For Research Use Only. Not for use in diagnostic procedures. © Copyright 2020 by Pacific Biosciences of California, Inc. All rights reserved. PN 101-916-900 Version 01 (April 2020)

Full-Length 16S Library Preparation Using

SMRTbell Express Template Prep Kit 2.0Sequel System v8.0 / Sequel Chemistry 3.0 / SMRT Link v8.0

Sequel II System v8.0 / Sequel II Chemistry 2.0 / SMRT Link v8.0

Full-Length 16S Library Preparation Using

SMRTbell Express Template Prep Kit 2.0

1. Full-Length 16S Amplicon Sample Preparation Workflow Details

2. Full-Length 16S Amplicon Sequencing Workflow Details

3. Full-Length 16S Amplicon Data Analysis Recommendations

4. Full-Length 16S Amplicon Library Example Performance Data

5. Technical Documentation & Applications Support Resources

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 PacBio protocol

document contains instructions for targeted PCR

amplification and construction of a multiplexed SMRTbell

library using Express Template Prep Kit 2.0 for sequencing

full-length 16S genes on the Sequel and Sequel II Systems.

APPLICATIONS

CHARACTERIZE MICROBIAL COMMUNITIES

FULL-LENGTH 16S WORKFLOW SUMMARY OVERVIEW

3. Sequencing Preparation (CCS Sequencing Mode)

- Perform Sample Annealing / Binding / Cleanup (A/B/C)

- Generate ≥3 million ≥Q20 16S reads per SMRT Cell 8M

1. 1-Step PCR Amplicon Generation with 16S Barcoded Forward and Reverse primers

and Sample Pooling

- Pool up to 96 barcoded 16S metagenomic samples to generate a single, pooled sample for SMRTbell

library construction

2. SMRTbell Express Template Prep Kit 2.0 Library Construction

- Perform single-tube, addition-only reactions

4. Analyze

- De-multiplex barcodes within SMRT Link GUI or on the command line

Barcoded amplicons are generated via a 1-step PCR by using 16S Barcoded

Forward and Reverse primer oligos. Once the PCR products are barcoded, they

can be pooled into a single tube for SMRTbell library construction.

~4 hours

Full-Length 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 and

Sequel II Systems.

- Document also provides the sequences of and ordering information

for 8 barcoded forward, and 12 barcoded reverse, 16S-specific

primers that can be combined for multiplexed analysis of up to 96

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 and Sequel II Systems

https://www.pacb.com/support/documentation/

FULL-LENGTH 16S DETAILED WORKFLOW OVERVIEW

1. 1-Step PCR Amplicon Generation with 16S Barcoded

Forward and Reverse primers and Sample Pooling

- PCR amplify 16S gene using 16S Barcoded Forward and

Reverse primers (order from any oligo vendor)

- Pool up to 96 barcoded 16S metagenomic samples to generate

a single, pooled sample for SMRTbell library construction

2. SMRTbell Express TPK 2.0 Library Construction

- Required input mass of pooled sample for library

construction is ≥500 ng

- Single-tube, addition-only reactions

- Typical library yield ≥40%

4. Analyze

- De-multiplex barcodes within SMRT Link GUI or on the command

line

- Assess community membership and function with third-party 16S

analysis tools like QIIME, DADA2, and MEGAN

3. Sequencing Preparation (CCS Sequencing Mode)

- Anneal Sequencing Primer v4, bind Polymerase, perform

AMPure PB Bead Complex Cleanup

- Generate ≥3 million ≥Q20 16S reads per SMRT Cell 8M

- 1-hour Pre-Extension Time; 10-hour movie collection time

(Sequel II System)

Amplify 16S Gene with Barcoded

Forward and Reverse Primers

QC and Pool Samples

AMPure PB Bead Purification

DNA Damage Repair

End Repair / A-Tailing

Adapter Ligation

Prepare for Sequencing

AMPure PB Bead Purification

(2 Rounds)

1

2

3

4

~4 hours

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 ThermoFisher Scientific Q33226

Qubit™ 1X dsDNA HS Assay Kit ThermoFisher Scientific Q33230

16S PCR Amplification

KAPA HiFi HotStart ReadyMix PCR Kit KAPA Biosystems KK2600 (or KK2601 or KK2602)

Barcoded 16S Primers Any Oligo VendorSee Table 1 in Procedure &

Checklist for 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

GENOMIC 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

BEST PRACTICES RECOMMENDATIONS FOR BACTERIAL GENOMIC

DNA QC

Bacterial Genomic DNA QC Recommendations

Bacterial gDNA from up to 96 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.

