Technical Overview: Multiplexed Microbial Library Preparation Using SMRTbell Express Template Prep Kit 2.0 Sequel System ICS v8.0 / Sequel Chemistry 3.0 / SMRT Link v9.0 Sequel II System ICS v9.0 / Sequel II Chemistry 2.0 / SMRT Link v9.0 Sequel IIe System ICS v10.0 / Sequel II Chemistry 2.0 / SMRT Link v10.0
For Research Use Only. Not for use in diagnostic procedures. © Copyright 2021 by Pacific Biosciences of California, Inc. All rights reserved. PN 101-742-600 Ver 2021-02-01-A (February 2021) Multiplexed Microbial Library Preparation Using SMRTbell Express Template Prep Kit 2.0
1. Multiplexed Microbial Sample Preparation Workflow Overview 2. Multiplexed Microbial Sample Preparation Workflow Details 3. Multiplexed Microbial Sequencing Workflow Details 4. Multiplexed Microbial Data Analysis Workflow Details 5. Multiplexed Microbial Library Example Performance Data 6. Technical Documentation & Applications Support Resources 7. Appendix: General Recommendations for High-Molecular Weight gDNA QC and Handling for Multiplexed Microbial SMRTbell Library Construction MULTIPLEXED MICROBIAL SEQUENCING: HOW TO GET STARTED
Application-Specific Application-Specific Application Consumable Library Construction, Best Practices Guide Procedure & Checklist Bundle Purchasing Guide Sequencing & Analysis
gDNA QC & Shearing ≥1 µg Input gDNA / Microbial Sample 10 kb – 15 kb Target DNA Shear Size
Library Construction Multiplex Up To 48 Microbial Samples with the Sequel II and IIe Systems using Barcoded Overhang Adapters (BOA)
SMRT Sequencing Use the Sequel, Sequel II or Sequel IIe Systems
Data Analysis (SMRT Analysis) Demultiplexing Analysis Application Brief: Microbial whole genome Procedure & Checklist – Preparing Multiplexed PacBio Application Consumable Bundle Microbial Assembly Analysis sequencing – Best Practices (BP101-013020) Microbial Libraries Using SMRTbell Express Purchasing Guide (PG100-051320) Template Prep Kit 2.0 (101-696-100) * Application Consumable Bundles include reagents for library Summary overview of application-specific sample Purchasing Guide enables users to easily order construction, primer annealing and polymerase binding. Core PacBio- preparation and data analysis workflow Technical documentation containing sample library required consumables needed to prepare a branded SMRT Sequencing consumables (SMRT Cells, Sequencing recommendations construction and sequencing preparation protocol SMRTbell library to run a specific type of Kits & SMRT Oil), plastics and other 3rd-party reagents are not included details application on the Sequel II and IIe Systems* in the application bundles 3 Multiplexed Microbial Sample Preparation Workflow Overview MULTIPLEXED MICROBIAL SAMPLE PREPARATION PROCEDURE DESCRIPTION
- Procedure & Checklist – Preparing Multiplexed Microbial Libraries Using SMRTbell Express Template Prep Kit 2.0 (PN 101-696-100) PacBio protocol document describes how to prepare multiplexed 10 kb – 15 kb SMRTbell libraries for sequencing on the Sequel, Sequel II and Sequel IIe Systems (Sequel Systems). - For each microbe, 1 μg of gDNA is required. For optimal results, input gDNA must migrate predominantly above 20 kb as determined by DNA sizing QC analysis. - This procedure includes instructions for multiplexing a minimum of 2 to a maximum of 16 genomes (up to a total sum of 30 Mb of genome sizes including plasmid elements) on the Sequel System. For the Sequel II and IIe Systems, PacBio recommends multiplexing a minimum of 32 to a maximum of 48 genomes (up to 200 Mb). - Options for size-selection using AMPure PB beads and the BluePippin System are also provided.
▪ BluePippin System size-selection may improve microbial genome assembly of microbes with long repeat regions of >6 kb. For this use-case, the average pooled library insert size should be approximately 12.5 kb – 15 kb. https://www.pacb.com/support/documentation/
APPLICATIONS WHOLE GENOME SEQUENCING Multiplexed Microbial De Novo Assembly 5 MULTIPLEXED MICROBIAL LIBRARY SAMPLE PREPARATION & SEQUENCING WORKFLOW OVERVIEW Workflow summary for constructing SMRTbell libraries suitable for sequencing on the Sequel, Sequel II and Sequel IIe Systems for multiplexed microbial genome de novo assembly applications
Genomic DNA QC & Shearing Sequencing ▪ Recommend ≥1 µg input gDNA per microbe ▪ Follow Quick Reference Cards for primer ▪ Shear gDNA to 10 kb – 15 kb target fragment size annealing, polymerase binding, complex cleanup and sample loading ▪ Movie collection time: 10 hours (Sequel System) or 15 hours (Sequel II and IIe Systems) SMRTbell Library Construction (4 – 6 hrs) ▪ Procedure & Checklist – Preparing Multiplexed Microbial Libraries Using SMRTbell Express Template Prep Kit 2.0 (PN 101-696-100) Data Analysis ▪ Multiplex up to 48 microbial genomes per Sequel ▪ Use SMRT Link II SMRT Cell 8M Microbial Assembly analysis application to generate de novo assemblies of small Optional: SMRTbell Library Size Selection genomes up to 10 Mb and companion ▪ BluePippin System size-selection may improve plasmids up to 220 assembly of microbes with long repeat regions of kb >6 kb (≥99% accuracy) 6 Multiplexed Microbial Sample Preparation Workflow Details PROCEDURE & CHECKLIST – PREPARING MULTIPLEXED MICROBIAL SMRTBELL LIBRARIES USING SMRTBELL EXPRESS TEMPLATE PREP KIT 2.0
- This document (PN 101-696-100) presents a workflow for preparing multiplexed microbial libraries using SMRTbell Express Template Prep Kit 2.0 and Barcoded Overhang Adapters for sequencing on the Sequel, Sequel II and Sequel IIe Systems (Sequel Systems) - Recommended pooling strategy: - Sequel System: A minimum of 2 to a maximum of 16 microbial genomes (up to a total sum of 30 Mb of genome sizes including plasmid elements)
- Sequel II and IIe Systems: A minimum of 32 to a maximum of 48 microbial genomes (up to 200 Mb). - Protocol document contains: 1. Recommendations for gDNA QC and quantification 2. Recommendations for shearing gDNA to the desired insert size 3. Enzymatic steps for barcoded SMRTbell library preparation 4. Size-selection options for the final SMRTbell library 5. Sample setup guidance for preparing multiplexed microbial SMRTbell libraries for sequencing on the Sequel Systems
https://www.pacb.com/support/documentation/ 8 MULTIPLEXED MICROBIAL LIBRARY SAMPLE PREPARATION WORKFLOW DETAILS
1. Genomic DNA QC & Shearing - Qubit dsDNA HS quantitation assay - CHEF Mapper, Femto Pulse or Pippin Pulse sizing QC - g-TUBE or Megaruptor shearing 1
2. Multiplexed SMRTbell Express TPK 2.0 Library Construction - Single-tube, addition-only reactions - Ligation with Barcoded Overhang Adapters ~4 – 6 hours (Depends on Multiplex Level) 3. Pool Barcoded SMRTbell Libraries 2 - Perform equimolar pooling of samples using the Microbial Multiplexing Calculator
4. SMRTbell Library Size Selection Options 3 - Perform no size selection; or - AMPure PB bead size selection (removes <3 kb SMRTbell Templates); or - BluePippin System size selection (>6 kb or >7 kb lower cutoff) 4
5. Sequencing Preparation - Anneal sequencing primer, bind polymerase, perform complex cleanup 5 - 2-hour Pre-Extension Time; 15-hour movie collection time (Sequel II or IIe Systems)
6. Data Analysis - Reference: Analysis Procedure – Multiplexed Microbial Assembly with SMRT Link and SMRTbell 6 9 Express Template Prep Kit 2.0 LIST OF REQUIRED MATERIALS AND EQUIPMENT
ITEM VENDOR PART NUMBER
DNA QC (One of the following)
Pulsed Field Gel Electrophoresis System: CHEF Mapper XA or Bio-Rad 170-3670
Pippin Pulse Electrophoresis Power Supply or Sage Science PP10200
Femto Pulse System Agilent P-0003-0817
2100 Bioanalyzer Instrument (sheared DNA only) Agilent G2939BA
DNA Quantitation
Qubit Fluorometer ThermoFisher Scientific Q33226
Qubit 1X dsDNA HS Assay Kit ThermoFisher Scientific Q33230
DNA Shearing (One of the following)
g-TUBE Covaris 10145
Megaruptor Diagenode B06010001
Long Hydropores Diagenode E07010002
Hydrotubes Diagenode C30010018
Megaruptor 3 Diagenode B06010003
Megaruptor 3 Shearing Kit Diagenode E07010003 10 LIST OF REQUIRED MATERIALS AND EQUIPMENT (CONT.)
