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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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