://www.sai-infusion.com/products/blunt-needles

- Simple, manual shearing method using 26G needles for fragmentation of gDNA >30

kb

- DNA solution is passed through the syringe needle multiple times (e.g., 5, 10, 20

times or more); the higher the number of passes, the smaller the resulting fragment

size

- Relatively inexpensive shearing method – but the shearing size distribution profile is

typically broader than that obtained with Megaruptor-treated samples

3. Diagenode Megaruptor® 2 System (3 – 75 kb)

- https://www.diagenode.com/en/p/megaruptor2-1-unit

- Simple, automated, and reproducible device for the fragmentation of gDNA from 3 kb - 75 kb

- Uses mechanical shearing to fragment DNA by pumping a DNA solution through a disposable

Hydropore shearing device containing a uniform array of pores

- Passage of the DNA molecules several times through the pores ensures that they will reach a

minimum and uniform length as compared to a single pass through the Hydropore; thus a relatively

tighter shearing size distribution profile is generated

- Large gDNA must be sheared carefully

- Test Shears help determine the best shearing parameters

- Evaluation with PFGE or AATI Femto Pulse is recommended

- The average library size depends on the fragment distribution post shearing

A B

Figure A. PFGE gel image of gDNA sheared

to various target fragment sizes using

Diagenode Megaruptor.

Figure B. Femto Pulse digital gel image of gDNA sheared

to various target fragment sizes using Diagenode

Megaruptor.

EXAMPLE: DNA FRAGMENTATION USING THE DIAGENODE

MEGARUPTOR SYSTEM

-Starting with high-quality, high-molecular-weight gDNA is required

-Perform Test Shears:

- Perform test shears to target a fragment size distribution between ~40 kb – 60 kb

- Shear by Megaruptor System or by Needle Shearing

-Verify fragment size distribution of test shears using pulsed-field gel

electrophoresis prior to carrying out large-scale shears

-Perform large-scale shears using predetermined test shear conditions

- Large-scale shears are carried out using the same concentration and conditions –

but in a larger volume – to achieve a similar shearing distribution profile as the test

shears

-Verify fragment size distribution of scale-up shears using pulsed-field gel

electrophoresis prior to starting SMRTbell library construction

PERFORMING TEST SHEARS IS HIGHLY RECOMMENDED FOR

CONSTRUCTING >30 KB SMRTBELL LIBRARIES

Shearing Methods

-MR = Megaruptor System

(Setting 50, 75)

- NS = Needle shearing

(26G needle, 125 ng/µL, 5-20 passes)

Shearing Results

- Both MR and NS generated shearedgDNA

samples with peak mode sizes >30 kb

K1

2gD

NA

E UBT-g

MR

_50

kb

MR

_75

kb

K1

2gD

NA

26

G_

12

5n

g/µ

L_5

x

26

G_

12

5n

g/µ

L_1

0x

26

G_

12

5n

g/µ

L_2

0x

40kb→

20kb→ 15kb→

10kb→

5kb→

MR MR NS NS NS

30kb

COMPARISON OF MEGARUPTOR VS. NEEDLE SHEARING METHODS

FOR CONSTRUCTING >30 KB SMRTBELL LIBRARIES

Recommended Materials

Video Demonstration of needle shearing available at: http://bit.ly/needle_shearing

NEEDLE SHEARING PROCEDURE FOR CONSTRUCTING >30 KB

LIBRARIES

Perform Test Shears

- 26G Blunt End Needle (SAI Infusion B26-150)

- 1 mL Luer-Lok Tip Syringe (BD 309628)

- Adjust sample conc. to 250 ng/µL

- Reserve 1 µL for QC at start of shearing

- Use 50 µL sample in 1.5 mL Lo-Bind tube

- Aspirate entire sample volume through the needle 5 times,

reserve 1 µL for QC

- Repeat aspiration/reservation for 10 to 20 times

- Run 1 µL reservations on PFGE to assess shearing

- See http://bit.ly/needle_shearing

Needle Shearing Video Demonstration

Video Demonstration of needle shearing available at: http://bit.ly/needle_shearing

Assess Test Needle Shears by PFGE

Perform Large-Scale Needle Shears

Video Demonstration of needle shearing available at: http://bit.ly/needle_shearing

- If sample appears under-sheared:

- Decrease DNA concentration (for example, try 125

ng/μl);

- And/or increase the number of passes until you

achieve a similar distribution of fragmented gDNA

- If sample appears over-sheared:

- Reduce the number of passes through the needle

(e.g., try 1 to 2 times)

