Procedure & Checklist – Multiplexing 2.5 kb Amplicons for ... · 4. For higher multiplexing (e.g. ≥96-plex), purifying and quantifying each individual PCR product may be difficult

Page 1 Part Number 101-921-300 Version DRAFT (June 2020)

Procedure & Checklist – Multiplexing 2.5 kb Amplicons for Whole Genome Sequencing of SARS-CoV-2

Before You Begin This document describes a workflow for sequencing SARS-CoV-2 using fourteen overlapping 2.5 kb amplicons (designed by John-Eden Sebastian of University Sydney, Australia) tiled across the 29 kb genome.

Each patient sample requires two multiplex PCR reactions (each generating 7 non-overlapping 2.5 kb amplicon products) using M13-tailed target-specific primers. The two multiplex PCR reactions are pooled for asymmetric barcoding by amplification (using the barcoded M13 primers). Once barcoded, the pooled PCR products tiling the entire 29 kb SARS-CoV-2 genome are constructed to a single SMRTbell® library using the SMRTbell Express Template Prep Kit 2.0 and subsequently sequenced on the Sequel® and Sequel II Systems. Alternatively, different barcoded samples can be pooled for library construction.

For any questions or additional information about this procedure, please contact [email protected].

With asymmetric barcoding, different barcodes are used on the forward (F) and reverse (R) PCR primers. Each barcode on the M13 forward PCR primer may be used with any or all barcodes on the M13 reverse PCR primer, and vice versa. Recommendations for barcode sequences are found in the “Second Round PCR: Recommendations for Asymmetric Barcoding” section.

The general workflow described in this procedure is summarized below:

1st PCR: Two Multiplex PCR Reactions using Target-Specific Primers Tailed with M13 Sequence (F or R)

2nd PCR – Barcoded M13 Primers (F/R)

Pool M13-tailed Amplicons

SMRTbell Library Construction

Sequencing

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Required Materials

Item Where Used Vendor Part Number Target-Specific F/R Primers tailed with M13 Sequences (Customer-supplied) PCR Amplification (1st-Round) Oligo Synthesis

Company N/A

F/R PacBio-Barcoded M13 Primers (Customer-supplied)* PCR Amplification (2nd-Round) Oligo Synthesis

Company N/A

Platinum™ SuperFi™ Green PCR Master Mix PCR Amplification Thermo Fisher Scientific

12359010

SMRTbell® Express Template Prep Kit 2.0 Library Prep PacBio 100-938-900

AMPure® PB Beads Library Purification PacBio 100-265-900

Sequel® System or Sequel II System Binding and Internal Control Kit

(Recommended kits are listed below)

Sequel® II Bind Kit 2.1 and Int Ctrl 1.0 Sequencing short (<3 kb) amplicon samples on the Sequel II System PacBio 101-843-000

Sequel® Binding and Internal Ctrl Kit 3.0 Sequencing amplicon samples on the Sequel System PacBio 101-626-600

Sequel® System or Sequel II System Sequencing Kit (Recommended kits are listed below)

Sequel® II Sequencing Kit 2.0 Supports 4 sequencing reactions on the Sequel II System PacBio 101-820-200

Sequel® Sequencing Kit 3.0 (8-rxn) Supports 8 sequencing reactions on the Sequel System PacBio 101-597-800

Sequel® Sequencing Kit 3.0 (4-rxn) Supports 4 sequencing reactions on the Sequel System PacBio 101-597-900

Sequel® System or Sequel II System SMRT® Cells SMRT® Cell 8M Tray Sequencing on the Sequel II System PacBio 101-389-001

SMRT® Cell 1M v3 Tray Sequencing on the Sequel System (Max. 10-hour movie collection time) PacBio 101-531-000

SMRT® Cell 1M v3 LR Tray Sequencing on the Sequel System (Max. 20-hour movie collection time) PacBio 101-531-001

8- or 12-Multichannel Pipettor High Throughput Pipetting Any Vendor-specific Qubit™ 4 Fluorometer DNA concentration measurement ThermoFisher Q33238 Qubit™ 1x dsDNA HS Assay Kit DNA concentration measurement ThermoFisher Q33230

Table 1. List of Required Materials and Equipment.

