Technical Overview: Full-Length 16S Library Preparation Using SMRTbell Express Template Prep Kit 2.0
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Technical Overview: Full-Length 16S 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 101-916-900 Ver 2021-02-01-A (February 2021)
Full-Length 16S Library Preparation Using SMRTbell Express Template Prep Kit 2.0 1. 16S Amplicon Sample Preparation Workflow Overview 2. 16S Amplicon Sample Preparation Workflow Details 3. 16S Amplicon Sequencing Workflow Details 4. 16S Amplicon Data Analysis Recommendations 5. 16S Amplicon Library Example Performance Data 6. Technical Documentation & Applications Support Resources
FULL-LENGTH 16S AMPLICON SEQUENCING: HOW TO GET STARTED
Application-Specific Application-Specific Application Consumable Library Construction,
Best Practices Guide Procedure & Checklist Bundle Purchasing Guide Sequencing & Analysis
16S Amplicon Generation
1-Step PCR Amplification of Full-length
16S Genes Using Barcoded F/R Primers
Library Construction
(SMRTbell Express TPK 2.0)
Multiplex Up To 192 Full-Length 16S
Samples per SMRT Cell 8M
HiFi Sequencing
Generate Up To 3.5 Million HiFi Reads
per SMRT Cell 8M
Data Analysis
Application Brief: Metagenomic sequencing with Procedure & Checklist – Amplification of Full-Length PacBio Application Consumable Bundle
Demultiplex Barcodes Using SMRT Link
HiFi reads - Best Practices (BP108-030220) 16S Gene with Barcoded Primers for Multiplexed Purchasing Guide (PG100-082620) 16S Analysis Using 3rd-Party Software
SMRTbell Library Preparation and Sequencing
Summary overview of application-specific sample Purchasing Guide enables users to easily order
(101-599-700) * Application Consumable Bundles include reagents for library
preparation and data analysis workflow required consumables needed to prepare a
construction, primer annealing and polymerase binding. Core PacBio-
recommendations Technical documentation containing sample library SMRTbell library to run a specific type of branded SMRT Sequencing consumables (SMRT Cells, Sequencing Kits
construction and sequencing preparation protocol application on the Sequel II and IIe Systems* & SMRT Oil), plastics and other 3rd-party reagents are not included in the
details application bundles 3
16S Amplicon Sample Preparation Workflow Overview
FULL-LENGTH 16S AMPLICON SAMPLE PREPARATION PROCEDURE DESCRIPTION
- Procedure & Checklist – Amplification of Full-Length 16S Gene with Barcoded Primers for Multiplexed
SMRTbell Library Preparation and Sequencing (PN 101-599-700) PacBio protocol document describes
methods for targeted PCR amplification and construction of a multiplexed library using SMRTbell Express
Template Prep Kit 2.0 for sequencing full-length 16S genes on the Sequel, Sequel II and Sequel IIe
Systems (Sequel Systems).
- This document contains instructions for:
1. 1-Step PCR amplification of asymmetrically barcoded full-length 16S genes (V1-V9 regions) from bacterial DNA
isolated from metagenomic samples.
2. Multiplexed SMRTbell library preparation and sequencing of 16S amplicons on the Sequel, Sequel II and Sequel IIe
systems
- We also provide the sequences of and ordering information for 8 barcoded forward, and 24 barcoded
reverse, 16S-specific primers that can be combined for multiplexed analysis of up to 192 samples (per
SMRT Cell 8M) using the asymmetric barcoding strategy described in this procedure.
- Note: Alternatively, can also use third-party Shoreline Biome microbiome assay kits for DNA sample
extraction and PCR amplification, followed by SMRTbell library construction as described in this https://www.pacb.com/support/documentation/
procedure
APPLICATIONS
CHARACTERIZE MICROBIAL COMMUNITIES
5
16S 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 16S metagenomics applications
16S Amplicon Generation
HiFi Sequencing
▪ Perform 1-Step PCR amplification of full-length
▪ Follow Quick Reference Cards for primer
16S genes using recommended barcoded
annealing, polymerase binding, complex cleanup
forward / reverse primers (PN 101-599-700)
and sample loading
▪ Pool up to 192 barcoded 16S amplicon samples
▪ Generate up to 3.5 million HiFi reads per SMRT
to generate a single, pooled sample for SMRTbell
Cell 8M for a 16S library
library construction
Data Analysis
SMRTbell Library Construction (4 hrs)
▪ Demultiplex
▪ Follow Procedure & Checklist – Amplification barcodes using
of Full-Length 16S Gene with Barcoded SMRT Link PacBio HiFi
Primers for Multiplexed SMRTbell Library reads achieve
▪ Perform 16S Analysis
Preparation and Sequencing (PN 101-599-700) >99.9% accuracy
Using Third-Party
▪ Purify final SMRTbell library using AMPure PB Software (DADA2)
beads
HiFi Read
6
16S Amplicon Sample Preparation Workflow Details
PROCEDURE & CHECKLIST – AMPLIFICATION OF FULL-LENGTH 16S GENE WITH
BARCODED PRIMERS FOR MULTIPLEXED SMRTBELL LIBRARY PREPARATION AND
SEQUENCING
- This document (PN 101-599-700) presents a workflow for amplifying full-length 16S
genes from bacterial gDNA isolated from metagenomic samples and constructing
multiplexed libraries using SMRTbell Express TPK 2.0 for sequencing on the
Sequel, Sequel II and Sequel IIe Systems (Sequel Systems).
- Document also provides the sequences of and ordering information for 8 barcoded
forward, and 24 barcoded reverse, 16S-specific primers that can be combined for
multiplexed analysis of up to 192 samples using the asymmetric barcoding strategy
described in this procedure.
- Protocol document contains:
1. Recommendations for metagenomic DNA extraction QC and quantification
2. Barcoded 16S Primer Sequences, ordering and storage Information
3. Instructions for amplification of full-length 16S gene from bacterial gDNA extracted from
metagenomic samples using barcoded primers in a single round of PCR
4. Enzymatic steps for preparation of barcoded 16S SMRTbell libraries
5. Sample setup guidance for preparing 16S SMRTbell libraries for sequencing on the
Sequel Systems
8
https://www.pacb.com/support/documentation/
FULL-LENGTH 16S DETAILED WORKFLOW OVERVIEW
1. 1-Step PCR Amplicon Generation with 16S Barcoded Forward and Amplify 16S Gene with Barcoded
Reverse primers and Sample Pooling 1 Forward and Reverse Primers
- PCR amplify 16S gene using 16S Barcoded Forward and Reverse primers
(order from any oligo vendor)
QC and Pool Samples
- Pool up to 192 asymmetrically-barcoded 16S metagenomic samples to generate
a single, pooled sample for SMRTbell library construction
AMPure PB Bead Purification
2. SMRTbell Express TPK 2.0 Library Construction (4 hours)
- Required input mass of pooled sample for library construction is ≥500 ng DNA Damage Repair
- Single-tube, addition-only reactions
2
- Typical library yield ≥40% End Repair / A-Tailing
3. Sequencing Preparation Adapter Ligation
- Anneal sequencing primer, bind polymerase, perform AMPure PB bead complex
cleanup
AMPure PB Bead Purification
- Generate ≥3 million HiFi 16S reads per SMRT Cell 8M (2 Rounds)
4. Data Analysis
3 Prepare for Sequencing
- 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
- De-multiplex barcodes within SMRT Link GUI or on the command line
4
- Assess community membership and function with third-party 16S analysis tools like 9
QIIME, DADA2, and MEGAN
LIST OF REQUIRED MATERIALS AND EQUIPMENT
ITEM VENDOR PART NUMBER
DNA QC
Lonza FlashGel System Lonza 57025
2100 Bioanalyzer Instrument Agilent G2939BA
NanoDrop UV/Vis Spectrophotometer System Thermo-Fisher ND-2000
DNA Quantitation
Qubit Fluorometer Thermo Fisher Scientific Q33226
Qubit 1X dsDNA HS Assay Kit Thermo Fisher Scientific Q33230
16S PCR Amplification
KAPA HiFi HotStart ReadyMix PCR Kit KAPA Biosystems KK2600 (or KK2601 or KK2602)
See Table 1 in Procedure & Checklist for
Barcoded 16S Primers Any Oligo Vendor
Ordering Info
SMRTbell Library Preparation
SMRTbell Express Template Prep Kit 2.0 PacBio 100-938-900
AMPure PB Beads PacBio 100-265-900
100% Ethanol, Molecular Biology Grade Any MLS
DNA LoBind tubes, 2.0 mL Eppendorf 022431048 10GENOMIC DNA EXTRACTION FROM METAGENOMIC SAMPLES FOR 16S DNA
SMRTBELL LIBRARY CONSTRUCTION
- Due to the harsh lysis methods required for some organisms, it may be difficult to extract large quantities of high
quality, intact genomic DNA (gDNA) from metagenomic samples.
- However, for most metagenomic samples, gDNA quality and quantity are likely sufficient for full-length 16S
amplification.
- 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.
11BEST PRACTICES RECOMMENDATIONS FOR BACTERIAL GENOMIC DNA QC
Bacterial Genomic DNA QC Recommendations
Bacterial gDNA from up to 192 metagenomic samples can be processed using this procedure. For best results,
characterize your bacterial gDNA samples thoroughly and normalize gDNA concentration before use.
- Bring gDNA to room temperature and mix well by pipetting to ensure sample homogeneity, then measure gDNA concentration using
Qubit dsDNA HS assay reagents.
- Assess sample purity by using a NanoDrop system. OD260/280 should be between 1.8 and 2.0.
- To ensure pipetting accuracy, aim to deliver 25 pg – 2.5 ng to each individual PCR reaction in a constant 5 μL volume. Normalize
sample gDNA concentration to 5 – 500 pg/μL in 10 mM Tris-HCl pH 8.0-8.5 prior to setting up PCR reactions. The recommended
total input gDNA per PCR reaction is 1 ng – 2 ng.
12BARCODED 16S PCR PRIMER SEQUENCES, ORDERING AND STORAGE INFORMATION
Table 1 in the Appendix of the procedure lists sequences for 32 barcoded16S gene-specific primers (8
Forward and 24 Reverse) that can be used in all possible asymmetric pairs for multiplexing up to 192 samples.
BARCODED
OLIGO SEQUENCE
FORWARD PRIMER
>16S_For_bc1005 /5Phos/GCATCCACTCGACTCTCGCGTAGRGTTYGATYMTGGCTCAG
>16S_For_bc1007 /5Phos/GCATCTCTGTATCTCTATGTGAGRGTTYGATYMTGGCTCAG Each primer oligo contains:
>16S_For_bc1008 /5Phos/GCATCACAGTCGAGCGCTGCGAGRGTTYGATYMTGGCTCAG
5’ Buffer Sequence
>16S_For_bc1012 /5Phos/GCATCACACTAGATCGCGTGTAGRGTTYGATYMTGGCTCAG
>16S_For_bc1015 /5Phos/GCATCCGCATGACACGTGTGTAGRGTTYGATYMTGGCTCAG
16-base Barcode Sequence
>16S_For_bc1020 /5Phos/GCATCCACGACACGACGATGTAGRGTTYGATYMTGGCTCAG Degenerate 16S gene-specific forward or
>16S_For_bc1022 /5Phos/GCATCCACTCACGTGTGATATAGRGTTYGATYMTGGCTCAG reverse primer sequences
>16S_For_bc1024 /5Phos/GCATCCATGTAGAGCAGAGAGAGRGTTYGATYMTGGCTCAG
BARCODED BARCODED
OLIGO SEQUENCE OLIGO SEQUENCE
REVERSE PRIMER REVERSE PRIMER
>16S_Rev_bc1033 /5Phos/GCATCAGAGACTGCGACGAGARGYTACCTTGTTACGACTT >16S_Rev_bc1076 /5Phos/GCATCGAGAGCGCGAGTGCACRGYTACCTTGTTACGACTT
>16S_Rev_bc1035 /5Phos/GCATCCAGAGAGTGCGCGCGCRGYTACCTTGTTACGACTT >16S_Rev_bc1082 /5Phos/GCATCGTGCTCTGTGTGTCACRGYTACCTTGTTACGACTT
>16S_Rev_bc1044 /5Phos/GCATCCGCGCGTCGTCTCAGCRGYTACCTTGTTACGACTT >16S_Rev_bc1083 /5Phos/GCATCTGCGTGTATGTCATATRGYTACCTTGTTACGACTT
>16S_Rev_bc1045 /5Phos/GCATCAGAGAGTACGATATGTRGYTACCTTGTTACGACTT >16S_Rev_bc1089 /5Phos/GCATCACGAGATACTCGCGCGRGYTACCTTGTTACGACTT
>16S_Rev_bc1054 /5Phos/GCATCTCTGTAGTGCGTGCGCRGYTACCTTGTTACGACTT >16S_Rev_bc1096 /5Phos/GCATCCTGTGTAGAGAGCACARGYTACCTTGTTACGACTT
>16S_Rev_bc1056 /5Phos/GCATCATGTGCGTGTGTGTCTRGYTACCTTGTTACGACTT >16S_Rev_bc1098 /5Phos/GCATCTGATGTGACACTGCGCRGYTACCTTGTTACGACTT
>16S_Rev_bc1057 /5Phos/GCATCCTCTCAGACGCTCGTCRGYTACCTTGTTACGACTT >16S_Rev_bc1100 /5Phos/GCATCACTACTGAGACATAGARGYTACCTTGTTACGACTT
>16S_Rev_bc1059 /5Phos/GCATCTATCTCAGTGCGTGTGRGYTACCTTGTTACGACTT >16S_Rev_bc1101 /5Phos/GCATCTATATCGCGTCGCTATRGYTACCTTGTTACGACTT
>16S_Rev_bc1060 /5Phos/GCATCTGTGTCTATACTCATCRGYTACCTTGTTACGACTT >16S_Rev_bc1105 /5Phos/GCATCGCGTACTGCGACTGTGRGYTACCTTGTTACGACTT
>16S_Rev_bc1062 /5Phos/GCATCTATAGACTATCTGAGARGYTACCTTGTTACGACTT >16S_Rev_bc1107 /5Phos/GCATCATATATGCACGCTCTARGYTACCTTGTTACGACTT
>16S_Rev_bc1065 /5Phos/GCATCGTATGTGAGAGAGCGCRGYTACCTTGTTACGACTT >16S_Rev_bc1110 /5Phos/GCATCCGCTGTATACACGCTCRGYTACCTTGTTACGACTT
>16S_Rev_bc1075 /5Phos/GCATCCACGCGACGCTCTCTARGYTACCTTGTTACGACTT >16S_Rev_bc1112 /5Phos/GCATCAGAGACTGTAGCGCACRGYTACCTTGTTACGACTT 13BARCODED 16S PCR PRIMER SEQUENCES, ORDERING AND STORAGE INFORMATION
(CONT.)
- Oligos must contain 5’ phosphates
- HPLC-purification is recommended, but not required
- Each oligo contains a 5’ buffer sequence (GCATC), a 16-base barcode, and degenerate 16S gene-specific forward or
reverse primer sequences
- Degenerate base identities are: R = A,G; Y = C,T; M = A,C
- Primers should be stored at high concentration in a buffered solution (e.g., 100 μM primer in 10 mM Tris-HCl pH 8.0-8.5)
at -20°C
- Avoid repeated freeze-thaw cycles
14PREPARATION OF 16S PRIMER STOCK SOLUTIONS
- Dilute Barcoded, 16S gene-specific forward and reverse PCR primers (see Table 1 in the Appendix of the procedure
for sequences and ordering information) to 2.5 μM in 10 Tris-HCl pH 8.0-8.5.
- If necessary, initially resuspend oligos at 100 μM in 10 Tris-HCl pH 8.0-8.5.
- Mix well by pipetting or vortexing, then dilute each primer individually to 2.5 μM in 10 Tris-HCl pH 8.0-8.5. For example, add 5 μL 100
μM primer to 195 μL 10 Tris-HCl pH 8.0-8.5. Mix well by pipetting. This volume of diluted oligo is sufficient for more than 50 PCR
reactions.
- For a 96-plex experimental design, each forward primer will be used in 12 separate reactions, and each reverse primer will be used
in 8 separate reactions.
- Note: Always mix primer stock solutions well before preparing dilutions, as concentration gradients may form during the freeze-thaw
process.
- Prior to use, verify diluted primer oligo concentrations by directly measuring the OD260 of each 2.5 μM primer solution using a
NanoDrop system.
15PREPARATION OF 16S GENE PCR AMPLIFICATION REACTIONS
A. Preparation of PCR Master Mix for 16S Gene Amplification Reactions
Prepare the PCR Master Mix of all common components outlined below in a 2.0 mL LoBind tube, including a 25%
overage. Always thaw the KAPA HiFi HotStart ReadyMix (2X) PCR reagent on ice and mix well before use.
Ensure that all other reagents are also thawed and mixed well prior to use.
COMPONENT 1 SAMPLE N FOR 96-PLEX* FOR 192-PLEX*
PCR-grade Water 1.5 µl 1.5 x N x 1.25 180.0 µl 360.0 µl
KAPA HiFi HotStart ReadyMix
12.5 µl 12.5 x N x 1.25 1500.0 µl 3000.0 µl
(2X)
Total Volume 14.0 µl 14.0 x N x 1.25 1680.0 µl 3360.0 µl
* Includes 25% overage
- Note: All KAPA HiFi HotStart (2X) reagents and reactions must be set up and kept on ice until ready for use in PCR; the high
proofreading activity of the enzyme will rapidly degrade primers at room temperature.
16B. Preparation of Barcoded Forward Primer Master Mix
Add Barcoded Forward Primers to the PCR Master Mix to generate eight different Forward Primer Master Mix
solutions
1. Label 8 LoBind tubes A, B, C, D, E, F, G, and H to assign a specific Barcoded Forward Primer to each tube (see table below).
TUBE BARCODED FORWARD PRIMER
A >16S_For_bc1005
B >16S_For_bc1007
C >16S_For_bc1008
D >16S_For_bc1012
E >16S_For_bc1015
F >16S_For_bc1020
G >16S_For_bc1022
H >16S_For_bc1024
17B. Preparation of Barcoded Forward Primer Master Mix (Cont.)
2. Depending on the desired level of multiplex (96-plex or 192-plex), transfer the appropriate volume of PCR Master Mix into each
of the eight tubes (see table below).
FOR 96-PLEX FORWARD PRIMER FOR 192-PLEX FORWARD PRIMER
COMPONENT
MASTER MIX MASTER MIX
PCR Master Mix 201.6 µL 403.2 µL
Barcoded Forward Primer (2.5 μM) 43.2 µL 86.4 µL
Total Volume per Tube 244.8 µL 489.6 µL
3. Then add the required volume of Barcoded Forward Primer (2.5 μM) to the appropriate tube.
4. Mix well by pipetting.
▪ The total volume of each tube for the 96-plex Forward Primer Master Mix is 244.8 μL
▪ The total volume of each tube for the 192-plex Forward Primer Master Mix is 489.6 μL
18C. Transfer of Barcoded Forward Primer Master Mixes into 96-Well Sample Plates
Transfer 17 μL aliquots of the Forward Primer Master Mix (tubes A-H) into each well of the appropriate row (A-H) of a
96-well plate.
▪ For a 96-plex experiment design, use one 96-well plate.
▪ For a 192-plex experiment design, use two 96-well plates.
BARCODED FORWARD PRIMER MASTER MIX TO ADD
1 2 3 4 5 6 7 8 9 10 11 12
>16S_For_bc1005 Master Mix (17 µL per well) A
>16S_For_bc1007 Master Mix (17 µL per well) B
>16S_For_bc1008 Master Mix (17 µL per well) C
>16S_For_bc1012 Master Mix (17 µL per well) D
>16S_For_bc1015 Master Mix (17 µL per well) E
>16S_For_bc1020 Master Mix (17 µL per well) F
>16S_For_bc1022 Master Mix (17 µL per well) G
>16S_For_bc1024 Master Mix (17 µL per well) H
Figure illustration of an example plate layout for setting up a 96-plex PCR design using eight different 16S
Barcoded Forward Primer Master Mixes. (For a 192-plex design, set up two PCR plates.) 19C. Transfer of Barcoded Reverse Primers into 96-Well Sample BARCODED REVERSE PRIMER TO ADD (3 µL PER WELL)
Plates
>16S_Rev_bc1033
>16S_Rev_bc1035
>16S_Rev_bc1044
>16S_Rev_bc1045
>16S_Rev_bc1054
>16S_Rev_bc1056
>16S_Rev_bc1057
>16S_Rev_bc1059
>16S_Rev_bc1060
>16S_Rev_bc1062
>16S_Rev_bc1065
>16S_Rev_bc1075
Add 3 µL of Barcoded Reverse Primers to appropriate wells
containing the 17 μL aliquots of Forward Primer Master Mix
dispensed in the previous step above.
▪ To each well of columns 1-12 of the 96-well plate(s), add 3 μL of
Barcoded Reverse Primers (2.5 μM).
1 2 3 4 5 6 7 8 9 10 11 12
▪ For a 96-plex experiment design, use the first 12 reverse primers
A
listed in Table 1 in Appendix A.
B
▪ For a 192-plex experiment design, use all 24 reverse primers
listed in Table 1 in Appendix A. C
▪ The total volume in each well is 20 μL and the final concentration of D
barcoded forward and reverse primers in each well is 0.375 μM. E
F
G
H
Figure illustration of an example plate layout for setting up a 96-
plex PCR design using twelve different 16S Barcoded Reverse
Primers (2.5 µM). For a 192-plex design, set up two PCR plates
using twenty-four different 16S Barcoded Reverse Primers. 20D. Sample Plate Final Preparations and PCR Thermal Cycling Conditions
Add the diluted bacterial gDNA samples to the 96-well plate(s) and perform PCR according to the recommended
conditions described below.
1. Add 5 μL (1-2 ng) of each diluted gDNA sample to a single well of the 96-well PCR plate. Mix reactions well by pipetting. Seal the plate
thoroughly with adhesive seal to prevent evaporation during PCR. Briefly spin the plate in a refrigerated centrifuge (4°C) to ensure
sample volume is at the bottom of each well.
2. Perform PCR using the thermal cycling parameters indicated in the Table below. Typical yield from each PCR reaction is ~500 ng.
STEP NUMBER STEP DESCRIPTION TEMPERATURE TIME
1 Initial Denature 95 C 3 minutes
2 Denature 95 C 30 seconds
3 Anneal* 57 C 30 seconds
4 Extend 72 C 60 seconds
Repeat steps 2 to 4 for a total of 20-27 cycles**
* Refer to manufacturer’s recommendations for your thermocycler to set the ramp rate for the annealing
step toEXAMPLE AGAROSE GEL DNA QC FOR 16S PCR AMPLICON PRODUCTS
- Spot check amplification results by directly loading 1.0 μL of one or more PCR reactions onto an agarose gel. A typical
result is shown in the example Figure below.
Agarose gel QC spot check of individual 16S gene PCR reactions. 1 μL from four
independent PCR reactions was analyzed per lane on a 1.2% agarose Lonza DNA Flash Gel
according to the manufacturer’s recommendations. The PCR products were of the expected size
(~1.5 kb) and of comparable quantity as determined by visual inspection of band intensity.
- The expected 16S amplicon size is ~1500 bp, and the amount of amplicon from each sample should be comparable as assessed by relative
band intensity of the ~1500 bp PCR product.
- If available, you may also use an Agilent Bioanalyzer or TapeStation system to spot check PCR product size and quantity.
22BEST PRACTICES RECOMMENDATIONS FOR PREPARING 16S DNA SMRTBELL
LIBRARIES
1. Ensure that the AMPure PB beads are at room temperature prior to performing the purification steps.
2. 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.
3. Measure gDNA concentration using a Qubit fluorometer and Qubit dsDNA High Sensitivity (HS) Assay Kit
reagents as recommended by the manufacturer.
23SMRTBELL EXPRESS TEMPLATE PREP KIT 2.0 REAGENT HANDLING
RECOMMENDATIONS
- Several reagents in the kit are sensitive to temperature LIST OF TEMPERATURE-SENSITIVE REAGENTS INCLUDED IN SMRTBELL
EXPRESS TPK 2.0.
and vortexing
REAGENT WHERE USED
- PacBio highly recommends: DNA Damage Repair Mix v2 DNA Damage Repair
▪ Never leaving reagents at room temperature
End Prep Mix End-Repair/A-tailing
▪ Working on ice at all times when preparing master mixes Overhang Adapter v3 Ligation
▪ Finger tapping followed by a quick-spin prior to use Ligation Mix Ligation
Ligation Additive Ligation
Ligation Enhancer Ligation
24EXAMPLE AGAROSE GEL DNA QC FOR A 96-PLEX POOLED 16S AMPLICON SAMPLE
BEFORE AND AFTER SMRTBELL LIBRARY CONSTRUCTION
- Verify the size of the final purified SMRTbell library on an agarose gel. A typical result is shown in the example Figure
below.
Agarose gel QC of pooled 16S PCR amplicon products and the final SMRTbell library.
Samples were loaded and run on a 1.2% Agarose Lonza DNA Flash Gel Cassette according to
the manufacturer’s recommendations. The pooled PCR products (Lane 1) and final SMRTbell
library (Lane 2) are of the expected size (~1.5 kb).
2516S Sequencing Workflow Details
SAMPLE SETUP RECOMMENDATIONS FOR 16S 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 16S amplicon samples for sequencing
27SAMPLE SETUP RECOMMENDATIONS FOR 16S 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 16S amplicon samples for sequencing
- For SMRT Link v10.0 (or higher): Select ‘Full-Length 16S rRNA Sequencing’ from the
Application field drop-down menu in the SMRT Link Sample Setup and SMRT Link Run
Design user interface
* PacBio recommends Sequel II Binding Kit 2.1
for sequencing 16S amplicons samples.
28IMPORTING THE BARCODE FASTA FILE INTO SMRT LINK FOR AUTOMATED
DEMULTIPLEXING OF POOLED 16S SMRTBELL 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_192_barcodes_v1.zip (contains a list of 8 Forward and 24 Reverse PacBio barcodes for use with Barcoded 16S
Primers)
EXAMPLE FASTA FILE CONTAINING A LIST
OF PACBIO 16-BASE PAIR BARCODES
29IMPORTING THE BARCODE FASTA FILE INTO SMRT LINK FOR AUTOMATED
DEMULTIPLEXING OF POOLED 16S SMRTBELL 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
30SMRT LINK RUN DESIGN SETUP PROCEDURE FOR AUTOMATED DEMULTIPLEXING OF
POLLED 16S SMRTBELL 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_96_barcodes_v1) 1
3. Same Barcodes on Both Ends of Sequence: No 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
31SMRT LINK RUN DESIGN SETUP PROCEDURE FOR AUTOMATED DEMULTIPLEXING OF
POOLED 16S SMRTBELL 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--bc1002 16S Sample 1
▪ Delete entire rows of barcodes not used 2 bc1001--bc1003 16S Sample 2
▪ Allowed characters: Alphanumeric; space; dot; bc1001--bc1004 16S Sample 3
underscore; hyphen. Other characters will be bc1001--bc1005 16S 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
32SMRT LINK RUN DESIGN SETUP PROCEDURE FOR AUTOMATED DEMULTIPLEXING OF
POOLED 16S SMRTBELL 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 3316S Data Analysis Recommendations
16S DATA ANALYSIS RECOMMENDATIONS
- 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 16S amplicon
sequencing data
- SMRT Link GUI Demultiplex Barcodes analysis application supports up to 10,000 barcodes per sample
- Refer to Barcoding Overview documents available on PacBio’s Multiplexing Resources website (https://www.pacb.com/products-
and-services/analytical-software/multiplexing/) for detailed information on QC metrics for evaluation of barcoding performance using
SMRT Link
- For metagenomic community profiling, PacBio recommends aiming for ≥8,000 HiFi reads per demultiplexed 16S
sample (See Overview – Sequel Systems Application Options and Sequencing Recommendations)
- Output data in standard file formats, (BAM and FASTA/Q) for seamless integration with downstream analysis tools
- HiFi reads are compatible with third-party 16S data analysis tools to assess community membership and function:
- QIIME
- DADA2
- MEGAN
- Note: If using third-party Shoreline Biome microbiome assay kits for PCR amplification and sample barcoding, can use
Shoreline’s SBanalyzer software to perform de-multiplexing and 16S data analysis
3516S Library Example Performance Data
MSA-1003 MOCK COMMUNITY SAMPLE DESCRIPTION
MSA-1003 is a controlled, pre-defined, standardized reference % MSA-1003 COMPONENT
material that can help with metagenomic analysis protocol 0.18 Acinetobacter baumannii (ATCC 17978)
development optimization, verification, and quality control 1.80 Bacillus cereus (ATCC 10987)
0.02 Bacteroides vulgatus (ATCC 8482)
- 20 Strain Staggered Mix Genomic Material (ATCC MSA-1003)
0.02 Bifidobacterium adolescentis (ATCC 15703)
https://www.atcc.org/products/all/MSA-1003.aspx
1.80 Clostridium beijerinckii (ATCC 35702)
- MSA-1003 sample is a mock microbial community that mimics mixed metagenomic 0.18 Cutibacterium acnes (ATCC 11828)
samples 0.02 Deinococcus radiodurans (ATCC BAA-816)
- MSA-1003 sample comprises genomic DNA prepared from fully sequenced, 0.02 Enterococcus faecalis (ATCC 47077)
characterized, and authenticated ATCC Genuine Cultures that were selected by 18.0 Escherichia coli (ATCC 700926)
ATCC based on relevant phenotypic and genotypic attributes, such as Gram stain, 0.18 Helicobacter pylori (ATCC 700392)
GC content, genome size, and spore formation 0.18 Lactobacillus gasseri (ATCC 33323)
- For the example data shown in this presentation, replicate MSA-1003 samples were 0.18 Neisseria meningitidis (ATCC BAA-335)
processed in parallel during library construction to generate a 192-plex 18.0 Porphyromonas gingivalis (ATCC 33277)
(asymmetrically barcoded) pooled 16S SMRTbell library 1.80 Pseudomonas aeruginosa (ATCC 9027)
18.0 Rhodobacter sphaeroides (ATCC 17029)
0.02 Schaalia odontolytica (ATCC 17982)
1.80 Staphylococcus aureus (ATCC BAA-1556)
18.0 Staphylococcus epidermidis (ATCC 12228)
1.80 Streptococcus agalactiae (ATCC BAA-611)
18.0 Streptococcus mutans (ATCC 700610)
https://www.atcc.org/products/all/MSA-1003.aspx 37EXAMPLE PRIMARY SEQUENCING PERFORMANCE RESULTS FOR A 192-PLEX 16S
LIBRARY (SEQUEL II SYSTEM)
PRIMARY RUN STATISTICS* FOR A 192-PLEX 16S LIBRARY1 RUN ON A SINGLE SEQUEL II SYSTEM SMRT CELL 8M2
PRODUCTIVITY
TOTAL RAW HIFI (≥Q20) READS
(%) MEAN POLYMERASE
LIBRARY NAME BASE YIELD
READ LENGTH (bp) HIFI READ MEAN HIFI READ HIFI BASE
P0 P1 P2 (Gb) MEAN QV
COUNT LENGTH (bp) YIELD (Gb)
192-Plex MSA-1003 16S 29.7 65.3 5.0 24572 128.51 2568971 1532 3.94 41
1 20 Strain Staggered Mix Genomic Material (ATCC MSA-1003) mock community sample
2 50 pM on-plate loading concentration; 10-hour movie collection time; 1-hour pre-extension time; Sequel II Binding Kit 2.1; Sequel II Chemistry 2.0
Base Yield Density Insert Read Length Density HiFi Read Length Distribution
Insert Read Length (bp)
20 kb
2.0 kb
5 kb Mean HiFi Read Length
(1.5 kb) is consistent
1.0 kb
with expected 16S
amplicon size
Polymerase Read Length (kb) Polymerase Read Length (bp) HiFi Read Length (bp)
38
* 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 192-PLEX 16S LIBRARY
SAMPLE (SEQUEL II SYSTEM)
Full-length 16S data were collected for MSA-1003 mock community replicate samples
on a single SMRT Cell 8M. Amplification was performed with a barcoded 16S primer /
1-step PCR approach to generate a 192-plex 16S SMRTbell library.