ITEM VENDOR PART NUMBER SMRTbell Library Preparation
SMRTbell Express Template Prep Kit 2.0 PacBio 100-938-900
Barcoded Overhang Adapter Kit 8A (8 adapters) PacBio 101-628-400
Barcoded Overhang Adapter Kit 8B (8 adapters) PacBio 101-628-500
96 Barcoded Overhang Adapters Any Oligo Synthesis Company
Eppendorf MiniSpin Plus or other equivalent benchtop centrifuge model Eppendorf 022620100
Magnetic Bead Rack ThermoFisher Scientific 12321D
0.2 mL PCR 8-strip tubes USA Scientific 1402-4708
Wide Orifice Tips (Tips LTS W-O 200 UL Fltr RT-L200WFLR) Rainin 17014294
AMPure® PB Beads PacBio 100-265-900
Elution Buffer PacBio 101-633-500
100% Ethanol, Molecular Biology Grade Any MLS
Thermomixer Any MLS
Vortex mixer Any MLS
96-well PCR Plate Eppendorf 951020303
BioRad MSB1001 96-well PCR Plate Seal ThermoFisher Scientific 4306311
Multi-channel Pipettes Rainin 17013810 / 17013808 / 17013807
ThermoFisher Scientific AM10027 Magnetic Plate 11 Permagen T480 LIST OF REQUIRED MATERIALS AND EQUIPMENT (CONT.)
ITEM VENDOR PART NUMBER
Size-selection (If not using AMPure PB Bead Size Selection)
BluePippin Size Selection System Sage Science BLU0001
0.75% Agarose Cassettes and S1 Marker Sage Science BLF7510
12 PACBIO BARCODED OVERHANG ADAPTERS A. Sequel System Recommendations - For the Sequel System, PacBio Barcoded Overhang Adapter Kit 8A (PN 101-628-400) or 8B (PN 101-628-500) are available for multiplexing up to 16 microbes. - 8 barcodes in each kit; 6 reactions per BOA tube - Compatible with SMRTbell Express TPK 2.0 (not compatible with Standard SMRTbell Template Prep Kit 1.0) - Includes Sequencing Primer v4 - Combined kits support up to a 16-plex pooled library
Barcoded Overhang Adapter Kit - 8A Barcoded Overhang Adapter Kit - 8B (PN 101-628-400) (PN 101-628-500) Tube # Description Tube # Description 1 TUBE, Bar Over Adapt - bc1001 1 TUBE, Bar Over Adapt - bc1015 2 TUBE, Bar Over Adapt - bc1002 2 TUBE, Bar Over Adapt - bc1016 3 TUBE, Bar Over Adapt - bc1003 3 TUBE, Bar Over Adapt - bc1017 4 TUBE, Bar Over Adapt - bc1008 4 TUBE, Bar Over Adapt - bc1018 5 TUBE, Bar Over Adapt - bc1009 5 TUBE, Bar Over Adapt - bc1019 6 TUBE, Bar Over Adapt - bc1010 6 TUBE, Bar Over Adapt - bc1020 7 TUBE, Bar Over Adapt - bc1011 7 TUBE, Bar Over Adapt - bc1021 8 TUBE, Bar Over Adapt - bc1012 8 TUBE, Bar Over Adapt - bc1022
- To download the barcode FASTA sequences for BOA Kit 8A/8B, visit PacBio’s Multiplexing Resources webpage
- FASTA filename: Sequel_16_Barcodes_v3.zip (Link) 13 PACBIO BARCODED OVERHANG ADAPTERS (CONT.) B. Sequel II and IIe System Recommendations - For the Sequel II and IIe Systems, a list of 48 barcoded overhang adapters is available in Appendix 2 of the procedure. - To download the barcode FASTA sequences and an oligo ordering sheet, visit PacBio’s Multiplexing Resources webpage: - FASTA filename: Sequel_384_barcodes_v1.zip (Link) - Oligo ordering sheet: Oligo-Ordering-Sheet-for-Microbial-Mplex_v2.xlsx (Link) ▪ Barcoded overhang adapter oligos may be ordered from your favorite oligo synthesis company (e.g., IDT).
Barcoded Overhang Adapters recommended for multiplexing on the Sequel II and IIe Systems.
14 SAMPLE INPUT REQUIREMENTS FOR MICROBIAL MULTIPLEXED LIBRARY CONSTRUCTION FOR SEQUEL AND SEQUEL II AND IIe SYSTEMS
- For each microbe, 1.0 µg of starting input gDNA (pre-shearing) is required - For optimal results, input gDNA must migrate predominantly above 20 kb as determined by pulsed-field gel electrophoresis - To ensure even coverage across the genome, be sure to isolate gDNA from bacterial cultures which have reached a stationary growth phase
20 kb
Example Femto Pulse sizing QC analysis of several bacterial gDNA samples for microbial multiplexed library construction. For all samples, 15 the majority of the gDNA migrates predominantly above 20 kb RECOMMENDED TOOLS FOR EVALUATION OF GENOMIC DNA QUALITY Assess gDNA size and integrity by pulsed field gel electrophoresis (PFGE) or an equivalent method before beginning library preparation.