* For multiplexing, a list of 32 x Forward and 32 x Reverse Barcoded M13 primers are available here.

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https://www.pacb.com/wp-content/uploads/Eden-SARS-CoV-2-Primers-with-PacBio-Barcoding-Options.xlsx


Designing Target Specific Primers Tailed with M13 Sequences for 1st-Round PCR The 14 pairs of primers tailed with M13 sequences are available here. Primers can be ordered from any oligo synthesis provider, following the guidelines below.

1. IMPORTANT: Add a 5’ block (e.g., 5AmMC6) to ensure that carryover amplicons from first-round PCR are not ligated to the SMRTbell adapters in SMRTbell library construction.

2. Desalted Primers are sufficient for amplification. 3. Use the following primer format:

Primer Type

M13 Sequences

Template- Specific Sequence

Primer to Order

Forward_Primer /5AmMC6/gtaaaacgacggccagt FOREXAMPLE1 /5AmMC6/gtaaaacgacggccagtFOREXAMPLE1

Reverse_Primer /5AmMC6/caggaaacagctatgac REVXAMPLE2 /5AmMC6/caggaaacagctatgacREVXAMPLE2

Table 2. Recommended Primer Format for Ordering Oligos

Best Practices for Generating High-Quality PCR Products Clean, target-specific PCR products are extremely important for obtaining high-quality sequence data. Non-specific products can represent a substantial percentage of the sequencing reads if they are not removed. To minimize the presence of non-specific products, consider the following recommendations for generating high-quality amplicons suitable for SMRTbell library preparation and sequencing.

1. Begin with high-quality nucleic acids and work in a clean environment. a. If extracted nucleic acids must be stored, freeze at high concentrations in appropriately-buffered

solutions. b. To minimize possible contamination and degradation caused by multiple freeze/thaw cycles, sub-

aliquot DNA into smaller volumes for storage. For DNA samples, DNAStable® Plus from Biomatrica may be used to help preserve extracted DNA.

c. Set up PCR reactions in an environment free from sources of non-specific primer and template contaminants; ideally a laminar flow hood, using dedicated pre-PCR pipettor, tips and reagents.

2. Use PCR reagents and conditions for generating target-specific, full-length amplicons. a. Use the highest fidelity polymerase compatible with your PCR amplification system. b. Use desalted or HPLC-purified oligos; damaged bases at the ends of the amplicons cannot be

repaired by DNA Damage Repair enzymes. c. Optimize PCR conditions to minimize total time spent at high (>65°C) temperatures, particularly

during denaturation. d. PCR extension time should be long enough to ensure complete extension, taking into consideration

the polymerase used and target amplicon size. For mixed samples with similar targets, it is important to complete extension at every step to avoid generating chimeric products in subsequent steps. As a general guideline, use extension times of one minute per 1000 base pairs (e.g. 3 minutes for a 3 kb product).

3. Use the lowest number of cycles required for obtaining adequate yields (ng) of PCR products to proceed with SMRTbell library construction. Avoid over-amplification.

4. If non-specific products are present, optimize PCR conditions or perform AMPure PB Bead-based size selection to enrich for PCR amplicons with the desired target size (see recommendations below).

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DNA Input Requirements for Library Construction When planning your amplification experiments, always consider the total input DNA required for SMRTbell library construction. In this procedure, 500 – 1000 ng of pooled PCR products are required. If using more than 1000 ng of total pooled PCR products, scale reaction volumes accordingly. Samples should be present at equimolar concentrations after pooling (see Best Practices for Equimolar Pooling below). If necessary, replicate PCR reactions should be set up to obtain the required total amount of DNA product needed for library construction. This approach also minimizes PCR sampling bias for samples containing heterogeneous templates. It is also important not to exceed the recommended DNA input amount when working with highly multiplexed samples. For high-multiplexing projects (e.g. ≥96-plex), it may be desirable to pool amplicons, purify with AMPure PB beads, measure the concentration, and then aliquot the appropriate amount for library construction.

