SHORELINE BIOME PacBio Complete StrainID Kit

Introduction

The Shoreline Complete™ kits are a complete solution for bacterial 16S microbiome profiling studies. Each kit contains reagents for comprehensive bacterial lysis, extraction and purification of DNA, individual sample barcoding for up to 96 samples, and amplification of either the complete 16S gene or the novel StrainID™ for microbiome analysis. DNA can be extracted from a wide variety of samples including fecal, swab, tissue, and saliva. DNA is amplified using dual-unique barcoded primers targeting the complete 16S rRNA gene (V1-V9) or StrainID™ (16S-ITS-23S). The single step PCR, made up of primers containing dual-unique barcodes and a diverse set of target-specific primers covering known variations in the primer sites, generates amplicons ready for SMRTbell® template prep and subsequent sequencing on the PacBio® Sequel System. The 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 Complete™ kits sequenced on the PacBio® platform.

Workflow:

Kit Components

Kit Contents for Shoreline Complete™ Lyse and Purify

1. Lysis-1, dried solution in plate/tubes
2. Purification Buffer

Kit Contents for Shoreline Complete™ Amplify

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

Important Parameters

Storage buffer

Most sample storage buffers are compatible with the lysis, however for buffers with pH < 5 we recommend adding 10 µL sample, to prevent neutralization of the Shoreline Biome Complete lysis buffer.

Sample Types and Input Quantities

Shoreline Complete™ kits have been validated with the sample types specified in Table 1, below.

Protocol for frozen/liquid/mouse fecal, OMNIgene® Gut

Liquid fecal stored in other buffers
DNA Genotek OMNIgene®•GUT
Tissue Samples
Mouse fecal pellet
Swabs|

Protocol for skin, swabs, cell pellets, saliva

Saliva
Pelleted cells

Table 1. Validated Sample Types
Table 2 (below) provides the recommended input quantities for each of the sample types. Please note that adding less than the recommended amount may not yield sufficient amounts of DNA, while using more than the maximum quantity may cause insufficient lysis.

Table 2. Sample Type Input Quantities

DNA Quantitation

The Shoreline Complete PCR has been optimized to handle a broad range of input DNA, therefore, it is not necessary to quantify DNA prior to amplification. However, if quantitation is desired, keep in mind that the Shoreline Biome lysis protocol results in single stranded DNA. We recommend the Qubit™ ssDNA Quantification kit. DNA quantification by NanoDrop™ is not recommended.

Target Reads per Sample

The required reads per sample are dependent on goals of each sequencing project. General guidelines recommend minimum of ~5000 reads per sample. We suggest running ~96 samples on one SMRT cell of Sequel system. This gives sufficient reads/sample to identify bacterial strains present at 1% with reasonable confidence on the 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 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 addition of SMRTbell® barcodes. Contact customer service for information on this application.

Amplicon Normalization

Using an equi-volume pooling approach for unpurified PCR products is the quickest and most convenient way to normalize amplicon libraries. Equi-volume pooling is rapid and generally provides good representation of samples when used in combination with visualization on a gel, Bioanalyzer, or equivalent. Other normalization methods are acceptable including equimolar pooling based on fluorometry or qPCR, however, these methods may be more expensive or labor- intensive. Equimolar pooling may be beneficial if processing multiple sample types and/or samples that are expected to have widely varying microbial loads1.

Microbiome Controls

Several controls are recommended to monitor the workflow including lysis and amplification efficiency, as well as environmental contamination. Recommended controls and control materials are provided in Table 3.

Table 3. Recommended controls and control materials

Safe stopping points

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

Sample Storage

Genomic DNA can be stored at +4°C for up to 10 days, or frozen at -20°C for 3 months. Avoid repeated freeze/thaws. Amplicons and library 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 for Lyse and Purify

·      96-well plates/0.2ml tubes

·      Plate sealing film

·      Sterile Laboratory Grade Water

·      70% ethanol

·      TE Buffer – 10mM Tris, 1mM EDTA, pH 8.0

·      0.4M KOH Solution

·      Magnetic Rack ( for plates : Invitrogen Dyna Mag 96 side magnetic rack, #12331D or Magnetic stand-96 #AM10027;

for tubes : Permagen: 0.2 mL PCR Strip Magnetic Separation Rack, Cat

MSR812)

Additional Items Required for Amplify:
·      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

·      Sterile inoculating loops

Suggested items for sample QC
·      0.8% agarose gel with ethidium bromide or other dsDNA stain

·      Gel loading dye

·      2kb DNA ladder

·      Bioanalyzer reagents or equivalent

Protocols

Shoreline Complete™ Lyse Protocol

1. Use the following calculation to make 0.4 M potassium hydroxide (KOH) solution. Make solution fresh daily.
   × 44.56 = 0.4

   • ℎ: = ,
     = ,

2. Make 70% Ethanol. Add 17.5 mL 100% ethanol to 7.5 mL sterile laboratory grade water

3. Bring Purification Buffer (white capped bottle) to room temperature.

4. Remove film from the 96-well plate/uncap the tubes containing dried Lysis-1.

5. Follow the instructions for the specific Sample Type in Table 4, below:
   CAUTION: Avoid over-mixing to prevent foaming of the Lysis-1 reagent.