PippinPulse System (Sage Science) A B
Resolves up to ~80 kb Requires ≥50 ng of sample ~16-hour analysis time
http://www.sagescience.com/products/pippin-pulse/
Femto Pulse System (Agilent Technologies)
Highly Recommended
Resolves up to ~165 kb Requires <1 ng of sample Lane 1: High MW gDNA <1.5-hour analysis time Lane 1: 8-48 kb Ladder (Bio-Rad) Lane 2: Degraded gDNA Lane 2: 5 kb Ladder (Bio-Rad) Lane 3: 165 kb Ladder https://www.agilent.com/en/product/automated-electrophoresis/femto-pulse-systems Lane 3: HMW gDNA Lane 4: Degraded gDNA
Evaluation of gDNA quality using A) Bio-Rad CHEF Mapper and B) Agilent Femto CHEF Mapper XA System (Bio-Rad) 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. Resolves up to ~10 Mb Lanes A4 and B2 show an example of a partially degraded gDNA sample where most of Requires ≥100 ng of sample the DNA migrates below ~50 kb. Depending on the application, partially degraded DNA ~16-h analysis time samples may still be utilized to construct a SMRTbell library. For example, if the majority of the isolated gDNA consists of fragments >20 kb, construction of a >10 – 15 kb SMRTbell library may suffice to assemble a microbial-sized genome (if it does not contain highly http://www.bio-rad.com/en-us/category/pulsed-field-gel-electrophoresis-systems 16 complex repeats; i.e., Class I/II microbes) to a single or a few contigs. BEST PRACTICES RECOMMENDATIONS FOR PREPARING MULTIPLEXED MICROBIAL GENOMIC DNA SMRTBELL LIBRARIES
1. When working with a large number of reactions, PacBio highly recommends using a multi-channel pipette along with a 96-well plate or 8-tube strips. 2. This procedure requires accurate pipetting of small volumes. For pipetting ≤2 μL, use a P2 pipette or a multi-channel pipette that has been recently calibrated and is in good working condition. 3. Never vortex tubes containing (unsheared) high-molecular weight genomic DNA samples. Mix by gentle pipetting or by flicking the tube. 4. For mixing the reactions with sheared gDNA samples in a 96-well plate, seal the plate and pulse-vortex followed by a quick spin- down. Mixing with a multichannel pipette using wide-bore pipette tips can also be performed. However, there may be some sample loss due to samples sticking to the wide-bore tips. 5. Ensure that the AMPure PB beads are at room temperature prior to performing the purification steps.
17 BEST PRACTICES RECOMMENDATIONS FOR PREPARING MULTIPLEXED MICROBIAL GENOMIC DNA SMRTBELL LIBRARIES (CONT.)
6. When performing AMPure PB bead purification steps, note that 80% ethanol is hygroscopic and should be prepared FRESH to achieve optimal results. Also, 80% ethanol should be stored in a tightly capped polypropylene tube for no more than 3 days. 7. For preparing 2- and 3-plex samples for sequencing on the Sequel System, PacBio recommends starting with twice the DNA input amount recommended for higher-plex samples (i.e., use 2.0 μg of input gDNA for 2- and 3-plexes to ensure sufficient quantity of final pooled SMRTbell library for sequencing). Throughout this procedure, use double the standard volumes of input DNA and master mix reagents at each step for 2- and 3-plexes, as indicated in Appendix 1 in the procedure. 8. Measure gDNA concentration using a Qubit fluorometer and Qubit dsDNA High Sensitivity (HS) assay reagents as recommended by the manufacturer.
18 GENOMIC DNA SHEARING RECOMMENDATIONS
- If the majority of the input gDNA sample consists of fragments >20 kb, construction of a >10 kb – 15 kb SMRTbell library may suffice to assemble a microbial-sized genome (if it does not contain highly complex repeats; i.e., Class I/II microbes) to a single or a few contigs - 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 using standard gel or field inversion agarose or capillary gel electrophoresis - Evaluate the size distribution of the sheared gDNA by standard gel or field inversion agarose or capillary gel electrophoresis. The average size of your sheared gDNA should be approximately 10 kb – 15 kb. - If performing BluePippin System size-selection is desired, the average pooled SMRTbell library insert size should be approximately 12.5 kb – 15 kb - The concentration of sheared and AMPure PB bead purified gDNA should be adjusted to 33 ng/μL for each microbe before proceeding with library construction
19 GENOMIC DNA SHEARING RECOMMENDATIONS (CONT.) - If the concentration of sheared and AMPure PB bead purified gDNA is <33 ng/μL for any microbe, choose one of the following options:
1. If available, shear additional gDNA, pool sheared gDNA, and perform another AMPure PB bead purification step. Be sure to elute sheared gDNA in an appropriate volume to achieve ≥33 ng/μL.
2. If additional gDNA is not available, normalize the concentration of all samples to the lowest sample concentration available and proceed with the instructions in this procedure. PacBio does not recommend starting with a total sum of input sheared gDNA amounts of <1000 ng for non-size-selected libraries or <2000 ng for size-selected libraries. For example, starting with 16.5 ng/μL sheared gDNA for each microbe in an 8-plex would give a total input DNA mass of 1927 ng (14.6 μL * 16.5 ng/μL * 8).
3. If additional gDNA is not available and the concentration of one or more samples after shearing and AMPure PB bead purification is <16.5 ng/μL, consider excluding that microbe from the pool, if possible.
4. If none of the above options is possible, proceed with the maximum amount(s) of the limiting sample(s) available and enter the DNA sample concentration values measured in Step 13 of the ‘Purify SMRTbell Library Using 0.45X AMPure PB Beads Before Pooling’ section (Page 13) into the Microbial Multiplexing Calculator. Note: Proceeding with this option may increase variation in relative coverage among the microbes in the pool.
20 EXAMPLE SIZE DISTRIBUTION PROFILES FOR MICROBIAL GENOMIC DNA SAMPLES SHEARED TO 10 KB AND 15 KB MEAN SIZE
A MICROBIAL SAMPLE ID# SAMPLE NAME 10 kb Microbe 1 Helicobacter pylori Microbe 2 Methanocorpusculum labreanum Microbe 3 Neisseria meningitidis Microbe 4 Treponema denticola Microbe 5 Rhodopseudomonas palustris Microbe 6 Bacillus subtilis Microbe 7 Burkholderia cepacia Microbe 8 Enterococcus faecalis Microbe 9 Bacillus cereus B Microbe 10 Klebsiella pneumoniae 15 kb Microbe 11 Staphylococcus aureus subsp. Aureus
Microbe 12 Listeria monocytogenes Microbe 13 Escherichia coli Microbe 14 Staphylococcus aureus HPV Microbe 15 PacBio E. coli K12
Figure: Femto Pulse DNA sizing QC analysis profiles for 15 microbial gDNA samples sheared to 10 kb (A) or 15 kb (B) mean fragment size. 21 SMRTBELL EXPRESS TEMPLATE PREP KIT 2.0 REAGENT HANDLING RECOMMENDATIONS
- Several reagents in the SMRTbell Express TPK 2.0 are LIST OF TEMPERATURE-SENSITIVE REAGENTS INCLUDED IN SMRTBELL sensitive to temperature and vortexing EXPRESS TPK 2.0. REAGENT* WHERE USED - PacBio highly recommends: DNA Prep Additive Remove Single-Strand Overhangs ▪ Never leaving reagents at room temperature DNA Prep Enzyme Remove Single-Strand Overhangs
▪ Working on ice at all times when preparing master mixes DNA Damage Repair Mix v2 DNA Damage Repair
▪ Finger tapping followed by a quick-spin prior to use End Prep Mix End-Repair/A-tailing
Ligation Mix Ligation
Ligation Additive Ligation
Ligation Enhancer Ligation
* Barcoded Overhang Adapters (not included with SMRTbell Express TPK 2.0) are also temperature- sensitive reagents.