Best Practices for Equimolar Pooling For studies targeting a single consensus sequence per sample, amplicons may be multiplexed to leverage the throughput capacity of a single SMRT Cell. However, pooling is generally recommended for amplicons of similar sizes (i.e., within +/-15% of the mean size). 1. Ideally, amplicons should be AMPure PB bead purified prior to pooling. 2. To obtain equal representation of each amplicon in the data, it is important to pool samples in

equimolar concentrations. To do this, purify with AMPure PB beads and quantify each sample using the Agilent® Bioanalyzer System, Agilent TapeStation, or Advanced Analytical Technologies Fragment Analyzer™ system.

3. Remove non-specific PCR products (contaminating bands) prior to pooling. The presence of non-specific products in the pool will impact sequencing data yield. a. If amplicons contain secondary bands that are <1.5 kb, it may be possible to remove them using

AMPure PB bead purification using an appropriate concentration of beads. b. If the contaminating bands are close in size to or are larger than the desired amplicon, or are

greater than 1.5 kb (i.e, they cannot be removed by AMPure PB bead purification), size selection using an automated size selection tool or other gel-based method may be necessary.

c. If removal of contaminating bands is not possible, we recommend re-optimization of the amplification reaction using more stringent PCR conditions.

d. Always determine the concentration of the amplicon target band or peak only and use this value to calculate the mass or volume of the amplicon sample to be used during pooling.

e. If presence of contaminating bands is determined to be acceptable (i.e., their presence has minimum impact on the sequencing yield of the desired target), you may choose to include the amplicon in the sample pool. In such cases, however, it may be necessary to increase the relative input amounts of such amplicons (containing non-specific products) during pooling in order to achieve adequate sequencing data yields for each amplicon in the sample pool.

4. For higher multiplexing (e.g. ≥96-plex), purifying and quantifying each individual PCR product may be difficult or impractical. A QC method that may work well is to load samples on an agarose gel to view the PCR products prior to pooling. This QC method may work well if PCR conditions are fully optimized to generate clean specific PCR products consistently. a. PCR products that show the same band intensity on a gel may be pooled by volume or mass. To do

this, include control fragments of known concentrations when loading samples to perform agarose gel electrophoresis. The pooled samples must meet the minimum DNA input requirements for SMRTbell library construction listed in Table 4.

b. For samples that show weak signals on a gel, increase the volume or mass used during pooling.

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




First Round PCR: Recommendations for Target Specific Primers Tailed with M13 Sequence

The Round 1 PCR recommendations below have been tested with a control sample, SARS-CoV-2 reference material from ATCC VR1986D (2019-ncoV/USA-Wa1/2020). In general, longer amplicons may be more difficult to obtain from samples with lower viral load or extensive viral RNA degradation.

We recommend using Platinum SuperFi Green Master Mix (Thermo Fisher Scientific) for amplification. Other high-fidelity polymerases may also be used for this procedure, but please optimize PCR conditions before proceeding to the second-round PCR step.

1. Prepare primer Set A and Set B pools (100 µM stock solutions) for multiplex PCR as follows:

PCR Primer Pool Set A Stock: A. Into a clean 1.5 mL Eppendorf tube add the following:

• Add 2 μL of each 100 µM M13-tailed forward primers (a total of 7 forward primers) • Add 2 μL of each 100 µM M13-tailed reverse primers (a total of 7 reverse primers)

PCR Primer Pool Set B Stock:

B. Into a clean 1.5 mL Eppendorf tube add the following the tube: • Add 2 μL of each 100 µM M13-tailed forward primers (a total of 7 forward primers) • Add 2 μL of each 100 µM M13-tailedreverse primers (a total of 7 reverse primers)

Note: The primer pools can be stored at 4°C or at -20°C for future use.

2. To set up the First-Round PCR reactions, the primer pool stocks for each set must first be diluted with nuclease-free water to a working concentration of 1 µM. Mix well and perform a quick spin.

