Metagenomics Shotgun Library Preparation Using Smrtbell

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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 Prep Kit 2.0 PacBio 100-938-900

Barcoded Overhang Adapter Kit 8A (8 adapters) or PacBio 101-628-400 Barcoded Overhang Adapter Kit 8B (8 adapters) PacBio 101-628-500

AMPure PB Beads PacBio 100-265-900

Elution Buffer PacBio 101-633-500

SMRTbell Enzyme Cleanup Kit PacBio 101-746-400

Sequencing Primer V2 PacBio 101-847-900

Eppendorf MiniSpin Plus or other equivalent benchtop centrifuge model Eppendorf 022620100

Wide Orifice Tips (Tips LTS W-O 200 UL Fltr RT-L200WFLR) Rainin 17014294

100% Ethanol, Molecular Biology Grade Any MLS

Rotator Any MLS

2.0 mL DNA Lo-Bind Tubes Any MLS 13 SMRTBELL EXPRESS TEMPLATE PREP KIT 2.0 AND SMRTBELL ENZYME CLEANUP KIT REAGENT HANDLING RECOMMENDATIONS

- Several reagents in the kit are sensitive to LIST OF TEMPERATURE-SENSITIVE REAGENTS INCLUDED IN SMRTBELL EXPRESS TPK 2.0 temperature and vortexing AND SMRTBELL ENZYME CLEANUP KIT. PACBIO KIT REAGENT WHERE USED - PacBio highly recommends: Remove Single-Strand DNA Prep Additive Overhangs ▪ Never leaving reagents at room Remove Single-Strand DNA Prep Enzyme temperature Overhangs ▪ Working on ice at all times when DNA Damage Repair Mix v2 DNA Damage Repair SMRTbell Express preparing master mixes End Prep Mix End-Repair/A-tailing Template Prep kit 2.0 ▪ Finger tapping followed by a quick-spin (PN 100-938-900) Overhang Adapter v3* Ligation prior to use Ligation Mix Ligation

Ligation Additive Ligation SMRTbell Express TPK 2.0 Ligation Enhancer Ligation

Enzyme A Nuclease Treatment

SMRTbell Enzyme Enzyme B Nuclease Treatment Cleanup Kit (PN 101-746-400) Enzyme C Nuclease Treatment Enzyme D Nuclease Treatment

* Barcoded Overhang Adapters (not included with SMRTbell Express TPK 2.0) are also temperature-sensitive reagents. 14 PACBIO BARCODED OVERHANG ADAPTERS FOR MULTIPLEXED METAGENOMICS SHOTGUN LIBRARY CONSTRUCTION

- PacBio Barcoded Overhang Adapter Kit 8A (PN 101-628- Barcoded Overhang Adapter Kit - 8A 400) or 8B (PN 101-628-500) are available for multiplexing (PN 101-628-400) up to 4 metagenomics shotgun samples per SMRT Cell 8M Tube # Description for sequencing on the Sequel II and IIe Systems: 1 TUBE, Bar Over Adapt - bc1001 2 TUBE, Bar Over Adapt - bc1002 - PacBio Barcoded Overhang Adapter Kit 8A, PN 101-628-400; 3 TUBE, Bar Over Adapt - bc1003 and 4 TUBE, Bar Over Adapt - bc1008 5 TUBE, Bar Over Adapt - bc1009 - PacBio Barcoded Overhang Adapter Kit 8B, PN 101-628-500) 6 TUBE, Bar Over Adapt - bc1010 7 TUBE, Bar Over Adapt - bc1011 - Each Barcoded Overhang Adapter (BOA) Kit 8A or 8B 8 TUBE, Bar Over Adapt - bc1012 contains sufficient reagents to perform 2 ligation reactions for each BOA to support metagenomics shotgun library Barcoded Overhang Adapter Kit - 8B construction. (PN 101-628-500) Tube # Description - Each PacBio barcode sequence is 16 bp in length 1 TUBE, Bar Over Adapt - bc1015 2 TUBE, Bar Over Adapt - bc1016 - To download the barcode FASTA sequences for BOA Kit 3 TUBE, Bar Over Adapt - bc1017 8A/8B, visit PacBio’s Multiplexing Resources webpage 4 TUBE, Bar Over Adapt - bc1018 5 TUBE, Bar Over Adapt - bc1019 - FASTA filename: Sequel_16_Barcodes_v3.zip (Link) 6 TUBE, Bar Over Adapt - bc1020 7 TUBE, Bar Over Adapt - bc1021 8 TUBE, Bar Over Adapt - bc1022