SMRTbell Express TPK 2.0
22 MICROBIAL MULTIPLEXING CALCULATOR FOR SAMPLE POOLING - Microbial Multiplexing Calculator provides the following: - Master Mix Volumes: Master mix volumes for library construction for Remove SS Overhangs, DNA Damage Repair, and Ligation reactions - Pooling Volumes: Calculates volumes needed for equimolar pooling after ligation - Maximum number of sample entries allowed = 96
Example calculation output for sample pooling volumes for a representative 32-plex microbial library using the Microbial Multiplexing Pooling Calculator. 23 MICROBIAL MULTIPLEXING POOLING CALCULATOR (CONT.) - Pool samples according to the volumes provided by the Microbial Multiplexing Calculator - The minimum recommended pooling volume is 100 μL - The pooling volume to enter in the pooling calculator depends on the number of samples being pooled. As a rule of thumb, scale up by 50 μL for every 8-plex (starting from 100 μL). For example, use 250 μL for 32-plex, 350 μL for 48- plex, etc. MULTIPLEX LEVEL TOTAL POOL VOLUME (µL) 8 100 16 150 24 200 32 250 40 300 48 350
- If unequal amounts of sheared DNA, or less than 500 ng of sheared DNA for any sample, were input into the DNA Prep Master Mix reaction to remove single-stranded overhangs, be sure to also enter the DNA sample concentration values measured in Step 13 of the ‘Purify SMRTbell Library Using 0.45X AMPure PB Beads Before Pooling’ section (Page 13) into the Optional Sample Concentration column of the Pooling Calculator to maintain equimolar pooling. - Store any unused, non-pooled sample at -20°C for future use - If one or more samples in a pool have insufficient sequencing yield to generate the desired assembly, stored ligated samples may be re-pooled at a lower plex level or at a higher relative amount 24 EXAMPLE SIZE DISTRIBUTION PROFILE FOR A 48-PLEX MICROBIAL LIBRARY SAMPLE BEFORE AND AFTER POOLING
Single, Pooled Library (15 kb) 48 Individual Libraries Overlaid
Residual Barcoded Adapter Oligos
Artifacts
Example Femto Pulse sizing QC analyses of a 48-plex (15 kb) microbial library sample before after before pooling. Left: Femto Pulse sizing profile overlay of 48 barcoded microbial libraries after 0.45X AMPure PB bead purification of each individual library prior to pooling. Right: Femto Pulse sizing profile of the 48-plex microbial library sample after pooling and 0.45X AMPure PB bead purification of the pooled sample (replicate samples are overlaid).
25 OPTIONAL: AMPURE PB BEAD SIZE SELECTION
- This optional AMPure PB bead purification step removes SMRTbell templates <3 kb - AMPure PB beads are first diluted to 40% (volume/volume) with Elution Buffer. - For effective size-selection, the DNA concentration of SMRTbell libraries to be size selected must be 0.5 – 10 ng/μL - Using higher concentrations (>15 – 100 ng/μL) decreases the efficiency of short insert SMRTbell template reduction. Adjust the sample concentration so that the DNA concentration is within the recommended range. - The final AMPure PB bead concentration is critical to the success of this procedure. - Therefore, accurate pipetting is of utmost importance to achieve a final 40% AMPure PB bead in EB
26 OPTIONAL: BLUEPIPPIN SYSTEM SIZE SELECTION - BluePippin System size-selection may improve assembly of microbial genomes with long repeat regions of >6 kb - For size-selection of Multiplexed Microbial pool samples, average pooled library insert size should be approximately 12.5 – 15 kb - Size-selection cut-off recommendations (BPstart values) are provided based on the quantity of the pooled library
POOLED SMRTBELL LIBRARY AMOUNT (µg) BP START (BASES)
>2 7000
≤2 6000
- For the latest BluePippin System User Manual and guidance on size-selection protocols, please contact Sage Science (www.sagescience.com) - Visit Sage's website to verify that your BluePippin System software is up-to-date. (Current version as of January 2021 is v6.31) - Perform size-selection using the 0.75% DF Marker S1 High-Pass 6 kb – 10 kb v3 run protocol and S1 marker. Enter the BPstart values indicated in the table above based on the size-distribution and quantity of your pooled sample - Use Lane 4 for Size Markers
27 Multiplexed Microbial Sequencing Workflow Details SAMPLE SETUP RECOMMENDATIONS FOR MULTIPLEXED MICROBIAL LIBRARIES – SEQUEL SYSTEM (CHEMISTRY 3.0 / SMRT LINK V9.0)
- Follow SMRT Link v9.0 Sample Setup instructions using the recommendations provided in the Quick Reference Card – Loading and Pre-Extension Time Recommendations for the Sequel System for preparing multiplexed microbial library samples for sequencing
29 SAMPLE SETUP RECOMMENDATIONS FOR MULTIPLEXED MICROBIAL LIBRARIES – SEQUEL II SYSTEM (CHEMISTRY 2.0 / SMRT LINK V9.0)
- SMRT Link v9.0 Sample setup requirements for microbial multiplexing on the Sequel II System are summarized below:
SMRT LINK V9.0 SAMPLE SETUP PARAMETERS PARAMETER VALUE / REAGENT KIT TO USE
Sequencing Primer Sequencing Primer V4
Sequencing Primer:Template Ratio 20:1
Polymerase Binding Kit Sequel II Binding Kit 2.0
Polymerase:Template Ratio 10:1
Polymerase Binding Time 4 hours
- To enable preparation of binding reactions with a 10:1 polymerase-to-template ratio, enter the following parameter information below in SMRT Link v9.0 Sample Setup:
SMRT LINK V9.0 SAMPLE VALUE TO SELECT PURPOSE SETUP FIELD Enables calculation of 10:1 polymerase to template ratio in SMRT Sequencing Primer “Sequencing Primer V2” Link v9.0 Sample Setup Enables calculation of 10:1 polymerase to template ratio in SMRT Sequencing Mode “CCS” Link v9.0 Sample Setup
- After entering all the required parameter information, follow the instructions in SMRT Link v9.0 Sample Setup to anneal the microbial multiplexing samples with Sequencing Primer V4 and to bind the samples with Sequel II Binding Kit 2.0 30 SAMPLE SETUP RECOMMENDATIONS FOR MULTIPLEXED MICROBIAL LIBRARIES – SEQUEL II AND IIe SYSTEMS (CHEMISTRY 2.0 / SMRT LINK V10.0)
- Follow SMRT Link v10.0 Sample Setup instructions using the recommendations provided in the Quick Reference Card – Loading and Pre-Extension Time Recommendations for the Sequel II/IIe Systems for preparing multiplexed microbial library samples for sequencing
→ For SMRT Link v10.0 (or higher): Select ‘Microbial Assembly’ from the Application field drop-down menu in the SMRT Link Sample Setup and SMRT Link Run Design user interface
31 IMPORTING THE BARCODE FASTA FILE INTO SMRT LINK FOR AUTOMATED DEMULTIPLEXING OF POOLED MICROBIAL LIBRARY SAMPLES
- Note: SMRT Link v9.0 (and higher) software installations by default come pre-bundled with a FASTA file containing a list of PacBio barcodes recommended for use with multiplexed SMRT sequencing applications - If your SMRT Link installation does not already include an appropriate barcode FASTA file, the following steps describe how to import such a file for use in automated demultiplexing (refer to “Importing Data” section in the SMRT Link User Guide):
1. Download the FASTA file containing the relevant barcode EXAMPLE FASTA FILE CONTAINING A LIST sequences from PacBio’s Multiplexing website, for example: OF PACBIO 16-BASE PAIR BARCODES ▪ Sequel_16_Barcodes_v3.zip → Contains 16 PacBio barcodes for use with Barcoded Overhang Adapter Kit 8A/8B (PN 101-628-400/500) ▪ Sequel_384_barcodes_v1.zip → Contains 48 PacBio barcodes for use with customer-supplied Barcoded Overhang Adapters ordered using PacBio’s Barcoded Overhang Adapter Oligo Ordering Sheet
32 IMPORTING THE BARCODE FASTA FILE INTO SMRT LINK FOR AUTOMATED DEMULTIPLEXING OF POOLED MICROBIAL LIBRARY SAMPLES (CONT.)