BARCODED 16S PCR PRIMER SEQUENCES, ORDERING AND

STORAGE INFORMATION

- Table 1 lists sequences for twenty (20) barcoded,16S gene-specific primers (8 Forward and 12

Reverse) that can be used in all possible asymmetric pairs for multiplexing up to 96 samples.

BARCODED FORWARD PRIMER

>16S_For_bc1005 /5Phos/GCATCCACTCGACTCTCGCGTAGRGTTYGATYMTGGCTCAG

>16S_For_bc1007 /5Phos/GCATCTCTGTATCTCTATGTGAGRGTTYGATYMTGGCTCAG

>16S_For_bc1008 /5Phos/GCATCACAGTCGAGCGCTGCGAGRGTTYGATYMTGGCTCAG

>16S_For_bc1012 /5Phos/GCATCACACTAGATCGCGTGTAGRGTTYGATYMTGGCTCAG

>16S_For_bc1015 /5Phos/GCATCCGCATGACACGTGTGTAGRGTTYGATYMTGGCTCAG

>16S_For_bc1020 /5Phos/GCATCCACGACACGACGATGTAGRGTTYGATYMTGGCTCAG

>16S_For_bc1022 /5Phos/GCATCCACTCACGTGTGATATAGRGTTYGATYMTGGCTCAG

>16S_For_bc1024 /5Phos/GCATCCATGTAGAGCAGAGAGAGRGTTYGATYMTGGCTCAG

BARCODED REVERSE PRIMER

>16S_Rev_bc1033 /5Phos/GCATCAGAGACTGCGACGAGARGYTACCTTGTTACGACTT

>16S_Rev_bc1035 /5Phos/GCATCCAGAGAGTGCGCGCGCRGYTACCTTGTTACGACTT

>16S_Rev_bc1044 /5Phos/GCATCCGCGCGTCGTCTCAGCRGYTACCTTGTTACGACTT

>16S_Rev_bc1045 /5Phos/GCATCAGAGAGTACGATATGTRGYTACCTTGTTACGACTT

>16S_Rev_bc1054 /5Phos/GCATCTCTGTAGTGCGTGCGCRGYTACCTTGTTACGACTT

>16S_Rev_bc1056 /5Phos/GCATCATGTGCGTGTGTGTCTRGYTACCTTGTTACGACTT

>16S_Rev_bc1057 /5Phos/GCATCCTCTCAGACGCTCGTCRGYTACCTTGTTACGACTT

>16S_Rev_bc1059 /5Phos/GCATCTATCTCAGTGCGTGTGRGYTACCTTGTTACGACTT

>16S_Rev_bc1060 /5Phos/GCATCTGTGTCTATACTCATCRGYTACCTTGTTACGACTT

>16S_Rev_bc1062 /5Phos/GCATCTATAGACTATCTGAGARGYTACCTTGTTACGACTT

>16S_Rev_bc1065 /5Phos/GCATCGTATGTGAGAGAGCGCRGYTACCTTGTTACGACTT

>16S_Rev_bc1075 /5Phos/GCATCCACGCGACGCTCTCTARGYTACCTTGTTACGACTT

Each primer oligo contains:

5’ Buffer Sequence

16-base Barcode Sequence

Degenerate 16S gene-

specific forward or reverse

primer sequences

BARCODED 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

BEST PRACTICES RECOMMENDATIONS FOR PREPARATION OF 16S

PRIMER STOCK SOLUTIONS

- Dilute Barcoded, 16S gene-specific forward and reverse PCR primers (see Table 1 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.