15 PacBio barcode sequence FASTA files for Sequel Systems can be obtained here: https://www.pacb.com/smrt-science/smrt-sequencing/multiplexing/ GENOMIC DNA EXTRACTION FROM METAGENOMIC SAMPLES FOR METAGENOMICS SHOTGUN SMRTBELL LIBRARY CONSTRUCTION - Starting Input genomic DNA (gDNA) for metagenomics shotgun library construction should be >15 kb - Due to the harsh lysis methods required for some organisms, it may be difficult to extract large quantities of high quality, intact gDNA from metagenomic samples. - However, for most metagenomic samples, gDNA quality and quantity are likely sufficient for ~10-12 kb SMRTbell library construction - It is important to note that the relative abundance of gDNA may be impacted by the extraction method used. - Example method for isolating gDNA from human intestinal microbiome samples: - Morita et al. (2007) An Improved DNA Isolation Method for Metagenomic Analysis of the Microbial Flora of the Human Intestine. Microbes and Environments. Vol. 22 Pages 214-222.

DNA QUALITY AND QUANTITY REQUIREMENTS FOR METAGENOMICS SHOTGUN LIBRARY CONSTRUCTION USING SMRTBELL EXPRESS TPK 2.0.

AMOUNT OF INPUT gDNA PACBIO INSTRUMENT REQUIRED INPUT gDNA TARGET LIBRARY REQUIRED FOR (MULTIPLEX LEVEL) gDNA QUALITY SHEARING METHOD INSERT SIZE (MODE) SHEARING Megaruptor System; Sequel System 1500 ng Majority of gDNA >15 kb or 10 kb – 12 kb (1 Sample) g-Tube Sequel II System 1500 ng for 1 sample; or Megaruptor System; (Up to 4 Multiplexed >750 ng per sample for Majority of gDNA >15 kb or 10 kb – 12 kb Samples) multiplexed samples g-Tube 16 RECOMMENDED TOOLS FOR GENOMIC DNA QUANTIFICATION AND QUALIFICATION DNA Quantification - For quantification of gDNA to be used with the metagenomics shotgun library preparation workflow, we recommend using the Qubit fluorometer and Qubit dsDNA High Sensitivity (HS) Assay Kit reagents. Measure the gDNA sample concentration as recommended by the manufacturer.

Qubit™ dsDNA HS Assay Kit Qubit 4 Fluorometer

17 https://www.thermofisher.com/order/catalog/product/Q33230#/Q33230 RECOMMENDED TOOLS FOR GENOMIC DNA QUANTIFICATION AND QUALIFICATION (CONT.) DNA Sizing - Three commercially available systems that may be used to evaluate gDNA size distribution are listed below with links to recommended procedures.

We highly recommend the use of the Femto Pulse System (Agilent) for low DNA input applications because of its ability to evaluate size distributions using only ~200 – 500 picograms of DNA

Femto Pulse System GENOMIC DNA SIZE EVALUATION METHODS AND PROCEDURES. METHOD COMMENTS PROCEDURE

▪ Highly recommended Femto Pulse System (Agilent) Agilent Femto Pulse Website ▪ Requires 200 – 500 pg

Procedure & Checklist - Using the BIO-RAD® CHEF Mapper CHEF Mapper XA PFGE System (Bio-Rad ) ▪ Requires >50 ng XA Pulsed Field Electrophoresis System