METRIC VALUE
Movie Collection Time 10 hours
P1 Reads 5,234,175
HiFi Reads 2,568,971
HiFi Reads / P1 49%
With the single-step 16S PCR protocol, the
Mean HiFi Read QV Q41 yield of HiFi reads is highly
Barcoded HiFi Reads 2,468,174 consistent across all barcodes (HiFi
Reads per Barcode CV isEXAMPLE TAXONOMIC CLASSIFICATION RESULTS FOR 192-PLEX 16S LIBRARY
SAMPLE (SEQUEL II SYSTEM)
PacBio 16S Sequencing Faithfully Represents a Known Mock Community Sample
16S ANALYSIS OF THE MSA-1003 MOCK COMMUNITY MSA-1003 SAMPLE DESCRIPTION
- 20 Strain Staggered Mix Genomic Material (ATCC®
MSA-1003™) https://www.atcc.org/products/all/MSA-
1003.aspx
16S HiFi sequencing data reproduces the
expected composition of the MSA-1003
mock community sample
Download and explore this Sequel II
System 16S HiFi dataset further
Full-length (V1-V9) 16S amplicon samples were pooled at 192-Plex and sequenced on a single SMRT Cell 8M (Sequel II System Chemistry 2.0). PacBio 40
results shown in bar graph reflect the average abundance values derived from the pooled MSA-1003 replicate samples.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 42
insert size. See Application Brief: Metagenomic sequencing with HiFi reads – Best PracticesBEST PRACTICES: METAGENOMIC SEQUENCING WITH HIFI READS
(SEQUEL II CHEMISTRY 2.0) (CONT.)
43
Application Brief: Metagenomic sequencing with HiFi reads – Best PracticesTECHNICAL DOCUMENTATION AND APPLICATIONS SUPPORT RESOURCES FOR 16S
LIBRARY PREPARATION, SEQUENCING & DATA ANALYSIS
Sample Preparation Literature
- Application Brief: Metagenomic sequencing with HiFi reads – Best Practices (PN BP108-030220)
- Procedure & Checklist – Amplification of Full-Length 16S Gene with Barcoded Primers for Multiplexed SMRTbell Library Preparation
and Sequencing (PN 101-599-700)
- 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/IIe Systems (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 Overview: Full-Length 16S Library Preparation Using SMRTbell Express Template Prep Kit 2.0 (PN 101-916-900)
Data Analysis Resources
- 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
44TECHNICAL DOCUMENTATION AND APPLICATIONS SUPPORT RESOURCES FOR 16S
LIBRARY PREPARATION, SEQUENCING & DATA ANALYSIS (CONT.)
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
Metagenomic Profiling & Assembly 20 Strain Mock Microbial Community – ATCC MSA-1003 – Shotgun HiFi Reads Sequel II System
Viral Sequencing SARS-CoV-2 – ATCC Control with Eden Primers HiFi Reads Sequel II System
Posters
- PacBio AGBT 2020 Poster: Unbiased characterization of metagenome composition and function using HiFi sequencing on the PacBio
Sequel II System
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
45TECHNICAL DOCUMENTATION AND APPLICATIONS SUPPORT RESOURCES FOR 16S
LIBRARY PREPARATION, SEQUENCING & DATA ANALYSIS (CONT.)
Publications
- Graf et al. (2021) Tracking Closely Related Enteric Bacteria at High Resolution in Fecal Samples of Premature Infants Using a Novel rRNA
Amplicon. MedRxiv Preprint. doi: https://doi.org/10.1101/2020.09.26.20201608. (Now accepted for publication in mBio.)
- Callahan, Benjamin J et al. (2019) High-throughput amplicon sequencing of the full-length 16S rRNA gene with single-nucleotide resolution.
Nucleic Acids Research. 47(18): e103.
- Martijn, Joran et al. (2019) Confident phylogenetic identification of uncultured prokaryotes through long read amplicon sequencing of the
16S-ITS-23S rRNA operon. Environmental Microbiology. 21(7), 2485–2498.
- Tedersoo, Leho et al. (2019) Towards PacBio-based pan-eukaryote metabarcoding using full-length ITS sequences. Environmental
Microbiology Reports. 11(5):659-668.
- See the PacBio Complex Populations Applications website for a list of other publications.
46www.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