2. Import the desired FASTA file into SMRT Link. i. On the SMRT Link Home Page, select Data Management. ii. Click Import Data and follow the steps below: A. Specify whether to import data from the SMRT Link Server, or from a Local File System. (Note: Only references and barcodes are available if you select Local File System.) B. Select the data type to import: Barcodes – FASTA (.fa or .fasta), XML (.barcodeset.xml), or ZIP files containing barcodes. C. Navigate to the appropriate file and click Import. The selected barcode filed is imported and becomes available for viewing in the SMRT Link Data Management module home screen.
A B C
33 SMRT LINK RUN DESIGN SETUP PROCEDURE FOR AUTOMATED DEMULTIPLEXING OF POLLED MICROBIAL LIBRARY SAMPLES - Open the Run Design module in SMRT Link and click New Run Design. - Fill in the Sample Information section, then click the small arrow to open Barcoded Sample Options. - Specify the following options:
1. Sample is Barcoded: Yes 2. Barcode Set:
3. Same Barcodes on Both Ends of Sequence: Yes 2 4. Assign a Biological Sample Name to each barcoded 3 sample using one of two ways: or From a CSV File or Interactively (SMRT Link v10.0 or higher only) 4
34 SMRT LINK RUN DESIGN SETUP PROCEDURE FOR AUTOMATED DEMULTIPLEXING OF POOLED MICROBIAL LIBRARY SAMPLES (CONT.) Barcode Selection and Bio Sample Name Specification Using a CSV File: 1
1. Click the From a File button, then click Download File. 2. Edit the file and enter the biological sample names associated with the barcodes in the second column, then save the file. Barcode Bio Sample Name bc1001_BAK8A_OA--bc1001_BAK8A_OA Microbial Sample 1 ▪ Delete entire rows of barcodes not used 2 bc1002_BAK8A_OA--bc1002_BAK8A_OA Microbial Sample 2 ▪ Allowed characters: Alphanumeric; space; dot; bc1003_BAK8A_OA--bc1003_BAK8A_OA Microbial Sample 3 underscore; hyphen. Other characters will be bc1008_BAK8A_OA--bc1008_BAK8A_OA Microbial Sample 4 automatically removed. 3. Browse for the Barcoded Sample File you just edited and click on Open. 3 4. You see Upload Was Successful appear on the line below, assuming the file is formatted correctly..
- Refer to “Run Design” section in the SMRT Link User Guide for further details 4
35 SMRT LINK RUN DESIGN SETUP PROCEDURE FOR AUTOMATED DEMULTIPLEXING OF POOLED METAGENOMICS SHOTGUN LIBRARY SAMPLES (CONT.) Interactive Method for Barcode Selection and Bio Sample Name Specification (SMRT Link v10.0 Only): 1. Click the Interactively button, then drag barcodes from the Available Barcodes column to the Included Barcodes column. 2. (Optional) Click a Bio Sample field to edit the Bio Sample Name associated with a barcode. 3. (Optional) Click Download as a file for later use. 4. Click Save to save the edited barcodes/bio sample names. You see Success on the line below, assuming the file is formatted correctly.
2
1
3 4 36 Multiplexed Microbial Data Analysis Workflow Details ANALYSIS PROCEDURE – MULTIPLEXED MICROBIAL ASSEMBLY WITH SMRT LINK AND SMRTBELL EXPRESS TEMPLATE PREP KIT 2.0
- Analysis Procedure document includes: 1. How to import a FASTA file containing PacBio barcode sequences into SMRT Link for sample demultiplexing analysis 2. How to set up a Run Design on the Sequel, Sequel II and Sequel IIe Systems to enable auto-demultiplexing of barcoded microbial sequencing data 3. How to review the resulting demultiplexed microbial data sets using the SMRT Link Data Management module 4. How to launch the SMRT Link Microbial Assembly analysis application on the microbial data sets 5. Updated microbial assembly troubleshooting guidance
https://www.pacb.com/support/documentation/ 38 REVIEWING DEMULTIPLEXED MICROBIAL DATA SETS IN SMRT LINK DATA MANAGEMENT If barcoded sample options are configured correctly in SMRT Link Run Design, then demultiplexing of the data should complete automatically - Once the run is complete, open the SMRT Link Data Management module - The barcoded samples are automatically demultiplexed - Click the number in the Demultiplexed Subsets column (20 in this example) to view the barcoded Data Sets in a separate table:
39 REVIEWING DEMULTIPLEXED MICROBIAL DATA SETS IN SMRT LINK DATA MANAGEMENT (CONT.)
- From the Data Management module home page, click the Data Set name of the parent sample (My microbial samples in the above example) to view the Data Set details
- The Barcodes section displays the number of reads per barcode and several graphs to assess the quality of the demultiplexing results
- Refer to the Demultiplex Barcodes Application section in the SMRT Link User Guide for further details on interpreting this report
- If review of the demultiplexing results reveals that any demultiplexing parameters need to be corrected (such as barcode set, barcode layout, or minimum barcode score), launch the Demultiplex Barcodes analysis application manually from the SMRT Analysis module, using the same Data Set as input
- Refer to Analysis Procedure – Multiplexed Microbial Assembly with SMRT Link and SMRTbell Express Template Prep Kit 2.0 documentation for further details
40 LAUNCHING MICROBIAL ASSEMBLY ANALYSES ON DEMULTIPLEXED MICROBIAL DATA SETS USING SMRT LINK SMRT Link Microbial Assembly analysis application provides de novo assembly, circularization, and plasmid identification for microbial genomes To launch one assembly analysis for each barcoded sample, open the SMRT Analysis module. Click Create New Analysis. Specify the following options: 1. Analysis Name: Enter an analysis name to persist across each analysis. 2. Data Type: Select Sequel Data 3. Data Sets: Locate the parent Data Set of the demultiplexed set (My microbial samples in this example). Click the number in the Demultiplexed Subsets column to view the child data sets. Click the checkbox in the column header to select all children.
41 LAUNCHING MICROBIAL ASSEMBLY ANALYSES ON DEMULTIPLEXED MICROBIAL DATA SETS USING SMRT LINK (CONT.) 4. Analysis Type: Select Analysis 5. Analysis of Multiple Data Sets: Select One Analysis per Data Set – Custom Parameters 6. Click Next. 7. Analysis Application: Select Microbial Assembly 5. For the first Data Set, edit the Genome Length parameter if desired. 6. Click Start and Create Next. This launches the first assembly analysis on the first Data Set and proceeds to the launch page of the next Data Set, preserving any edits to the parameters made in the previous page. 7. Edit the Genome Length parameter as appropriate for the second Data Set. 8. Click Start and Create Next. Repeat the process for all Data Sets. After launching the last analysis, you are redirected to the SMRT Analysis module home page, which displays the status of each launched analysis.
42 LAUNCHING MICROBIAL ASSEMBLY ANALYSES ON DEMULTIPLEXED MICROBIAL DATA SETS USING SMRT LINK (CONT.) 9. When an analysis completes, click on the analysis job name to view the results.
43 TROUBLESHOOTING MICROBIAL ASSEMBLY RESULTS FOR DEMULTIPLEXED MICROBIAL DATA SETS If the Microbial Assembly analysis generates a microbial genome assembly with >5 contigs, below are some additional suggestions to help improve the assembly, which can be tried individually or together.
1. To ensure sufficient coverage for assembly, set the Genome Length parameter to a value equal to (or slightly above) the actual genome size for the provided sample. 2. Increasing the coverage for assembly may help when the library quality is less than ideal. ▪ In the Create New Analysis page, click Advanced Parameters and change the Coverage parameter from the default of 30 to a higher value (such as 40). 3. If the input subread length is relatively short (e.g., under 6000 bp) the minimum threshold for preassembled read length may need to be lowered. This can also help for longer insert libraries for which the quality is less than ideal. ▪ In the Create New Analysis page, click Advanced Parameters and edit the Advanced options by adding the following line, which modifies the overlap filtering options by specifying a smaller “--min-len" value of 2000 bp (default is 4000 bp): stage1.ovl_filter_opt = --max-diff 80 --max-cov 100 --min-cov 1 -- bestn 20 --min-len 2000 --gapFilt --minDepth 4; 4. In case the Microbial Assembly analysis is taking a long time to complete, try modifying the block size. ▪ In the Create New Analysis page, click Advanced Parameters and edit the Advanced options by adding the following line, which changes the block size for the overlap process to a smaller value such as 200 Mb (default is 1024 Mb): stage1.block_size = 200; stage2.block_size = 200; ▪ Note that the block sizes for both stages do not have to be identical, but in general, the same rule of thumb applies for both stages. 5. In case the Microbial Assembly analysis stops due to an out-of-memory problem or overly high memory consumption in general, reducing the block size for the overlap process can help. Refer to the previous point for details on how to set the block size. 6. Optionally you can use the auto-analysis option when designing the sequencing run to set up analysis for automatic execution once the sequence data is available on the SMRT Link server. For more information, see the SMRT Link User Guide. 44 Multiplexed Microbial Library Example Performance Data EXAMPLE SEQUENCING PERFORMANCE* RESULTS FOR 10 KB & 15 KB 48-PLEX MICROBIAL LIBRARIES (SEQUEL II SYSTEM)
SMRTBELL LIBRARY PRODUCTIVITY YIELD OF MEAN POLYMERASE N50 POLYMERASE TYPE (P1) BARCODED READS READ LENGTH READ LENGTH
10 kb 48-plex 36% 59% Up to 45 kb Up to 97 kb Microbial Library
15 kb 48-plex 35% 52% Up to 38 kb Up to 88 kb Microbial Library
Data shown were generated using a Sequel II System v8.0 / Sequel II Chemistry 2.0; 15-h movie collection time; 2-h pre-extension time.
46 * Read lengths, reads/data per SMRT Cell and other sequencing performance results vary based on sample quality/type and insert size. EXAMPLE BARCODE DEMULTIPLEXING RESULTS FOR A 15 KB 48-PLEX MICROBIAL LIBRARY (SEQUEL II SYSTEM) Polymerase Reads 120,000
100,000
80,000
60,000
40,000
Barcoded Polymerase Reads Polymerase Barcoded 20,000
0
8 9 1 4 7 0 3 4 6 7 0 2 3 4 5 8 9 0 1 2 5 6 8 0 4 6 7 8 9 1 2 3 4 7 1 2 4 5 9 1 2 6 9 1 7 8 9 6
1 1 3 3 3 4 4 4 4 4 5 5 5 5 5 5 5 6 6 6 6 6 6 7 7 7 7 7 7 8 8 8 8 8 9 9 9 9 9 0 0 0 0 1 1 1 1 2
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 1 1
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c
b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b
------
------
8 9 1 4 7 0 3 4 6 7 0 2 3 4 5 8 9 0 1 2 5 6 8 0 4 6 7 8 9 1 2 3 4 7 1 2 4 5 9 1 2 6 9 1 7 8 9 6
1 1 3 3 3 4 4 4 4 4 5 5 5 5 5 5 5 6 6 6 6 6 6 7 7 7 7 7 7 8 8 8 8 8 9 9 9 9 9 0 0 0 0 1 1 1 1 2
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 1 1
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c
b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b
47 Data shown were generated using a Sequel II System v8.0 / Sequel II Chemistry 2.0; 15-h movie collection time; 2-h pre-extension time EXAMPLE SEQUENCING COVERAGE RESULTS FOR 10 KB & 15 KB 48-PLEX MICROBIAL LIBRARIES (SEQUEL II SYSTEM) Both 10 kb and 15 kb 48-plex microbial libraries achieved ≥200-fold total subread base coverage per barcoded bacterial genome sample
1600 1400 10Kb insert 1200 1000 800 600 400 200
0
B B sub B sub B sub
R.pal R.pal R.pal
Listeria… Listeria…
Neisseria… Neisseria… Neisseria… Neisseria…
Klebsiella… Klebsiella… Klebsiella… Klebsiella…
Treponema… Treponema… Treponema… Treponema… Treponema…
Burkholderia… Burkholderia… Burkholderia… Burkholderia…
Enterococcus… Enterococcus… Enterococcus… Enterococcus…
Bacillus cereus… Bacillus cereus… Bacillus
Staphylococcus… Staphylococcus… Staphylococcus… Staphylococcus… Staphylococcus…
PacBio E coliK12 E PacBio coliK12 E PacBio
Methanocorpuscul… Methanocorpuscul…
Helicobacter pylori… Helicobacter pylori… Helicobacter pylori… Helicobacter pylori… Helicobacter pylori… Helicobacter
E coli W (ATCC 9637) Ecoli W (ATCC 9637) Ecoli W (ATCC 9637) Ecoli
1000 900 15Kb insert 800 700 600 500 400 300 200 100
0
B sub B
R. pal R. pal R. pal R. pal R.
Listeria… Listeria… Listeria… Listeria…
E coli W coli E
E coli W coli E W coli E
Neisseria… Neisseria… Neisseria…
Klebsiella… Klebsiella… Klebsiella…
Treponema… Treponema… Treponema… Treponema…
Helicobacter… Helicobacter… Helicobacter… Helicobacter…
Burkholderia… Burkholderia…
Enterococcus… Enterococcus… Enterococcus… Enterococcus…
Bacillus cereus… Bacillus cereus… Bacillus cereus… Bacillus
E coli W (ATCC… coli E
Staphylococcus… Staphylococcus… Staphylococcus… Staphylococcus… Staphylococcus… Staphylococcus…
Methanocorpuscu…
Methanocorpuscu… Methanocorpuscu… coliK12 PacBio E coliK12 PacBio E coliK12 PacBio E 48 Data shown were generated using a Sequel II System v8.0 / Sequel II Chemistry 2.0; 15-h movie collection time; 2-h pre-extension time EXAMPLE DE NOVO ASSEMBLY RESULTS FOR A 15 KB 48-PLEX MICROBIAL LIBRARY (SEQUEL II SYSTEM)
Chromosomal contigs Sum of Contig Lengths
6 10 )
9 b
M (
5
s 8
s
g
h
i t
t 7 g
n 4
n o
6 e
C
L
f
o 3 5
g
i
t
r e
4 n b
2 o C
m 3
f
u
o N 2
1 m
1 u
0 0 S
* *
s s s s
3 2 2 2 9 9 9 9 e e e s s s s s s s
1 1 1 F F F F Z Z Z
8 8 8
V V V A A A A
i i i i
a a
W W W W e e e e
2 1 1 1 9 9 9 9 r r r r u u u
7 7 7 i i a a a
1 1 1
R R R R i i i t t t t P P P
i i i i
a a a a
J J J J
n n n n
c c l l l l e e e
9 9 9 m m m l l l l
K K K
1 1 1 1 n n n s s s s r r r
W
M M M
H H H
i i i i
e e e e
a a
i i i o o o o u u u o o o o
l l l r r r r o o o u u u u
s s s u u u
G G G G l l l l
A A A
g g g g
c c c c
s p p n n n c c c c
s s s
i i i i
i o o o o
u u u o o o a a a
a a a a
l l l l
o o o o t t t t
l m m m F F F
e e a a a
i O O O O u u u
t t t t
c c c a a a a
e e e . . .