Procedure & Checklist - Using the Sage Science Pippin Pippin Pulse System (Sage Science) ▪ Requires >50 ng Pulse Electrophoresis Power Supply System

18 EXAMPLE FEMTO PULSE SIZING QC ANALYSES OF MOCK COMMUNITY AND EXTRACTED METAGENOMIC DNA SAMPLES Metagenomic DNA samples are often degraded, but in most cases the isolated gDNA can likely still be sheared to ~10-12 kb target fragment size distribution

MSA 1003 21 kb MSA 1002 21 kb

Fecal Sample 1 Fecal Sample 2 21 kb 21 kb

19 BEST PRACTICES RECOMMENDATIONS FOR PREPARING METAGENOMICS SHOTGUN DNA SMRTBELL LIBRARIES

1. Metagenomics samples often contain impurities that may affect subsequent enzymatic reactions. Before proceeding with library construction, determine DNA purity by measuring the A260/280 and A260/230 ratios using a spectrophotometer instrument (e.g., NanoDrop). If ratios are lower than the expected values (ideally A260/280 = ~1.8, A260/230 = 2.0 – 2.2 for pure DNA), performing a 0.45X AMPure PB bead purification is necessary to remove contaminants. 2. Ensure that the AMPure PB beads are at room temperature prior to performing the purification steps. 3. 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. 4. For accurate quantification results, measure DNA concentration using a Qubit fluorometer and Qubit dsDNA High Sensitivity (HS) Assay Kit reagents as recommended by the manufacturer.

20 DNA SHEARING RECOMMENDATIONS FOR METAGENOMICS SHOTGUN LIBRARY CONSTRUCTION For constructing metagenomics shotgun libraries, it is necessary to shear the genomic DNA to allow for increased yield of HiFi reads and efficient detection of the barcodes in the SMRTbell templates (for multiplexed samples) - Shearing metagenomic DNA samples to a target size distribution mode between 10 kb - 12 kb using either the Megaruptor System (Diagenode) or g-Tubes (Covaris) is recommended - To shear gDNA using the Megaruptor System or g-Tubes, generally follow the manufacturer’s recommendations. g-TUBE - It is important to perform small-scale test shears (for example, using 150 ng of DNA in a 150 μL volume [1 ng/μL] to evaluate the response of each gDNA sample to shearing parameters. - After shearing the gDNA samples, proceed with the ‘Concentrate sheared gDNA Using AMPure PB Beads’ step and then evaluate the size distribution using a PFGE or capillary electrophoresis system

Megaruptor 2 System Megaruptor 3 System 21 DNA SHEARING RECOMMENDATIONS FOR METAGENOMICS SHOTGUN LIBRARY CONSTRUCTION (CONT.)

RECOMMENDED SHEARING SHEARED GENOMIC DNA TARGET SHEARING TOOL CONDITIONS SIZE DISTRIBUTION MODE Megaruptor 1 System 10 kb or 15 kb Target Size Setting 10 – 12 kb 4600 RPM in MiniSpin Plus Centrifuge g-TUBE 10 – 12 kb for 2 min.; invert and repeat spin

g-Tube

23498 bp

Megaruptor

Example Femto Pulse sizing QC analyses for metagenomic DNA samples sheared to a 10-kb mode size with the Megaruptor 1 System or g- Tubes. It is highly recommended that test shears be performed to determine optimal shearing conditions for each sample to be processed. 22 SAMPLE POOLING BEST PRACTICES RECOMMENDATIONS FOR MULTIPLEXED METAGENOMICS SHOTGUN LIBRARY CONSTRUCTION - Always quantify libraries before pooling. PacBio recommends using the Qubit dsDNA High Sensitivity (HS) Assay Kit for performing DNA concentration measurements. - Equal-molar pooling of barcoded libraries is recommended in order to generate even sequencing read coverage for each metagenomic shotgun sample. - After pooling, perform AMPure PB bead size-selection to remove SMRTbell templates <3 kb

