WAVEV19 Shoreline Wave V1-V9 Kit Instruction Manual

: June 6, 2024
: SHORELINE

Table of Contents

WAVEV19 Shoreline Wave V1-V9 Kit
Introduction to Shoreline Wave™ Kits
Kit Components
Important Parameters
Protocols
Amplicon QC
Post Amplification Clean-up
Pooled Amplicon QC
SMRTbell® Library Prep
Sequencing
Analysis
References
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WAVEV19 Shoreline Wave V1-V9 Kit

Introduction to Shoreline Wave™ Kits

The Shoreline Wave™ kits are an easy-to-use kit for 16S microbiome studies providing researchers a solution for long amplicon generation. Each kit contains reagents for amplification of either the complete 16S gene or the novel StrainID™ for microbiome analysis. DNA is amplified using dual-unique barcoded primers targeting the complete 16S gene (V1-V9) or StrainID™ (16S- ITS-23S). The single-step PCR, made up of primers that contain the barcode and target specific primer, generates amplicons ready for SMRTbell® template prep and subsequent sequencing on the PacBio® Sequel System. PacBio® long-read sequencing platform is powered by Single-Molecule, Real-Time (SMRT) Sequencing technology. The Shoreline Biome kits take advantage of the highly accurate Circular Consensus (ccs) reads produced on the Sequel System. After sequencing, investigators can use the point-and-click Shoreline Biome SBanalyzer software to sort (demultiplex) the ccs reads by sample, and map them to the Athena™ database, enabling high-resolution taxonomic classification. This document provides guidance for using the Shoreline Biome Wave™ kits sequenced on the PacBio® platform.
Workflow:

Kit Components

Kit Contents for Shoreline Wave™

Barcoded primers, dried in plate/tubes
2X PCR Premix
PCR sealing film (for plates)

Important Parameters

DNA Input Recommended genomic DNA input for the Shoreline Wave™ kit is 10ng, however successful amplification can be achieved from lower quantities. gDNA should be stored in TE buffer or equivalent. It is important to note that quality and quantity of genomic DNA is dependent on the extraction protocol used.

Target Reads per Sample The required reads per sample are dependent on the goals of each sequencing project. General guidelines recommend a minimum of ~5000 reads per sample. We suggest running ~96 samples on one SMRT cell of the Sequel I system. This gives sufficient reads/sample to identify bacterial strains present at 1% with reasonable confidence on Sequel I. In general, more reads will yield greater depth per sample, enabling identification of bacteria present at lower levels in the sample. For highly diverse samples where representation below 1% is needed, more than one Sequel I SMRT cell or a single Sequel II SMRTcell may be advisable. For projects with more than 112 samples, multiplexing can be achieved by the addition of SMRTbell® barcodes.

Multiplexing Barcodes The Shoreline Biome kits contain primers with dual- unique indexing. For V1-V9 and StrainID™ kits, two kits are available: 96 barcodes in plate format and 16 barcodes in tube format. These barcodes can be combined for a total of 112 barcodes. Further multiplexing can be achieved by the addition of SMRTbell® barcodes. Contact customer service for information on this application.

Amplicon Normalization Using an equi-volume pooling approach is the quickest and most convenient way to normalize amplicon libraries. It allows for library QC with little upfront equipment needed, while maintaining equal representation of each sample in sequencing. Other normalizations are acceptable including equimolar pooling based on fluorometry or qPCR. Equimolar pooling may be beneficial if processing multiple sample types and/or samples that are expected to have widely varying microbial loads1

Microbiome Controls

Control Type	Recommended Control Material
PCR Positive	Mock community DNA (commercially available)
PCR Negative	Water

Controls are recommended to monitor amplification efficiency and environmental contamination. Recommended controls and control materials are provided in Table 1.

Safe stopping points Safe stopping points, where the process can be paused, are marked with this symbol. Follow the storage recommendations.

Sample Storage Amplicons and libraries can be stored at 4°C for up to 1 week or -20°C for up to 1 month.

Equipment and reagents supplied by user

Additional Items Required

· Qiagen MinElute® PCR Purification Kit (Cat. No. 28004)

· Sterile Laboratory Grade Water

· Sterile 100% Ethanol

· PACBIO® SMRTbell® Express Template Prep Kit 2.0 (Part No. 100-938-900)

Suggested items for rapid processing
· Repeater Pipette with multi-volume tips

· Single Channel Pipettes

· Multichannel Pipette

Suggested items for sample QC

· 0.8% agarose gel with ethidium bromide or another dsDNA stain

· Gel loading dye

· 2kb DNA ladder

· Bioanalyzer reagents or equivalent

Protocols

Shoreline Wave™ Amplification Protocol

Remove plate seal/uncap tubes containing the Amplicon Primers.
Add 10 µL 2X PCR Premix (blue cap) to each well/tube.
Add 10ng purified genomic DNA to each well/tube. Adjust the remaining volume with water so that the PCR reaction final volume is 20 μL.
For plates, use the PCR sealing film provided with the kit to cover the plate and seal tightly with no gaps. For tubes, close tightly.
Primers will dissolve off the bottom. Observe that blue color is uniformly distributed throughout 20 µL reaction, if not, tap plate/tube gently until uniform blue color is achieved.
Spin briefly if the reaction mix is not completely at the bottom of the wells/tubes.
Transfer the plate/tubes to thermocycler; close and lock the lid.
Run the appropriate PCR protocol for the Shoreline Wave™ kit from Table 2 or Table 3 below.
V1-V9| | StrainID™
---|---|---
Heated lid @ 100°C| Heated lid @ 100°C
Temperature| Time| Ramp Speed| Cycles| Temperature| Time| Ramp Speed| Cycles
95°C| 3 min| | 1 cycle| 95°C| 3 min| | 1 cycle
95°C| 30 sec|