Frozen Solid Fecal Samples

| Add 50 µL Molecular Biology Grade water to each Lysis-1 well/tube. Using sterile inoculating loop, transfer ~3 mg of each fecal sample ( Figure 1 ) to the appropriate well/tube. Spin each loop to disperse the sample, and discard loop.

Figure 1.

Mouse Fecal Pellet

| Add 50 µL Molecular Biology Grade water to each Lysis-1 well/tube. Place up to 5 mg (1/4 mouse pellet) in each.
DNA Genotek

OMNIgene®•GUT Sample

Collection Tube

| Add 40 µL Molecular Biology Grade water to each Lysis-1 well/tube.

Add 10 µL of sample from the OMNIgene®•GUT Sample Collection Tube to Lysis-1.

Liquid Fecal Samples stored in other buffers| Add 40 µL Molecular Biology Grade water to each Lysis-1 well/tube. Add 10 µL of sample.

Tissue Samples

| Add 50 µL Molecular Biology Grade water to each Lysis-1 well/tube. Place 20mg tissue sample in each.

Saliva

| Centrifuge saliva samples at 5500 RCF for 10 minutes to pellet cells. Remove and discard supernatant.

Re-suspend in 50 µL of Molecular Biology Grade water, TE, or PBS.

Transfer 50 µL of cell suspension to Lysis-1 well/tube.

Skin Swab

| Moisten swab with buffer containing Tris-EDTA and 0.5% Tween 20.

Collect sample from skin. Place swab in 1.7 mL microcentrifuge tube. Break the swab handle off at designated location.

Centrifuge tube with swab for 5 minutes at 10000 RCF. Remove swab and transfer supernatant to a clean 1.7 mL tube.

Centrifuge tube with swab second time for 5 minutes at 10000 RCF.

Remove swab and combine supernatant with the first supernatant. Discard spent tube and swab.

Transfer 50 µL of the supernatant to Lysis-1 well/tube.

Cell Pellet

| Add 50 µL water, TE, or PBS to cells. Pipette to mix.

Transfer 50 µL of cell suspension to Lysis-1 well/tube.

Table 4. Sample type-specific instructions for lysis

• Add 50 µL 0.4M KOH to each well/tube. Mix briefly by pipetting up and down. Do not over-mix to avoid foaming. A precipitate will form.
• Cover the sample plate with plate film (or close the tubes tightly), load the plate/tubes into the thermocycler, close and lock the lid, and heat samples to 95°C for 5 minutes. Precipitate will dissolve.

Shoreline Complete™ Purify Protocols

Follow the purification protocol from Table 5 below based on the sample type being used.

Protocol for frozen/liquid/mouse fecal, OMNIgene® Gut| Protocol for skin, swabs, cell pellets, saliva
---|---
·      Remove the plate/tubes from the thermocycler

·      Place plate/tubes on ice for 2 minutes. (Precipitate will re-form)

·      Vortex Purification Buffer (white cap) to re-suspend any brown magnetic beads that may have settled.

·      Add 50 µL of Purification Buffer to new

plate/tube for sample purification

·      After incubating sample plate/tube on ice, centrifuge to pellet precipitate to bottom of wells/tubes. For plates , centrifuge at 2000 RCF for 3 minutes (or 400 RCF for 7 minutes) (see Figure 2 below) For tubes , centrifuge 1 minute in mini-centrifuge. (see Figure 3 below)

·      Carefully transfer 50 µL of supernatant into the plate/tube with Purification Buffer prepared as per the instructions above; and pipette to mix.

·      Cover the sample plate with a new

plate film (or close the tubes tightly).

| ·      Remove the plate/tubes from the thermocycler

·      Vortex Purification Buffer (white cap) to re-suspend any brown magnetic beads that may have settled.

·      Add 100 µL of Purification Buffer directly to each well/tube of the sample and pipette to mix.

·      Cover the sample plate with a _new _plate film (or close the tubes tightly).