23 AMPURE PB BEAD SIZE SELECTION FOR MULTIPLEXED METAGENOMICS SHOTGUN LIBRARY CONSTRUCTION - AMPure PB bead size-selection purification step removes SMRTbell templates <3 kb - AMPure PB bead stock solution is first diluted to 35% (vol./vol.) with Elution Buffer and subsequently used for purification - For effective size-selection, the DNA concentration of the SMRTbell library to be size-selected must be 0.5-10 ng/μL. - 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 35% (v/v) AMPure PB bead working solution in EB

24 EXAMPLE FEMTO PULSE SIZING QC ANALYSES OF METAGENOMICS SHOTGUN SMRTBELL LIBRARY BEFORE AND AFTER AMPURE PB BEAD SIZE SELECTION

Sheared gDNA

Final SMRTbell library after AMPure PB bead size selection

SMRTbell library before AMPure PB bead size selection

Example Femto Pulse sizing QC analysis of a metagenomics shotgun SMRTbell library before and after AMPure PB bead size selection. The final SMRTbell library sample after AMPure PB bead size selection shows a ~13 kb size mode. AMPure PB size-selection purification step effectively 25 removes SMRTbell templates <3 kb. ESTIMATED RECOVERY YIELDS FOR METAGENOMICS SHOTGUN LIBRARY CONSTRUCTION WORKFLOW STEPS

ESTIMATED LIBRARY CONSTRUCTION WORKFLOW STEP YIELD Average Post-Shearing Yield ~70 % (Starting from input gDNA) Average SMRTbell Library Construction Yield ~50 % (Starting from sheared gDNA input into the first enzymatic reaction [Remove ssDNA Overhangs step])

Average Yield of Size-Selected Library ~ 40% – 60%* (Starting from DNA input into AMPure PB bead size-selection)

* Library yield after size selection is highly dependent on the final SMRTbell library size distribution. Size-selection recovery yield range shown in the Table above was obtained for gDNA samples sheared to 10 – 12 kb.

26 Metagenomic Shotgun Sequencing Workflow Details SAMPLE SETUP RECOMMENDATIONS FOR METAGENOMIC SHOTGUN LIBRARIES – SEQUEL SYSTEM (CHEMISTRY 3.0)

- Follow SMRT Link Sample Setup instructions using the recommendations provided in the Quick Reference Card – Loading and Pre-Extension Time Recommendations for the Sequel System for preparing metagenomics shotgun library samples for sequencing

28 SAMPLE SETUP RECOMMENDATIONS FOR METAGENOMIC SHOTGUN LIBRARIES – SEQUEL II AND IIe SYSTEMS (CHEMISTRY 2.0)

- Follow SMRT Link 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 metagenomics shotgun library samples for sequencing

- For SMRT Link v10.0 (or higher): Select ‘Shotgun Metagenomic Profiling or Assembly’ from the Application field drop-down menu in the SMRT Link Sample Setup and SMRT Link Run Design user interface

29 IMPORTING THE BARCODE FASTA FILE INTO SMRT LINK FOR AUTOMATED DEMULTIPLEXING OF POOLED METAGENOMICS SHOTGUN 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 sequences from PacBio’s Multiplexing website, for example: ▪ Sequel_16_Barcodes_v3.zip (contains a list of 16 PacBio barcodes for use with Barcoded Overhang Adapters)

EXAMPLE FASTA FILE CONTAINING A LIST OF PACBIO 16-BASE PAIR BARCODES

30 IMPORTING THE BARCODE FASTA FILE INTO SMRT LINK FOR AUTOMATED DEMULTIPLEXING OF POOLED METAGENOMICS SHOTGUN 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

31 SMRT LINK RUN DESIGN SETUP PROCEDURE FOR AUTOMATED DEMULTIPLEXING OF POLLED METAGENOMICS SHOTGUN 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: