Technical Overview: Metagenomics Shotgun Library Preparation Using SMRTbell Express Template Prep Kit 2.0
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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
* Application Consumable Bundles include reagents for library
preparation and data analysis workflow Technical documentation containing sample library required consumables needed to prepare a
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
PacBio HiFi
▪ Multiplex up to 4 samples for sequencing on a ▪ Perform metagenomic
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
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/
10METAGENOMICS 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 (removesLIST 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 12LIST 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 13SMRTBELL 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
AND SMRTBELL ENZYME CLEANUP KIT.
temperature and vortexing
PACBIO KIT REAGENT WHERE USED
- PacBio highly recommends: Remove Single-Strand
DNA Prep Additive
▪ Never leaving reagents at room
Overhangs
Remove Single-Strand
temperature DNA Prep Enzyme
Overhangs
▪ Working on ice at all times when DNA Damage Repair Mix v2 DNA Damage Repair
preparing master mixes SMRTbell Express
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.
14PACBIO 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
(PN 101-628-500)
construction.
Tube # Description
- Each PacBio barcode sequence is 16 bp in length 1
2
TUBE, Bar Over Adapt - bc1015
TUBE, Bar Over Adapt - bc1016
- To download the barcode FASTA sequences for BOA Kit 3 TUBE, Bar Over Adapt - bc1017
4 TUBE, Bar Over Adapt - bc1018
8A/8B, visit PacBio’s Multiplexing Resources webpage
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
16RECOMMENDED 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#/Q33230RECOMMENDED 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
18EXAMPLE 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 121 kb Fecal Sample 2
21 kb
19BEST 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.
20DNA 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
21
Megaruptor 2 System Megaruptor 3 SystemDNA 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. 22SAMPLE 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
AMPURE PB BEAD SIZE SELECTION FOR MULTIPLEXED METAGENOMICS SHOTGUN LIBRARY CONSTRUCTION - AMPure PB bead size-selection purification step removes SMRTbell templates
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 templatesESTIMATED 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.
26Metagenomic 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
28SAMPLE 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
29IMPORTING 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
30IMPORTING 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
31SMRT 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: (e.g.,
Sequel_16_barcodes_v3) 1
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
32SMRT LINK RUN DESIGN SETUP PROCEDURE FOR AUTOMATED DEMULTIPLEXING OF
POOLED METAGENOMICS SHOTGUN LIBRARY SAMPLES (CONT.)
Barcode Selection and Bio Sample Name
1
Specification Using a CSV File:
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 Metagenomics Shotgun Sample 1
▪ Delete entire rows of barcodes not used 2 bc1002_BAK8A_OA--bc1002_BAK8A_OA Metagenomics Shotgun Sample 2
▪ Allowed characters: Alphanumeric; space; dot; bc1003_BAK8A_OA--bc1003_BAK8A_OA Metagenomics Shotgun Sample 3
underscore; hyphen. Other characters will be bc1008_BAK8A_OA--bc1008_BAK8A_OA Metagenomics Shotgun 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
33SMRT 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 34Metagenomics Shotgun Data Analysis Recommendations
METAGENOMICS SHOTGUN DATA ANALYSIS RECOMMENDATIONS
HiFi reads are compatible with third-party metagenomics data analysis tools
- Utilize SMRT Link to generate highly accurate and long single-molecule reads (HiFi reads) using the Circular
Consensus Sequencing (CCS) analysis application or perform CCS analysis on-instrument using the Sequel IIe System
- PacBio highly recommends upgrading to SMRT Link v9.0 or higher to perform de-multiplexing of your metagenomics
shotgun sequencing data
- Refer to Barcoding Overview documents available on PacBio’s Multiplexing Resources website for detailed information on QC
metrics for evaluation of barcoding performance using SMRT Link
- Output data in standard file formats, (BAM and FASTA/Q) for seamless integration with downstream analysis tools
- Perform taxonomic classification and functional gene profiling using QIIME and MEGAN
- Perform gene prediction and discovery using FragGeneScan and Prodigal
- Perform metagenomic shotgun assembly directly with HiFi reads using HiCanu
36Metagenomics Shotgun Library Example Performance Data
EXAMPLE PRIMARY SEQUENCING PERFORMANCE RESULTS FOR A 10 KB
METAGENOMICS SHOTGUN LIBRARY (SEQUEL II SYSTEM CHEMISTRY 2.0)
PRIMARY RUN STATISTICS* FOR A SINGLE-SAMPLE (NON-MULTIPLEXED) 10-KB METAGENOMICS SHOTGUN LIBRARY1 RUN ON A SINGLE SEQUEL II
SYSTEM SMRT CELL 8M2
UNIQUE MEAN MEAN LONGEST
TOTAL BASES POLYMERASE
LIBRARY NAME MOLECULAR POLYMERASE LONGEST SUBREAD N50 P0 P1 P2
(Gb) RL N50 (bp)
YIELD (Gb) RL (bp) SUBREAD (bp) (bp)
MSA1002 10 kb Shotgun 458.87 58.22 82511 159579 11610 14099 25% 69% 5%
1 20 Strain Even Mix Genomic Material [ATCC MSA-1002] mock community sample
2 44 pM on-plate loading concentration; 30-hour movie collection time; 2-hour pre-extension time
Base Yield Density Read Length Density HiFi Read Length Distribution
20 kb
5 kb
38
* Read lengths, reads/data per SMRT Cell and other sequencing performance results vary based on sample quality/type and insert size.EXAMPLE HIFI READ YIELD RESULTS FOR HUMAN FECAL METAGENOMICS
SHOTGUN SAMPLES (SEQUEL II SYSTEM CHEMISTRY 2.0)
The median read QV and read length of HiFi data outperforms many metagenome assembly quality metrics
HIFI READS PER HIFI BASES PER MEAN HIFI READ
SAMPLE MEAN HIFI READ QV
SMRT CELL 8M SMRT CELL 8M LENGTH (bp)
Human fecal 1 2,485,902 21,892,869,069 8,806 Q39
Human fecal 2 2,634,276 24,359,683,697 9,247 Q37
Human fecal 3 2,371,437 20,325,373,131 8,570 Q39
Human fecal 4 2,133,478 21,557,465,918 10,104 Q36
Human fecal 5 2,037,230 19,855,203,301 9,746 Q37
Human fecal 6 2,230,353 19,784,876,972 8,870 Q39
Human fecal 7 2,796,697 22,710,850,840 8,120 Q40
Human fecal 8 1,977,870 17,034,971,133 8,612 Q40
Human fecal 9 2,529,830 21,908,484,087 8,660 Q39
39METAGENOMICS SHOTGUN SEQUENCING ON THE SEQUEL II SYSTEM FAITHFULLY
RECAPITULATES THE MSA-1003 MOCK COMMUNITY
100% Rhodobacter sphaeroides
Escherichia coli
90% Porphyromonas gingivalis
Successful detection of species down
Streptococcus mutans
to 0.018 % abundance
80% Staphylococcus epidermidis
Pseudomonas aeruginosa Download and explore this MSA-1003
70% Bacillus cereus Mock Community shotgun data set
Clostridium beijerinckii
60% Streptococcus agalactiae
Staphylococcus aureus
50% Acinetobacter baumannii
Propionibacterium acnes
40%
Neisseria meningitidis
Helicobacter pylori
30%
Lactobacillus gasseri
Bacteroides vulgatus
20%
Deinococcus radiodurans
Enterococcus faecalis
10%
Actinomyces odontolyticus
0% Bifidobacterium adolescentis
Sequel II of
Proportion System
Reads Expected
Proportion Expected
Data Composition
MSA-1003 mock community sample was sequenced on a single Sequel II System SMRT Cell 8M (Chemistry 2.0)
40PACBIO METAGENOMICS SHOTGUN SEQUENCING IS FREE FROM GC BIAS,
ENABLING ACCURATE REPRESENTATION OF SPECIES DIVERSITY
Species Abundance of MSA-1003
100
%GC 50% 69% 36% ATCC Abundance
10 HiFi Shotgun Abundance
16S Abundance (Avg of 96)
Log Abundance
1
0.1
60% 38%
43% 67% 65%
0.01
0.001
0.0001
41LONG READS + HIGH ACCURACY MEANS GENE DISCOVERY CAN BE DONE DIRECTLY
ON HIFI READS, WITHOUT ASSEMBLY
NUMBER OF MEAN GENE MEAN PREDICTED CLUSTERED
SAMPLE
PREDICTED GENES LENGTH (bp) GENES / READ GENES (99% ID)
Human fecal 1 19,639,322 1,005 7.9 1,012,982
Human fecal 2 22,064,417 1,001 8.4 1,141,123
Human fecal 3 18,059,181 1,024 7.6 1,154,341
Human fecal 4 19,844,033 978 9.3 1,250,711
Human fecal 5 18,396,237 970 9.0 1,087,015
- 30-70% of short read data will not map to a metagenome assembly, and are therefore not useful for gene finding
- With HiFi sequencing, error-free genes can be found even from species with too little coverage for assembly
- High accuracy means existing NGS tools and pipelines can be used without modification
42AS WITH ISOLATE SEQUENCING, HIGHLY ACCURATE LONG READS IMPROVE BOTH
GENOME ASSEMBLY AND ANNOTATION
Short Read Technology PacBio Sequel System
Cost Normalized FragGene FragGene
Prodigal Prodigal
Scan Scan
# Proteins 29,325 32,009 127,750 117,145
After error penalties 17,573 26,074 - 84,656
De-replicated 24,195 21,143 80,705 49,658
After error penalties 14,499 17,222 - 35,886
- Goal: Mine soil metagenomes for gene clusters that synthesize bio-active compounds
- The combination of HiFi data and a highly contiguous assembly permits recovery of approximately twice as many
predicted proteins as short read technology
- SMRT sequencing is cost-effective
Work done in collaboration with Second Genome. 43
Jain, S. (2019). Comparison of sequencing approaches applied to complex soil metagenomes to resolve proteins of interest. ASM Microbe poster presentation.HIFI READS CAN BE ASSEMBLED WITH Canu TO PRODUCE NOVEL REFERENCES
FOR UNCULTURABLE SPECIES
2,000,000 8,000
N50 contig length
1,800,000
7,000
1,600,000 Number of contigs
Contig N50 Length (bp)
6,000
Number of Contigs
1,400,000
5,000
1,200,000
1,000,000 4,000
800,000
3,000
600,000
2,000
400,000
1,000
200,000
0 0
Human Fecal Samples
44CONTIGS PRODUCED BY Canu CAN BE BINNED BY SPECIES WITH TOOLS LIKE
PATRIC RAST BINNING SERVICE (RBS)
140
120
100
Number of Bins
80
60
40
20
0
Human Fecal Samples
45
Analyses performed at https://patricbrc.org/ on the combined Canu contigs and unassembled reads for each sample.PACBIO METAGENOME DATA CAN PROVIDE REFERENCE QUALITY ASSEMBLIES OF
UNCULTURABLE STRAINS
Uncover mechanisms with a highly resolved, functional view of your metagenome
- Closed, complete or nearly complete B. breve genomes from preterm neonate gut microbiome samples, with
sequencing coverage between 7- to 115-fold
- The B. breve strains, associated with healthy gut development, possess diverse carbohydrate metabolism capabilities,
including a “bifid shunt” that can convert human milk oligosaccharides (HMO) to short chain fatty acids (SCFAs)
46
McComb, E. (2019). High-resolution evaluation of gut microbiota associated with intestinal maturation in early preterm neonates. ASM Microbe poster presentation.Technical Documentation & Applications Support Resources
BEST PRACTICES: METAGENOMIC SEQUENCING WITH HIFI READS
(SEQUEL II CHEMISTRY 2.0)
* Read lengths, reads/data per SMRT Cell 8M and other sequencing performance results vary based on sample quality/type and 48
insert size. See Application Brief: Metagenomic sequencing with HiFi reads – Best PracticesBEST PRACTICES: METAGENOMIC SEQUENCING WITH HIFI READS
(SEQUEL II CHEMISTRY 2.0) (CONT.)