Reagent Volume

Notes

Primer Pool Set A Stock (100 µM) 1.0 μL

Nuclease Free Water 99.0 μL Total Volume 100.0 μL

Reagent Volume

Notes Primer Pool Set B Stock (100 µM) 1.0 μL

Nuclease Free Water 99.0 μL

Total Volume 100.0 μL

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3. For each sample to be processed, prepare two separate multiplex PCR reactions using working solutions of Primer Pool sets A and B as follows:

Multiplex 1st-Round PCR Reaction with Primer Pool Set A Working Solution:

Reagent Stock Conc. Final Conc. 1X volume

Notes Nuclease Free Water 3.0 μL Platinum SuperFi Green Master Mix (2X) 2X 1X 10.0 μL cDNA* 1-5 ng/μL 1.0 μL

Primer Pool Set A Working Solution (1 µM) 1 μM 0.3 μM 6.0 μL

Total Volume

20.0 μL *cDNA from ATCC RNA Control (~8000 copies/ul)

Multiplex 1st-Round PCR Reaction with Primer Pool Set B Working Solution


Notes Nuclease Free Water 3.0 μL Platinum SuperFi Green Master Mix (2X) 2X 1X 10.0 μL cDNA* 1-5 ng/μL 1.0 μL Primer Pool Set B (1 µM) Working Solution 1 μM 0.3 μM 6.0 μL

Total Volume

20.0 μL *cDNA from ATCC RNA Control (~8000 copies/ul)

4. Slowly aspirate and dispense several times to mix contents. Quick-spin the tubes (or plate) to bring contents to the bottom.

5. Place the PCR reactions in a thermocycler and run the following program (set the heated lid at 105°C). The PCR reactions may be held at 4°C overnight.

*Optimization is highly recommended

6. Note: The first-round PCR products should be checked on an agarose gel for visual inspection before proceeding with the second-round PCR. If a 2.5 kb band is visible, proceed to AMPure PB bead purification and pooling. You need at least 1 ng of the 2.5 kb PCR product for 2nd round PCR. If PCR product is not visible, perform additional cycles until a 2.5 kb band appears.

Step Temperature Time

1 98ºC 2 minutes

2 98ºC 15 seconds

3 65ºC 30 seconds

4 72ºC 2 minutes

5 Repeat steps 2 to 5 (25* cycles total) -

6 72ºC 5 minutes

7 4ºC Hold

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STEP

Purification with AMPure PB Beads Notes

1 Bring each PCR reaction to a total volume of 100 µL with 1X Elution Buffer (EB) before AMPure PB bead purification. Add 60 µL (0.60X) of resuspended, room-temperature AMPure PB beads to the 100 μL. Pipette mix 10 times. Perform a quick spin to collect all liquid from the sides of the tube. Note that the beads must be brought to room temperature before use and all AMPure PB bead purification steps should be performed at room temperature. Before using, mix the bead reagent well until the solution appears homogenous. Pipette the reagent slowly since the bead mixture is viscous and precise volumes are critical to the purification process.

2 Mix the bead/DNA solution thoroughly by pipette mixing 15 times. It is important to mix well.

3 Quickly spin down the tube (for 1 second) to collect the beads.

4 Incubate the samples on a bench top for 5 minutes at room temperature.

5 Spin down the tube (for 1 second) to collect beads.

6 Place the tube in a magnetic bead rack to collect the beads to the side of the tube.

7 Slowly pipette off cleared supernatant and save (in another tube). Avoid disturbing the beads.

8 Wash beads with freshly prepared 80% ethanol. 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.

– Do not remove the tube from the magnetic rack. – Use a sufficient volume of 80% ethanol to fill the tube (1.5 mL for a 1.5 mL

DNA LoBind tube) – Slowly dispense the 80% ethanol against the side of the tube opposite the

beads. – Do not disturb the beads. – After 30 seconds, pipette and discard the 80% ethanol.

9 Repeat step 8.

10 Remove residual 80% ethanol. – Remove tube from magnetic bead rack and spin. Both the beads and any

residual 80% ethanol will be at the bottom of the tube. – Place the tube back on magnetic bead rack and allow beads to separate. – Pipette off any remaining 80% ethanol.

11 Check for any remaining droplets in the tube. If droplets are present, repeat step 10.

12 Immediately add 12 μL of Elution Buffer volume to your beads. Pipette mix 15 times. It is important to mix well.

– Elute the DNA by letting the mix incubate at 37 ºC for 15 minutes. This is important to maximize recovery of high molecular weight DNA.

