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Multiplexed Microbial Library Preparation Using Smrtbell 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
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