- Scale-up input mass of gDNA to shear remainder

of sample material

- Increase the volume while maintaining same DNA

concentration and ideal number of passes through

the needle

Lane 1: 1 kb Extension DNA Ladder

Lane 2: Input gDNA

Lane 3: g-TUBE sheared

Lane 4: Input gDNA

Lane 5: 26G_250ng/ul_5x shears

Lane 6: 26G_250ng/ul_10x shears

Lane 7: 26G_250ng/ul_20x shears

Lane 8: Bio-Rad 5 kb DNA ladder

MEGARUPTOR 2 DNA SHEARING SYSTEM OVERVIEW

- Uses mechanical shearing to fragment DNA

- As the DNA in solution is pumped through a Hydropore shearing device, it passes through an

array of uniform pores.

- The resulting turbulent flow stretches and breaks the DNA strands.

- The length of the resulting fragments is dependent on the fluid flow rate and the size of the

pores

- Passage of the DNA molecules several times through the pores ensures that they will reach

a minimum and uniform length as compared to a single pass through the Hydropore

device.

- Enables processing of 2 samples in series with walk-away capability

The Megaruptor 2 is designed to provide researchers with a simple, automated,

and reproducible device for the fragmentation of DNA from 3 kb – 75 kb.

https://www.diagenode.com/en/p/megaruptor2-1-unit

Hydropores

Collection Tube

- Two Hydropore shearing devices available:

- Short Fragment Hydropore: 2 kb – 9 kb

- Large Fragment Hydropore: 10 kb – 75 kb

- They each produce narrow distributions of

fragments with the majority of molecules lying

within a 2- to 3-fold size distribution

- Hydropore devices are disposable to eliminate

cross-contamination

- Sample Volumes Accepted: 50 – 400 µl (Max.)

- Sample Concentration: Up to 50 ng/µl

- Time per sample: 10 - 20 min (depending on

target size); includes sample processing and

system washing

https://www.diagenode.com/files/products/shearing_technology/megaruptor/megaruptor-V2-manual.pdf

MEGARUPTOR SYSTEM TECHNICAL SPECIFICATIONS

- To demonstrate shearing performance of the Megaruptor, a high molecular weight human

genomic DNA sample was sheared to 10 kb, 20 kb, 30 kb, 40 kb, 50 kb, 60 kb, 75 kb

- In this example, 30 kb, 40 kb and 50 kb shears were determined as the best conditions for

constructing >30 kb libraries

MEGARUPTOR SHEARING EXAMPLE: HUMAN GENOMIC DNA

Femto Pulse analysis of Megaruptor test shears ranging

from 10 kb to 75 kb shear settings.

Label Peak Max Average10 kb 11304 12245

20 kb 21245 24123

30 kb 36399 46143

40 kb 49495 60600

50 kb 78351 75416

60 kb 134525 92133

75 kb 149225 104512

Genomic DNA Test Shearing Bulk Shearing

- In this example, a plant gDNA was sheared to 40 and 50 kb

- While the 40 kb shear generated a good size distribution, the 50 kb shear condition was

selected for the large-scale shear because it provided slightly larger fragments

MEGARUPTOR SHEARING EXAMPLE: PLANT GENOMIC DNA

SPECIAL HANDLING RECOMMENDATIONS FOR

PREPARING >15 KB AND >30 KB SMRTBELL

LIBRARIES

1. For small numbers of samples, use DNA Lo-Bind 1.5 mL microcentrifuge tubes for all

enzymatic and AMPure PB bead purification steps.

2. Multi-channel pipettes and PCR strip tubes may be used to efficiently process large

numbers of samples.

3. Use wide-bore tips for all pipette mixing steps when preparing >15 kb and >30 kb size-

selected libraries.

4. Never vortex tubes containing high molecular weight DNA samples. Mix by gentle pipetting

or by flicking the tube.

5. Always follow best practices for DNA quantitation using a Qubit system:

- Use the dsDNA high sensitivity reagent kit.

- Prepare the Qubit working solution by diluting the Qubit reagent 1:200 in Qubit buffer. Prepare 200 μl of

working solution for each standard and sample. Always prepare fresh standards for each assay.

- Set up two 190 μl assay tubes for the standards and one 199 μl assay tube for each sample. Add 10 μl

standard (from kit) and 1 μl sample to the respective assay tubes. Both the standard and sample

DNAs should be at room temperature.

- Vortex all tubes for 2 seconds.

- Incubate the tubes for 2 minutes at room temperature prior to measurement.

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 Advanced Analytical Technologies.

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

www.pacb.com

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