– Spin the tube down, then place the tube back on the magnetic bead rack. – Let beads separate fully. Then without disturbing the beads, transfer supernatant

to a new 1.5 ml Lo-Bind tube. – Discard the beads.

13 Use 1 µL to perform DNA quantitation using the Qubit dsDNA HS kit.

14 Proceed to “Pooling of Round 1 PCR for 2nd-Round PCR Reaction”.

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Pooling of Round 1 PCR Products for 2nd-round PCR Reaction. PCR products from the two multiplex PCR reactions may now be pooled for 2nd-round PCR. For best results, we recommended normalizing Round 1 PCR products from Primer Pool Set A and Primer Pool Set B for pooling . Without using equivalent molar concentrations or mass across samples, there is a risk of generating uneven coverage in the resulting sequencing data.

Second Round PCR: Recommendations for Asymmetric Barcoding

Barcoded M13 Primers are ordered from any Oligo Synthesis providers. Sequences of the 32 x Forward and 32 x Reverse M13 Barcoded Primers are available here. For demultiplexing, the FASTA file is available here.

1. Dilute PacBio-barcoded M13 primers with nuclease-free buffer (10mM Tris-HCl pH 7.5) to a concentration of 3.0 µM and aliquot into a 96-well plate as shown in the example layout below (or use a different configuration that facilitates aliquoting the desired combinations of forward and reverse barcoded primers). Note that QC analysis of barcoded primers is highly recommended, as oligos with missing 5’ bases may appear to produce the expected amounts of PCR products but generate low (de-multiplexed) sequencing data yields if they lack the complete barcode sequence.

2. Example Plate Layout for 32 Forward and 32 Reverse PacBio-Barcoded M13 Primers.

1 2 3 4 5 6 7 8 9 10 11 12 A 1001 1009 1017 1025 x x 1049 1057 1065 1073 x x B 1002 1010 1018 1026 x x 1050 1058 1066 1074 x x C 1003 1011 1019 1027 x x 1051 1059 1067 1075 x x D 1004 1012 1020 1028 x x 1052 1060 1068 1076 x x E 1005 1013 1021 1029 x x 1053 1061 1069 1077 x x F 1006 1014 1022 1030 x x 1054 1062 1070 1078 x x G 1007 1015 1023 1031 x x 1055 1063 1071 1079 x x H 1008 1016 1024 1032 x x 1056 1064 1072 1082 x x

3. Columns 1-4 are M13 forward primers tailed with PacBio barcode 1001 to barcode 1032.

4. Columns 7-10 are M13 reverse primers tailed with PacBio barcode 1049 to barcode 1079 and barcode 1082.

5. Any/all of the 32 forward primers may be combined with any/all of the 32 reverse primers to create asymmetrically barcoded pairs. Plan out a specific barcoding strategy to use prior to preparing PCR reaction mixes.

6. For each sample to be processed, prepare the following Round 2 PCR reaction using the pooled Round 1 PCR products:


Notes

Nuclease Free Water 10.6 μL Platinum SuperFi Green Master Mix (2X) 2X 1X 15.0 μL Round 1 PCR products 1

ng/μL 1.0 μL

M13 Forward Barcoded Primer 3 μM 0.17 μM 1.7 μL M13 Reverse Barcoded Primer 3 μM 0.17 μM 1.7 μL Total Volume

30.0 μL

7. Slowly aspirate and dispense several times to mix contents. Quick spin the tube or plate.

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https://www.pacb.com/wp-content/uploads/Sequel_96_M13_Asymmetric.zip


8. Place in a thermocycler and run the following program (lid 105°C). The PCR reactions may be held

at 4°C overnight.

Step Temperature Time

1 98ºC 2 minutes

2 98ºC 20 seconds

3 60ºC 15 seconds

4 72ºC 2 minutes

5 Repeat steps 2 to 4 -

6 98ºC 20 seconds

7 65ºC 15 seconds

8 72ºC 2 minutes

9 Repeat steps 6 to 8 (up to 20 cycles total*) -

10 72ºC 5 minutes

11 4ºC Hold

*Optimization is highly recommended

9. After amplification, perform visual inspection of the Round 2 PCR products on an agarose gel to ensure 2.5 kb products are visible.