4°C/sec

|

34

cycles

| 95°C| 30 sec|

4°C/sec

|

34

cycles

63°C| 45 sec| 59°C| 45 sec
72°C| 90 sec| 72°C| 120 sec
72°C| 3 min| | 1 cycle| 72°C| 3 min| | 1 cycle
4°C| hold| | hold| 4°C| hold| | hold

Table 2. PCR Protocol for V1-V9 Kits

Table 3. PCR Protocol for StrainID™ Kits

Amplicons can be stored at 4°C for up to 1 week or -20°C for up to 1 month

Amplicon QC

Check individual reactions on gel: Run 1.5 µL of the sample with 5 µL diluted gel loading dye on 0.8% agarose gel in TBE, 150V for approximately 45 minutes, with DNA ladder. Band should be present at ~1500bp for V1-V9 and ~2500 StrainID™. Agilent Bioanalyzer or equivalent can also be used.

Post Amplification Clean-up

Qiagen MinElute® PCR Purification Protocol

Follow MinElute instructions for adding Ethanol (96% – 100%) to PE buffer, and label tubes.
Pool 5 µL of each amplified sample into a 1.5 mL microcentrifuge tube. (If the amplicon QC gel shows variation in the amount of PCR product, volume adjustments can be made here, during pooling.) Save 2 µL of pooled unpurified DNA for gel comparison with purified DNA in the last step below.
Combine 80 µL of the pooled sample with 400 µL of Qiagen PB buffer.
Transfer all of the resulting solutions into the MinElute column with provided 2 mL collection tube.
Centrifuge the MinElute column at 17,900 RCF for 1 minute or until the solution has passed through the column. Discard the flow-through in the collection tube and return the column into the empty tube.
Add 750 µL Buffer PE to the MinElute column and centrifuge at 17,900 RCF for 1 minute; discard flow-through and return MinElute column to the collection tube. Centrifuge for 1 minute at 17,900 RCF to remove residual Ethanol.
Place MinElute column in a clean 1.5 mL microcentrifuge tube.
Add 50 µL EB Buffer (10mM Tris-Cl, pH 8.5) or water directly onto the center of the MinElute membrane. Incubate column for 1 minute at RT, and then centrifuge column at 17,900 RCF for 1 minute. Pooled undiluted samples can be stored at 4°C for up to 1 week or -20°C for up to 1 month
Quantify the pooled amplicons by a fluorometric method (e.g. Qubit).
Proceed to SMRTbell® library prep for sequencing on the PacBio® Sequel system.

Pooled Amplicon QC

QC the amplicon pool by running on Agilent Bioanalyzer or equivalent to determine the size distribution. Below is an example of the library traces for StrainID™ (Figure 1). Note that because of the variability in the internally transcribed spacer region of the StrainID™, amplicons can differ in size between and within organisms. This results in a smear at ~2500bp rather than a distinct band. Depending on composition of the bacterial population, StrainID™ traces can result in single or multiple bands.

SMRTbell® Library Prep

Use the Express Template Prep Kit 2.0 for sequencing Shoreline Biome V1-V9 or StrainID™ on the Sequel and Sequel II Systems. Users should refer to the “Procedure & Checklist – Amplification of Full-Length 16S Gene with Barcoded Primers for Multiplexed SMRTbell® Library Preparation and Sequencing” document2. Please note that you will be beginning the procedure with an amplicon pool, therefore, start the protocol at the
“AMPure® PB Bead Purification” step using the pooled Shoreline Biome PCR products, then proceed “SMRTbell® Library Construction” step. Note that additional materials are required for SMRTbell® library prep (SMRTbell® Express Template Prep 2.0 PacBio® (PacBio® cat#100-938-900) and AMPure® PB beads PacBio® (PacBio® cat#100-265-900). Alternatively, PacBio® service providers can provide the library prep service.
https://www.pacb.com/products-and-services/service-providers

Sequencing

Sequencing of the Shoreline Biome Complete V1-V9 and StrainID™ kits, can be performed on either PacBio® Sequel or Sequel II system (see Multiplexing Barcodes.) For the preparation of SMRTbell® templates for sequencing, refer to SMRT Link Sample Setup3. In the SMRT Link Software, select CCS Sequencing Mode using default parameters (minPasses=3 and minPredictedAccuracy=0.99). Note that barcode sequences are not required since demultiplexing will be performed by the SBanalyzer™ software. For details on sequencing with the Sequel or Sequel II system refer to the “Quick Reference Card – Diffusion Loading and Pre-Extension Time Recommendations for the Sequel or Sequel II System” document and follow the recommendations for amplicons < 3 kb4,5.

Analysis

Post-sequencing demultiplexing and analysis is performed with the Shoreline Biome SBanalyzer™ software. Refer to Shoreline Biome SBanalyzer™ Software Installation and Quick Start Guide for instructions. Note that CCS reads from the Sequel instrument are required data input for the software.