t p p p p
y y y y
i i i i n n n n
c c u u u
r r r e e e
y y y y e e e
s s s
s s s s p p p
b p p p p r r r
i i i
h h h h
a a a r r r
i i i i e e e e e e e
e e e c c c c s s s s
i i i
a a l l l l s s s
c c c c b b b
d d d
u i i r r r r u u u
c c c n n n i i i d d d d
i i i i o o o o a a a a
r r h h h
a a a a
b b b
s r r r r a a a
t t t
e e e e a a a
p p p i i i
c c c n n n n l l l n n n n
c c c c t t t t
s s s e e u u u a a a a
i i i
e e e e
g g g
s r r r c c c c o o o o o o o o s s s
e e e e s s s
u u u d d a a a
l l l l l h h h h
l l l
n n n m m m m
u a a a a m m m
l l l e e e l l l
a a a a u u u
l i i i
i i i c c c c m m m m m m m m
o o
f f f f s s s
l e e e e
b b b b u u u c c c
i h h h e e e
n n n
c c c s s s s i i i l l l
h h
o o o o u u u
n n n n
c c c c c c
c s s s s a a a a
o o o o
e e e
a a a s s s i i i i u u u
k k E E E E
d d d d e e e
o o o o
s s s c c c c o o o
a u u u u r r r r
r r i i i i c c c r r r
b b b
B B B u u u u
l l l l m m m c c c p p p p
c c c c
E E E e e e e
B u u s s s u u u
e e e
t t t t e e e e
e e e e o o o
c c c c l l l e e e e
u u u a a a l l l a a a
B B s s s s s s s s r r r r
i i i
o o o o i i i i
H H H H K K K y y y
p p p r r r
p p p p T T T T
s s s
c c c c L L L L r r r
h h h
e e e
o o o o
u u u
o o o o o o o
p p p
s s s
r r r r
d d d d
c c c
c c c
s s s
a a a
e e e e
i i i o o o o c c c
o o o t t t
t t t t
e e e h h h h o o o
n n n
S S S
n n n n
c c c
a a a N N N R R R R
E E E E
o o o
h h h l l l
t t t
y y y
e e e
h h h
p p p
M M M
a a a
t t
* Burkholderia cepacia has three chromosomes t
S S S 49 Data shown were generated using a Sequel II System v8.0 / Sequel II Chemistry 2.0; 15-h movie collection time; 2-h pre-extension time EXAMPLE ASSEMBLY QUALITY RESULTS FOR A 15 KB 48-PLEX MICROBIAL LIBRARY (SEQUEL II SYSTEM)
QV By Comparison With a Published Reference 70
60
50
40
30
Average QV Average 20
10
0
- The QV shown is the average value of all replicate microbial samples sequenced in the 48-plex pooled library - Microbes without a published, high-quality reference are excluded from this analysis 50 EXAMPLE PLASMID RECOVERY PERFORMANCE FOR A 15 KB 48-PLEX MICROBIAL LIBRARY (SEQUEL II SYSTEM) ORGANISM EXPECTED PLASMIDS ASSEMBLED PLASMIDS Bacillus cereus 971 1 plasmid (15 kb) 0 Bacillus cereus 971 1 plasmid (15 kb) 0 Bacillus cereus 971 1 plasmid (15 kb) 1 complete Burkholderia cepacia 1 plasmid (209 kb) 1 partial: 204,166 bp Burkholderia cepacia 1 plasmid (209 kb) 1 partial: 204,170 bp Escherichia coli W 2 plasmids (5, 102 kb) 1 complete (102 bp) | 1 missing (5 kb) Escherichia coli W 2 plasmids (5, 102 kb) 2 complete (5; 102 bp) Escherichia coli W 2 plasmids (5, 102 kb) 1 complete (102 bp) | 1 missing (5 kb) Escherichia coli W 2 plasmids (5, 102 kb) 2 complete (5; 102 bp) Klebsiella pneumoniae 4 plasmids (2, 85, 118, 141 kb) 3 complete (85, 118, 141 kb) | 1 missing (2 kb) Klebsiella pneumoniae 4 plasmids (2, 85, 118, 141 kb) 3 complete (85, 118, 141 kb) | 1 missing (2 kb) Klebsiella pneumoniae 4 plasmids (2, 85, 118, 141 kb) 3 complete (85, 118, 141 kb) | 1 missing (2 kb) Listeria monocytogenes no recorded plasmid in NCBI none Listeria monocytogenes no recorded plasmid in NCBI new circular plasmid! 94,380 bp Listeria monocytogenes no recorded plasmid in NCBI new circular plasmid! 94,378 bp Listeria monocytogenes no recorded plasmid in NCBI new circular plasmid! 94,380 bp Staphylococcus aureus HPV 1 plasmid (24 kb) 1 complete: 32,188 bp Staphylococcus aureus HPV 1 plasmid (24 kb) 1 complete: 32,188 bp Staphylococcus aureus HPV 1 plasmid (24 kb) 1 complete: 32,188 bp Staphylococcus aureus subsp. aureus 1 plasmid (27 kb) 1 complete Staphylococcus aureus subsp. aureus 1 plasmid (27 kb) 1 complete Staphylococcus aureus subsp. aureus 1 plasmid (27 kb) 1 complete - Good plasmid recovery performance was observed for most replicate microbial samples sequenced in the 48-plex pooled library that were expected to contain plasmids 51 - Some small plasmids were not detected possibly due to loss during AMPure PB bead purification steps Technical Documentation & Applications Support Resources BEST PRACTICES: MICROBIAL WHOLE GENOME SEQUENCING
Template Preparation with SMRTbell Express Template Prep Kit 2.0 - Start with recommended input of high-quality gDNA per microbial sample (1.0 μg) and construct a 10 – 15 kb SMRTbell library - Reduce costs by multiplexing samples to assemble most bacterial genomes into 5 contigs or fewer, exclusive of plasmids - Simplify equimolar pooling with Microbial Multiplexing Calculator - Adjust multiplexing depth to balance cost per genome with genome completeness - Note: Closure of Class III complexity genomes with large repeat regions may require 20–30 kb library preparations & size- selection and may not be suitable for multiplexing
Sequence on the Sequel, Sequel II or Sequel IIe Systems - Maximize output and turn-around-time with adjustable sequencing parameters* - Multiplex up to 48 isolates per Sequel II SMRT Cell 8M for $70/sample† with a 15-hour movie collection time - Recommend generating ≥30-fold unique molecular coverage (UMC) per microbial genome
Data Analysis Solutions with the PacBio Analytical Portfolio - Use SMRT Link for fully automated demultiplexing, assembly, circularization, and polishing of both chromosomes and plasmids to produce gold standard references - Achieve high-quality consensus accuracies >99.999% - Detect and annotate active m6A and m4C Restriction-Modification system motifs with the ‘Base Modification and Motif Analysis’ application in SMRT Analysis [m4C (≥25-fold coverage per strand) / m6A (≥25-fold coverage per strand)]
* Read lengths, reads/data per SMRT Cell 8M and other sequencing performance results vary based on sample quality/type and insert size. † Prices, listed in USD, are approximate and may vary by region. Pricing includes library and sequencing reagents run on a Sequel II System and does not include instrument amortization or other reagents. 53 Application Brief: Microbial whole genome sequencing – Best Practices (PN BP101-013020) TECHNICAL DOCUMENTATION AND APPLICATIONS SUPPORT RESOURCES FOR MULTIPLEXED MICROBIAL LIBRARY PREPARATION, SEQUENCING & DATA ANALYSIS
Sample Preparation Literature
- Application Brief: Microbial Whole Genome Sequencing – Best Practices (PN BP101-013020) - Procedure & Checklist – Preparing Multiplexed Microbial SMRTbell Libraries Using SMRTbell Express Template Prep Kit 2.0 (PN 101- 696-100) - Microbial Multiplexing Pooling Calculator - Quick Reference Card – Loading and Pre-extension Recommendations for the Sequel System (PN 101-461-600) - Quick Reference Card – Loading and Pre-extension Recommendations for the Sequel II System (PN 101-769-100) - Overview – Sequel Systems Application Options and Sequencing Recommendations (PN 101-851-300) - Application Consumable Bundles Purchasing Guide (PN PG100-051320) - Product Note: SMRTbell Express Template Prep Kit 2.0 for Microbial Multiplexing (PN PN104-022119) - Technical Note: Preparing DNA for PacBio HiFi sequencing – Extraction and quality control (PN TN101-061920) - Technical Overview: Multiplexed Microbial Library Preparation Using SMRTbell Express Template Prep Kit 2.0 (PN 101-742-600)
54 TECHNICAL DOCUMENTATION AND APPLICATIONS SUPPORT RESOURCES FOR MULTIPLEXED MICROBIAL LIBRARY PREPARATION, SEQUENCING & DATA ANALYSIS (CONT.)
