Technical Overview: Metagenomics Shotgun 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 II 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 101-894-900 Ver 2021-02-01-A (February 2021) Metagenomics Shotgun Library Preparation Using SMRTbell Express Template Prep Kit 2.0 1. Metagenomics Shotgun Sample Preparation Workflow Overview 2. Metagenomics Shotgun Sample Preparation Workflow Details 3. Metagenomics Shotgun Sequencing Workflow Details 4. Metagenomics Shotgun Data Analysis Recommendations 5. Metagenomics Shotgun Library Example Performance Data 6. Technical Documentation & Applications Support Resources METAGENOMICS SHOTGUN SEQUENCING: HOW TO GET STARTED Application-Specific Application-Specific Application Consumable Library Construction, Best Practices Guide Procedure & Checklist Bundle Purchasing Guide Sequencing & Analysis Genomic DNA QC & Shearing Start With Input gDNA ≥15 kb 10 kb Target DNA Shear Size Library Construction (SMRTbell Express TPK 2.0) Multiplex Up To 4 Samples per SMRT Cell 8M for Metagenomic Profiling HiFi Sequencing Generate Up To 2.4 Million HiFi Reads per SMRT Cell 8M Data Analysis Demultiplex Barcodes Using SMRT Link Application Brief: Metagenomic sequencing with Procedure & Checklist – Preparing 10 kb Library PacBio Application Consumable Bundle Perform Metagenomic Profiling or HiFi reads - Best Practices (BP108-030220) Using SMRTbell Express Template Prep Kit 2.0 for Purchasing Guide (PG100-082620) Assembly Using 3rd-Party Software Metagenomics Shotgun Sequencing (101-800-800) Summary overview of application-specific sample Purchasing Guide enables users to easily order preparation and data analysis workflow Technical documentation containing sample library required consumables needed to prepare a * Application Consumable Bundles include reagents for library construction, primer annealing and polymerase binding. Core PacBio- recommendations construction and sequencing preparation protocol SMRTbell library to run a specific type of branded SMRT Sequencing consumables (SMRT Cells, Sequencing Kits details application on the Sequel II and IIe Systems* & SMRT Oil), plastics and other 3rd-party reagents are not included in the application bundles 3 Metagenomics Shotgun Sample Preparation Workflow Overview METAGENOMICS SHOTGUN SAMPLE PREPARATION PROCEDURE DESCRIPTION - Procedure & Checklist – Preparing 10 kb Library Using SMRTbell Express Template Prep Kit 2.0 for Metagenomics Shotgun Sequencing (PN r 101-800-800) protocol document contains instructions for constructing 10 – 12 kb insert libraries using SMRTbell Express Template Prep Kit 2.0 for metagenomic shotgun sequencing on the Sequel, Sequel II and Sequel IIe Systems (Sequel Systems). - The size distribution of the starting genomic DNA is critical for shearing and PacBio recommends working with samples where the majority of the input gDNA is greater than 15 kb whenever possible. - Depending on project goals and coverage requirement needs, either a single metagenomic sample can be sequenced on the Sequel System or up to 4 (barcoded) samples can multiplexed and sequenced on the Sequel II and IIe Systems https://www.pacb.com/support/documentation/ APPLICATIONS CHARACTERIZE MICROBIAL COMMUNITIES 5 METAGENOMIC SHOTGUN LIBRARY SAMPLE PREPARATION & SEQUENCING WORKFLOW OVERVIEW Workflow summary for constructing SMRTbell libraries suitable for HiFi sequencing on the Sequel, Sequel II and Sequel IIe Systems for metagenomics shotgun applications Genomic DNA QC & Shearing HiFi Sequencing ▪ Majority of input gDNA should be >15 kb ▪ Follow Quick Reference Cards for preparing ▪ Shear gDNA to a 10 kb – 12 kb target mode size samples for sequencing ▪ Generate up to 2.4 million HiFi reads per SMRT Cell 8M for a 10 kb library SMRTbell Library Construction (4 – 7 hrs) ▪ Follow Procedure & Checklist – Preparing 10 kb Library Using SMRTbell Express Template Prep Kit 2.0 for Metagenomics Shotgun Sequencing (PN 101-800-800) Data Analysis ▪ Perform metagenomic PacBio HiFi ▪ Multiplex up to 4 samples for sequencing on a reads achieve single SMRT Cell 8M using barcoded adapters profiling or assembly >99.9% accuracy ▪ Perform AMPure PB bead size selection to analysis using third- remove SMRTbell templates <3 kb party software HiFi Read 6 HOW MANY METAGENOMICS SHOTGUN SAMPLES CAN BE MULTIPLEXED ON A SINGLE SEQUEL II SMRT CELL 8M? The Overall Goals of Your Project Will Determine the Needed Coverage Depth Question 1: What is the estimated abundance of the rarest species you want to observe? Example: “I want to see species present at 1% abundance." With 1 SMRT Cell 8M, you can expect ~24,000 HiFi reads