BEST PRACTICES RECOMMENDATIONS FOR PREPARATION OF 16S

GENE PCR AMPLIFICATION REACTIONS

Preparation of PCR Master Mix for 16S Gene Amplification Reactions

1. 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.

- 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.

COMPONENT 1 SAMPLE FOR 96-PLEX*

PCR-grade Water 1.5 µl 180.0 µl

KAPA HiFi HotStart ReadyMix (2X) 12.5 µl 1500.0 µl

Total Volume 14.0 µl 1680.0 µl

* Includes 25% overage

Preparation of PCR Master Mix for 16S Gene Amplification Reactions (Cont.)

2. Add Barcoded Forward Primers (2.5 µM) to aliquots of the Master Mix solution and transfer 17-μL

aliquots of the Forward Primer Master Mix across the desired rows of the 96-well plate as described in

the example 96-plex PCR design layout shown on Page 4 of the procedure.

Figure illustration of an example plate layout for setting

up a Master Mix with Barcoded 16S Forward Primers for a

96-plex PCR design.

A

B

C

D

E

F

G

H

1 2 3 4 5 6 7 8 9 10 11 12BARCODED FORWARD PRIMER

>16S_For_bc1005

>16S_For_bc1007

>16S_For_bc1008

>16S_For_bc1012

>16S_For_bc1015

>16S_For_bc1020

>16S_For_bc1022

>16S_For_bc1024

BARCODED REVERSE PRIMER

>16S

_R

ev_bc1033

>16S

_R

ev_bc1035

>16S

_R

ev_bc1044

>16S

_R

ev_bc1045

>16S

_R

ev_bc1054

>16S

_R

ev_bc1056

>16S

_R

ev_bc1057

>16S

_R

ev_bc1059

>16S

_R

ev_bc1060

>16S

_R

ev_bc1062

>16S

_R

ev_bc1065

>16S

_R

ev_bc1075

Preparation of PCR Master Mix for 16S Gene Amplification Reactions (Cont.)

3. Add 3 µl of Barcoded Reverse Primers (2.5 µM) to each well of the appropriate columns of the 96-well

plate as described in the example 96-plex PCR design layout shown on Page 5 of the procedure

Figure illustration of an example plate layout for setting

up a Master Mix with Barcoded 16S Reverse Primers for a

96-plex PCR design.

A

B

C

D

E

F

G

H

1 2 3 4 5 6 7 8 9 10 11 12

BEST PRACTICES RECOMMENDATIONS FOR PREPARATION OF 16S

GENE PCR AMPLIFICATION REACTIONS (CONT.)

16S Gene PCR Sample Plate Preparation and Thermal Cycling Conditions

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 to <= 3 C per second.

** Refer to the Table below to determine the number of cycles of PCR to perform based on input gDNA amount. A

higher number of cycles may be required for samples contaminated with significant amounts of non-bacterial DNA.

INPUT gDNA N CYCLES

25 – 100 pg 27

100 – 500 pg 23

500 – 2500 pg 20

EXAMPLE AGAROSE GEL DNA QC FOR 16S PCR AMPLICON

PRODUCTS

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.

- 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.

- 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.

BEST 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.

SMRTBELL EXPRESS TEMPLATE PREP KIT 2.0 REAGENT

HANDLING RECOMMENDATIONS

- Several tubes in the kits are sensitive to temperature and vortexing

- PacBio highly recommends:

- Never leaving tubes at room temperature

- Working on ice at all times when preparing master mixes

- Finger tapping followed by a quick-spin prior to use

EXAMPLE AGAROSE GEL DNA QC FOR A 96-PLEX POOLED 16S

AMPLICON SAMPLE BEFORE AND AFTER SMRTBELL LIBRARY

CONSTRUCTION

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).

- Verify the size of the final purified SMRTbell library on an agarose gel. A typical result is

shown in the example Figure below.