49
Application Brief: Metagenomic sequencing with HiFi reads – Best PracticesTECHNICAL DOCUMENTATION AND APPLICATIONS SUPPORT RESOURCES FOR
METAGENOMIC SHOTGUN LIBRARY PREPARATION, SEQUENCING & DATA ANALYSIS
Sample Preparation Literature
- Application Brief: Metagenomic sequencing with HiFi reads - Best Practices (PN BP108-030220)
- Procedure & Checklist – Preparing 10 kb Library Using SMRTbell Express Template Prep Kit 2.0 for Metagenomics Shotgun Sequencing
(PN 101-800-800)
- 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)
- Technical Note: Preparing DNA for PacBio HiFi sequencing – Extraction and quality control (PN TN101-061920)
- Technical Overview: Metagenomics Shotgun Library Preparation Using SMRTbell Express Template Prep Kit 2.0 (PN 101-894-900)
Videos & Webinars
- PacBio Webinar (2020): Bioinformatics lunch & learn – A quick guide to metagenomic analysis with PacBio HiFi reads
- PacBio Webinar (2020): A HiFi View – Sequencing the gut microbiome with highly accurate long reads
50TECHNICAL DOCUMENTATION AND APPLICATIONS SUPPORT RESOURCES FOR
METAGENOMIC SHOTGUN LIBRARY PREPARATION, SEQUENCING & DATA ANALYSIS
(CONT.)
Data Analysis Resources
- 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
COMPLEX POPULATIONS
DATASET DATA TYPE PACBIO SYSTEM
APPLICATION
Full-length 16S Sequencing 20 Strain Mock Microbial Community – ATCC MSA-1003 – 16S HiFi Reads Sequel II System
Metagenomics Shotgun Profiling &
20 Strain Mock Microbial Community – ATCC MSA-1003 – Shotgun HiFi Reads Sequel II System
Assembly
51TECHNICAL DOCUMENTATION AND APPLICATIONS SUPPORT RESOURCES FOR
METAGENOMIC SHOTGUN LIBRARY PREPARATION, SEQUENCING & DATA ANALYSIS
(CONT.)
Posters
- PacBio AGBT 2020 Poster: Unbiased characterization of metagenome composition and function using HiFi sequencing on the PacBio Sequel
II System
Publications
- Xie, H. et al. (2020) PacBio Long Reads Improve Metagenomic Assemblies, Gene Catalogs, and Genome Binning. Frontiers in Genetics.
Volume 11, Article 516269.
- Somerville, V. (2019) Long-read based de novo assembly of low-complexity metagenome samples results in finished genomes and reveals
insights into strain diversity and an active phage system. BMC Microbiology. 19:143.
- Hiraoka, Satoshi et al. (2019) Metaepigenomic analysis reveals the unexplored diversity of DNA methylation in an environmental prokaryotic
community. Nature Communications. 10:159.
- Beaulaurier, John et al. (2018) Metagenomic binning and association of plasmids with bacterial host genomes using DNA methylation.
Nature Biotechnology. 36:61–69.
- Driscoll, C.B. et al. (2017) Towards long-read metagenomics: complete assembly of three novel genomes from bacteria dependent on a
diazotrophic cyanobacterium in a freshwater lake co-culture. Standards in Genomic Sciences. 12:9.
- See the PacBio Complex Populations Applications website for a list of other publications.
52www.pacb.com
For Research Use Only. Not for use in diagnostic procedures. © Copyright 2021 by Pacific Biosciences of California, Inc. All rights reserved. Pacific Biosciences, the Pacific Biosciences logo, PacBio, SMRT, SMRTbell, Iso-Seq, and Sequel are trademarks of Pacific
Biosciences. Pacific Biosciences does not sell a kit for carrying out the overall No-Amp Targeted Sequencing method. Use of these No-Amp methods may require rights to third-party owned intellectual property. FEMTO Pulse and Fragment Analyzer are trademarks
of Agilent Technologies Inc.
All other trademarks are the sole property of their respective owners.You can also read