Data Analysis Resources
- Analysis Procedure – Multiplexed Microbial Assembly with SMRT Link v8.0 and SMRTbell Express Template Prep Kit 2.0 (PN 101-855- 300) - SMRT Analysis Barcoding Overview (v9.0) (PN 101-923-200) - Contains detailed information on barcoding experimental design options and describes QC metrics for evaluation of barcoding performance using SMRT Link - PacBio Multiplexing Resources Website: https://www.pacb.com/smrt-science/smrt-sequencing/multiplexing/ - Barcoding Overview documents for different SMRT Link software versions - PacBio barcode sequence files (compressed FASTA) for use with Sequel, Sequel II and Sequel IIe Systems - Barcoded oligo ordering sheets
Example PacBio Data Sets
WHOLE GENOME SEQUENCING DATASET DATA TYPE PACBIO SYSTEM APPLICATION
Multiplexed Microbial Assembly Microbial Multiplexing Data Set – 48 plex Long Reads Sequel II System
55 TECHNICAL DOCUMENTATION AND APPLICATIONS SUPPORT RESOURCES FOR MULTIPLEXED MICROBIAL LIBRARY PREPARATION, SEQUENCING & DATA ANALYSIS (CONT.)
Posters - AGBT 2020 Poster: New advances in SMRT Sequencing facilitate multiplexing for de novo and structural variant studies. - ABRF 2019 Poster: Streamlined SMRTbell library generation using addition-only, single tube strategy for all library types reduces time to results.
Webinars - PacBio Webinar (2020): Bioinformatics lunch & learn – Better assemblies of bacterial genomes and plasmids with the new microbial assembly pipeline in SMRT Link v8.0 [Webinar Recording]
Publications - Davies, Mark R et al. (2019) Atlas of group A streptococcal vaccine candidates compiled using large-scale comparative genomics. Nature Genetics. 51(6): 1035–1043. - Ashton, P M et al. (2019) Three phylogenetic groups have driven the recent population expansion of Cryptococcus neoformans. Nature Communications. 10(1):2035. - See the PacBio Microbial Whole Genome Sequencing Applications website for a list of other publications. 56 General Recommendations for High-Molecular Weight gDNA QC and Handling for Multiplexed Microbial SMRTbell Library Construction GENERAL RECOMMENDATIONS FOR ISOLATING HIGH-MOLECULAR WEIGHT GENOMIC DNA 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) 58 DNA EXTRACTION AND QC FOR SMRT SEQUENCING WHOLE GENOME DE NOVO ASSEMBLY PROJECTS Starting with high-quality, high molecular weight (HMW) genomic DNA (gDNA) will result in longer libraries and better performance during sequencing.
- Technical Note: DNA Prep (TN101-061920) is intended to give recommendations, tips and tricks for the extraction of genomic DNA, as well as assessing and preserving the quality and size of your DNA sample to be used for PacBio HiFi sequencing for de novo assembly - Topics covered include: ▪ DNA Extraction - Commercially available kits across a wide variety of input sample types - Resource for alternative DNA extraction methods ▪ DNA Quality Control (QC) - DNA quantification, purity, size, and damage - Use of nucleic acid stabilizers - DNA storage and shipping ▪ Best Practices for DNA Extraction for PacBio Sequencing - This technical note also includes an example dataset for a California Redwood tree DNA sample that was isolated using a commercial DNA extraction kit Technical Note TN101-061920: Preparing DNA for PacBio HiFi sequencing – Extraction and 59 quality control DNA SAMPLE PREPARATION ONLINE RESOURCE
Literature resource for sample collection and DNA extraction protocol references
www.ExtractDNAforPacBio.com
PacBio does not assume responsibilities/guarantees for these external publications/protocols, but we are happy to help as best as we can to guide / connect. Please contact [email protected] for more discussions around your particular species & sequencing project! 60 METHODS FOR EVALUATION OF GENOMIC DNA QUALITY Starting with high-quality genomic DNA will result in longer libraries and better de novo assembly performance
- Input genomic DNA must be carefully QC’d to assess integrity - PFGE/FIGE or Femto Pulse sizing tool is highly recommended - High-quality, high-molecular weight DNA → longer read lengths - Low-quality, degraded/damaged DNA → shorter read lengths, lower library synthesis yields (dependent on size selection method and parameter settings employed) - 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
61 A. Genomic DNA Sizing Characterization Recommended methods for determining gDNA size distribution: A B
PippinPulse System (Sage Science)
Resolves up to ~80 kb Requires ≥50 ng of sample ~16-hour analysis time
http://www.sagescience.com/products/pippin-pulse/
Femto Pulse System (Agilent Technologies)
Highly Recommended Lane 1: 8-48 kb Ladder (Bio-Rad) Lane 1: High MW gDNA Resolves up to ~165 kb Lane 2: 5 kb Ladder (Bio-Rad) Lane 2: Degraded gDNA Requires <1 ng of sample Lane 3: HMW gDNA Lane 3: 165 kb Ladder <1.5-hour analysis time Lane 4: Degraded gDNA
https://www.agilent.com/en/product/automated-electrophoresis/femto-pulse-systems Evaluation of gDNA quality using A) Bio-Rad CHEF Mapper System and B) Femto Pulse System. Lanes 3A and 1B are examples of high quality, high-molecular weight genomic DNA. Lanes 4A and 2B are CHEF Mapper XA System (Bio-Rad) examples of degraded gDNA.
Resolves up to ~10 Mb Requires ≥100 ng of sample ~16-h analysis time 62 http://www.bio-rad.com/en-us/category/pulsed-field-gel-electrophoresis-systems B. DNA Purity Determination - 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 PB bead 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 63 C. DNA Quantification - 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 fluorometer tool and Qubit dsDNA High Sensitivity (HS) Assay Kit (Thermo Fisher Scientific) 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
- If the measured NanoDrop concentration is significantly different (>50%) from the Qubit measurement, PacBio recommends doing an AMPure PB bead purification step (as specified by your chosen library preparation protocol), followed by a re-measurement with both methods. Typically, a single AMPure PB bead purification step resolves the discrepancy.
▪ If the agreement does not improve after three rounds of AMPure PB bead purification, try using either a commercial kit, isopropanol precipitation, or a new DNA extraction method to obtain a cleaner DNA sample..
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