Full-Length 16S 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

SAMPLE SETUP RECOMMENDATIONS FOR 16S LIBRARIES –

SEQUEL II SYSTEM (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 System

IMPORTING THE BARCODE FASTA FILE INTO SMRT LINK FOR

AUTOMATED DEMULTIPLEXING OF 16S SMRTBELL LIBRARY

SAMPLES

- Note: SMRT Link v8.0 (and higher) software installations by default come pre-bundled with

a FASTA file containing a list of 96 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 v8.0):

1. Download the FASTA file containing the relevant barcode sequences, for example:

https://www.pacb.com/wp-content/uploads/Sequel_RSII_96_barcodes_v1.fasta_.zip (contains a list of

96 PacBio barcodes for use with Barcoded Primers)

EXAMPLE FASTA FILE CONTAINING A LIST

OF PACBIO 16-BASE PAIR BARCODES

IMPORTING THE BARCODE FASTA FILE INTO SMRT LINK FOR

AUTOMATED DEMULTIPLEXING OF 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.

iii. 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.)

iv. Select the data type to import: Barcodes – FASTA (.fa or .fasta), XML (.barcodeset.xml), or ZIP files containing

barcodes.

v. 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.

SMRT LINK RUN DESIGN SETUP PROCEDURE FOR AUTOMATED

DEMULTIPLEXING OF 16S SMRTBELL LIBRARY SAMPLES

1. Sample is Barcoded: Yes

2. Barcode Set: <Select Barcode File Name> (e.g., Sequel_96_barcodes_v1)

3. Same Barcodes on Both Ends of Sequence: No

4. Autofilled Barcode Name File: Click Download

Name File

5. Barcoded Sample Name File: In the downloaded

Autofilled Barcode Name File, fill in the desired

values for Bio Sample Name, as shown here for

the first 4 barcodes. Save the file and upload it

using Browse and Upload File

6. Save the Run Design and launch the

sequencing run.

- Refer to “Run Design” section in the SMRT

Link User Guide v8.0 for further details

- Open the Run Design module in SMRT Link. Click New Run Design. Fill in the Sample

Information section, then click the small arrow to open Barcoded Sample Options. Specify

the following options:

- Barcoded Sample Name File notes:

- Delete entire rows of barcodes not used

- Allowed characters: Alphanumeric; space; dot;

underscore; hyphen. Other characters will be

automatically removed.

Barcode Bio Sample Name

bc1005--bc1033 bc1005--bc1033_Sample_01

bc1005--bc1035 bc1005--bc1035_Sample_02

bc1005--bc1044 bc1005--bc1044_Sample_03

bc1023--bc1045 bc1005--bc1045_Sample_04

16S Data Analysis Recommendations

16S DATA ANALYSIS RECOMMENDATIONS

- PacBio highly recommends upgrading to SMRT Link v8.0 or higher to perform de-

multiplexing of your 16S amplicon sequencing data

- SMRT Link GUI Demultiplex Barcodes analysis application supports up to 384 barcodes per sample;

command-line supports >384 barcodes

- 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

- Utilize SMRT Link to generate highly accurate (≥Q20) single-molecule reads (HiFi reads)

using the Circular Consensus Sequencing (CCS) analysis application

- 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

- 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)

16S Library Example Performance Data

EXAMPLE SEQUENCING PERFORMANCE* RESULTS FOR 96-PLEX

16S LIBRARY SAMPLE (SEQUEL II SYSTEM)

Full-Length

16S Sample

Library

Total

Bases

Productivity

(P1)

Mean

Polymerase

Read Length

N50

Polymerase

Read Length

MSA-1003 188 Gb 80% 29.1 kb 54.7 kb

* Read lengths, reads/data per SMRT Cell and other sequencing performance results vary based on sample quality/type and insert size.

Data shown were generated using a Sequel II System v8.0 / Sequel II Chemistry

2.0 / Sequel II Binding Kit 2.1

MSA-1003 Mock Community Sample Description

- 20 Strain Staggered Mix Genomic Material (ATCC® MSA-1003™) https://www.atcc.org/products/all/MSA-1003.aspx

- MSA-1003 sample is a mock microbial community that mimics mixed metagenomic samples

- MSA-1003 sample comprises genomic DNA prepared from fully sequenced, characterized, and authenticated ATCC

Genuine Cultures® that were selected by ATCC based on relevant phenotypic and genotypic attributes, such as