Table 5. Purification protocols based on sample type

1. Load sample plate/tubes onto the thermocycler, close and lock the lid, and incubate solution for 5 minutes at 50˚C to allow DNA to bind to beads
2. After incubation, place on magnetic rack to pellet beads with bound DNA (~60 seconds).
3. Once all beads are pelleted, remove all supernatant SLOWLY and discard. Avoid aspirating beads.
4. Leave the plate/tubes on the magnet and add 200 µL 70% ethanol to each well/tube. After 30 seconds, remove the supernatant.
5. Repeat the wash from step 4 above.
6. Remove the ethanol completely and let air dry for 2 – 3 minutes. Over-drying will make eluting DNA more difficult, so watch to ensure ethanol is mostly gone but bead pellet does not begin to shrink and crack.
7. Remove plate/tubes from magnet.
8. Add 20 µL of 1X TE buffer to each and briefly pipette up and down to re-suspend beads.
9. Incubate at room temperature for 2 minutes.
10.  Place plate/tubes on magnetic rack for 30 – 60 seconds to pellet magnetic beads. DNA is now in solution.
11. Transfer supernatant containing eluted DNA into a clean plate/tube on ice.
12. Remove plate/tubes with pelleted beads from rack.
13. Add another 20 µL 1X TE buffer onto bead pellet, gently pipette up and down to re-suspend beads.
14. Incubate for 2 minutes at room temperature.
15. Return plate/tubes to magnetic rack. Allow beads to pellet to sides for 30 – 60 seconds.
16. Remove supernatant containing DNA and combine with first eluate, avoiding pelleted beads.
17.  Dilution:
18. For all fecal samples: Dilute 1:5 by adding 160 µl 1X TE buffer to 40 µl of combined eluted DNA for a total of 200 µl.
19. For all other samples: Do not dilute.
20. Proceed to Amplify step or store eluted DNA at +4°C.

Genomic DNA can be stored at +4°C for up to 10 days, or frozen at -20°C for 3 months

Shoreline Complete™ Amplify Protocol

1. Remove plate seal/uncap tubes containing the Amplicon Primers.
2. Add 10 µL 2X PCR Premix (blue cap) to each well/tube.
3. Add 10 µL Shoreline Biome DNA to each well/tube.
4. 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.
5. 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.
6. Spin briefly if reaction mix is not completely at the bottom of the wells/tubes.
7. Transfer the plate/tubes to thermocycler; close and lock the lid.
8. Run the appropriate PCR protocol for the Shoreline Complete™ kit from Table 6 or Table 7 below.

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

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

Amplicon QC

Check individual reactions on gel: Run 1.5 µL of 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

1. Follow MinElute instructions for adding Ethanol (96% – 100%) to PE buffer, and label tubes.

2. Pool 5 µL of each amplified sample into a clean 1.5 mL microcentrifuge tube. (If the amplicon QC gel shows variation in 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.

3. Combine 80 µL of pooled sample with 400 µL of Qiagen PB buffer.

4. Transfer all of the resulting solution into the MinElute column with provided 2 mL collection tube.

5. Centrifuge the MinElute column at 17,900 RCF for 1 minute or until solution has passed through the column. Discard the flow-through in the collection tube and return the column into the empty tube.

6.  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.

7. Place MinElute column in a clean 1.5 mL microcentrifuge tube.

8. Add 50 µL EB Buffer (10mM Tris-Cl, pH 8.5) or water directly onto 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 one (1) week or -20°C for up to one (1) month

9. Quantify the pooled amplicons by a fluorometric method (e.g. Qubit). Run on Agilent Bioanalyzer or equivalent to determine the size distribution.

10. 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 trace for StrainID™ (Figure 4). 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 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 performed by the SBanalyzer™ software. For details on sequencing with the Sequel or Sequel II system refer to “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 kb.4,5

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.

References

1.  https://www.pacb.com/wp-content/uploads/Procedure-Checklist-%E2%80%93-Amplification-of-Full-Length-16S-Gene-with-Barcoded-Primers-for-Multiplexed-SMRTbell-Library-Preparation-and-Sequencing.pdf
2. https://www.pacb.com/wp-content/uploads/Procedure-Checklist-%E2%80%93-Amplification-of-Full-Length-16S-Gene-with-Barcoded-Primers-for-Multiplexed-SMRTbell-Library-Preparation-and-Sequencing.pdf
3. https://www.pacb.com/wp-content/uploads/SMRT_Link_User_Guide_v700.pdf
4.  https://www.pacb.com/wp-content/uploads/Quick-Reference-Card-Loading-and-Pre-Extension-Recommendations-for-the-Sequel-System.pdf
5.  https://www.pacb.com/wp-content/uploads/Quick-Reference-Card-Loading-and-Pre-Extension-Recommendations-for-the-Sequel-II-System.pdf

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