from a 1% abundant species with an ‘average’ genome size Question 2: What is your goal? IN ORDER TO ACHIEVE… …YOU NEED Species detection ~100 HiFi reads Comprehensive gene profiling / 5-Fold coverage; ~3,000 HiFi reads discovery* Complete genome assembly* 20-Fold coverage; ~12,000 HiFi reads * # Reads Needed = Coverage x 5 Mb Genome / 8.5 kb Median HiFi Read Length 7 HOW MANY METAGENOMICS SHOTGUN SAMPLES CAN BE MULTIPLEXED ON A SINGLE SEQUEL II SMRT CELL 8M? (CONT.) EXAMPLE CALCULATION OF ESTIMATED COVERAGE LEVELS ACHIEVABLE FOR RARE SPECIES AT DIFFERENT MULTIPLEX LEVELS 1 SAMPLE / 2 SAMPLES / 3 SAMPLES / SMRT CELL SMRT CELL SMRT CELL Assignable HiFi Reads / 2.4 M 2.4 M 2.4 M SMRT Cell* HiFi Reads / Sample 2.4 M 1.2 M 800,000 1% of Reads assembly 24,000 assembly 12,000 profiling 8,000 0.2% of Reads profiling 4,800 detection 2,400 detection 1,600 * 99.5% of HiFi reads have recoverable barcodes - The average HiFi read length for metagenomics samples is 8.5 kb when following the recommended protocol with high- molecular weight genomic DNA - Choose your multiplex level depending on how many reads per rarest-OTU of interest you require for your analysis plan 8 Metagenomics Shotgun Sample Preparation Workflow Details PROCEDURE & CHECKLIST – PREPARING 10 KB LIBRARY USING SMRTBELL EXPRESS TEMPLATE PREP KIT 2.0 FOR METAGENOMICS SHOTGUN SEQUENCING - This protocol (PN 101-800-800) describes a workflow for preparing 10 kb libraries using SMRTbell Express Template Prep Kit 2.0 for metagenomics shotgun sequencing on the Sequel, Sequel II and Sequel IIe Systems (Sequel Systems) - Depending on project goals and coverage requirement needs, either a single metagenomic sample can be sequenced on the Sequel System or up to for 4 (barcoded) samples can multiplexed and sequenced on the Sequel II and IIe Systems. - Protocol document contains: 1. Recommendations for metagenomic DNA QC, shearing and quantification 2. Enzymatic steps for preparation of non-barcoded and barcoded metagenomics shotgun SMRTbell libraries 3. Instructions for size-selection of multiplexed metagenomics shotgun libraries using the AMPure PB Bead Size Selection method 4. Sample setup guidance for preparing metagenomics shotgun SMRTbell libraries for sequencing on the Sequel Systems https://www.pacb.com/support/documentation/ 10 METAGENOMICS SHOTGUN SAMPLE LIBRARY PREPARATION & SEQUENCING DETAILED WORKFLOW OVERVIEW 1. Genomic DNA QC & Shearing - Input gDNA should be >15 kb as assessed by CHEF Mapper, Femto Pulse or Pippin Pulse sizing QC - Shear gDNA to 10 kb – 12 kb using a Megaruptor or g-TUBEs 1 2. SMRTbell Express Library Construction - Single-tube, addition-only reactions with SMRTbell Express TPK 2.0 - For multiplexed samples, perform ligation with Barcoded Overhang Adapters - AMPure PB bead purification of barcoded SMRTbell libraries prior to pooling 3. Pool Barcoded SMRTbell Express Libraries ~4 – 7 h 2 - Pool up to four metagenomics shotgun samples at equimolar concentration 4. Size Selection & QC Final SMRTbell Library - Perform AMPure PB bead size selection (removes <3 kb SMRTbell templates) 5. Sequencing Preparation 3 - Anneal sequencing primer, bind polymerase, and perform AMPure PB bead complex cleanup - Generate up to 2.4 million HiFi reads per Sequel II SMRT Cell 8M* for a 10 kb library size 4 6. Analyze - De-multiplex barcodes within SMRT Link GUI or on the command line 5 - Analyze metagenomics shotgun HiFi reads using third-party analysis tools - Perform taxonomic classification and functional gene profiling using QIIME and MEGAN - Perform gene prediction and discovery using FragGeneScan and Prodigal 6 - Perform metagenomic shotgun assembly directly with HiFi reads using Canu 11 * Read lengths, reads/data per SMRT Cell 8M and other sequencing performance results vary based on sample quality/type and insert size. LIST OF REQUIRED MATERIALS AND EQUIPMENT ITEM VENDOR PART NUMBER DNA Sizing QC (One of the following) Femto Pulse System Agilent Technologies, Inc. M5330AA Pippin Pulse Electrophoresis Power Supply Sage Science PP10200 Pulsed Field Gel Electrophoresis System: CHEF Mapper XA Bio-Rad 170-3670 DNA Quantitation Qubit 4 Fluorometer ThermoFisher Scientific Q33226 Qubit 1X dsDNA HS Assay Kit ThermoFisher Scientific Q33230 DNA Shearing (One of the following) Megaruptor 2 System Diagenode B06010002 Long Hydropores Diagenode E07010002 Hydrotubes Diagenode C30010018 g-TUBE Covaris 10145 Eppendorf MiniSpin Plus or other equivalent benchtop centrifuge model Eppendorf 022620100 12 LIST OF REQUIRED MATERIALS AND EQUIPMENT (CONT.) ITEM VENDOR PART NUMBER SMRTbell Library Preparation SMRTbell Express Template