Gram stain, GC content, genome size, and spore formation

- Replicate MSA-1003 samples were processed in parallel during library construction to generate a 96-plex

(asymmetrically barcoded) pooled 16S SMRTbell library

1.5 kb

Primary Sequencing Performance Results

- Insert Read Length Density plot shows that most insert read lengths are

~1.5 kb, consistent with the expected size range of 16S amplicon products

EXAMPLE BARCODE DEMULTIPLEXING RESULTS FOR 96-PLEX

16S LIBRARY SAMPLE (SEQUEL II SYSTEM)

Data shown were generated using a Sequel II System v8.0 / Sequel II Chemistry 2.0 / Sequel II Binding Kit 2.1

Yielded an average of 38K HiFi (≥Q20) reads per 16S sample from a total of 3.6M

≥Q20 barcoded reads generated on a single Sequel II SMRT Cell 8M

16S Sample >Q20 BC reads>Q20 Read

Quality

BC Samples /

SMRT CellAvg reads / BC

MSA-1003 3,851,493 Q40 96 38,349

0

5,000

10,000

15,000

20,000

25,000

30,000

35,000

40,000

45,000

50,000

Reads per Barcode for 96 Pooled MSA-1003 Replicate 16S Samples

Full-length 16S data were collected for MSA-1003 mock community replicate samples on a single SMRT Cell 8M at 96-plex. Amplification was

performed with a barcoded 16S primer / 1-step PCR approach.

With the single-step 16S PCR protocol, the

yield of >99% accurate reads is

highly consistent across all barcodes

EXAMPLE TAXONOMIC CLASSIFICATION RESULTS FOR 96-PLEX

16S LIBRARY SAMPLE (SEQUEL II SYSTEM)

Full-length (V1-V9) 16S amplicon samples were pooled at 96-Plex and sequenced on a single SMRT Cell 8M (Sequel II System Chemistry 2.0).

PacBio results shown in bar graph reflect the average abundance values derived from the pooled MSA-1003 replicate samples.

Download and explore this Sequel II

System 16S HiFi dataset further

PacBio 16S Sequencing Faithfully Represents a Known Mock Community Sample

MSA-1003 SAMPLE DESCRIPTION

- 20 Strain Staggered Mix Genomic Material (ATCC®

MSA-1003™) https://www.atcc.org/products/all/MSA-

1003.aspx

16S ANALYSIS OF THE MSA-1003 MOCK COMMUNITY

Yield of >99% accurate 16S reads

matches the expected composition of

the MSA-1003 mock community sample

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

insert size. See Application Brief: Metagenomic sequencing with HiFi reads – Best Practices

BEST PRACTICES: METAGENOMIC SEQUENCING WITH HIFI

READS (SEQUEL II CHEMISTRY 2.0) (CONT.)

Application Brief: Metagenomic sequencing with HiFi reads – Best Practices

TECHNICAL DOCUMENTATION AND APPLICATIONS SUPPORT

RESOURCES FOR 16S LIBRARY PREPARATION, SEQUENCING &

DATA ANALYSIS

- 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

System (PN 101-769-100)

- Overview – Sequel Systems Application Options and Sequencing Recommendations (PN

101-851-300)

- Application Brief: Metagenomic sequencing with HiFi reads – Best Practices (PN BP108-

030220)

- SMRT Link Software download website: https://www.pacb.com/support/software-downloads/

- AGBT 2020 Poster: Unbiased characterization of metagenome composition and function

using HiFi sequencing on the PacBio Sequel II System

-Webinar: A HiFi View: Sequencing the Gut Microbiome with Highly Accurate Long Reads

(March 26, 2020)

- PacBio Applications Webpage: Characterize Microbial Communities

For Research Use Only. Not for use in diagnostic procedures. © Copyright 2020 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 this

method may require rights to third-party owned intellectual property. BluePippin and SageELF are trademarks of Sage Science. NGS-go and NGSengine are trademarks of GenDx. FEMTO Pulse and

Fragment Analyzer are trademarks of Agilent Technologies Inc.

All other trademarks are the sole property of their respective owners.

www.pacb.com