10. Proceed to AMPure PB bead purification.

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STEP

Purification with AMPure PB Beads Notes












9 Repeat step 8.








13 For library construction for 1 sample, proceed to SMRTbell library construction. For library construction for multiplexed samples, pool Round 2 PCR products from multiple samples for SMRTbell library construction. See next section “Normalization of Round 2 PCR Products for Pooling”.

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Normalization of Round 2 PCR Products for Pooling Multiple Samples For multiplexing multiple samples, pool round 2 PCR products for SMRTbell library preparation. 500 – 1000 ng of total pooled amplicon DNA is required for SMRTbell library construction. a. Pool samples in equimolar concentrations for SMRTbell library construction.

b. Alternatively, if samples have similar intensities on an agarose gel, it is acceptable to pool equal volumes of the PCR products. If some sample band intensities appear weak on an agarose gel, then sample volumes should be adjusted accordingly. See “Best Practices for Equimolar Pooling” section.

SMRTbell Library Construction The amount of DNA required for this step is 500-1000 ng.

DNA Damage Repair 1. Prepare the following reaction.

Reagent (Reaction Mix 1) Tube Cap Color Volume

Notes DNA Prep Buffer 7.0 μL

Pooled and Purified PCR Product 47.0 μL

NAD 1.0 μL

DNA Damage Repair Mix v2 2.0 μL


2. Pipette mix 10 times. It is important to mix well. 3. Spin down the contents of the tube with a quick spin in a microfuge. 4. Incubate at 37°C for 30 minutes, then return the reaction to 4°C. Proceed to the next step.

End-Repair/A-tailing 1. Prepare the following reaction.

2. Pipette mix 10 times. It is important to mix well. 3. Spin down the contents of the tube with a quick spin in a microfuge. 4. Incubate at 20°C for 30 minutes. 5. Incubate at 65°C for 30 minutes, then return the reaction to 4°C. Proceed to the next step.


Notes

Reaction Mix 1 57.0 μL

End Prep Mix 3.0 μL

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Adapter Ligation 1. Prepare the following reaction, adding the components below in the order listed.


Notes

Reaction Mix 2 60.0 μL

Overhang Adapter v3 5.0 μL

Ligation Mix 30.0 μL

Ligation Additive 1.0 μL

Ligation Enhancer 1.0 μL


1. Pipette mix 10 times. It is important to mix well. 2. Spin down the contents of the tube with a quick spin in a microfuge. 3. Incubate at 20°C for 60 minutes, then return the reaction to 4°C. Proceed to the next step.

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Purification of SMRTbell Templates STEP

First AMPure PB Bead Purification Notes












9 Repeat step 8.








13 Proceed to next step, “Second AMPure PB Bead Purification” recommendations.

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STEP

Second AMPure PB Bead Purification Notes 1 Add 45 µL (0.45X) of resuspended, room-temperature AMPure PB beads to the 100

μL. Pipette mix 10 times. Perform a quick spin to collect all liquid from the sides of the tube. Note that the beads must be brought to room temperature before use and all AMPure PB bead purification steps should be performed at room temperature. Before using, mix the bead reagent well until the solution appears homogenous. Pipette the reagent slowly since the bead mixture is viscous and precise volumes are critical to the purification process.











9 Repeat step 8.






– Spin the tube down, then place the tube back on the magnetic bead rack. – Let beads separate fully. Then without disturbing the beads, transfer supernatant to

a new 1.5 ml Lo-Bind tube. – Discard the beads.

13 Verify your DNA amount and concentration using a Qubit quantitation platform. – Measure the DNA concentration using a Qubit fluorometer. – Using 1 μL of the eluted sample, make a 1:10 dilution in EB. – Use 1 µL of this 1:10 dilution to measure the DNA concentration using a Qubit

fluorometer and the dsDNA HS Assay kit according to the manufacturer’s recommendations.

14 Actual recovered DNA SMRTbell concentration (ng/µl): __________________ Total recovered DNA SMRTbell amount (ng): _____________________

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Prepare for Sequencing Sequel System: • For primer annealing and polymerase binding, follow the instructions in SMRT Link Sample Setup

(SMRT Link v8.0). Select the following options:

Sample Setup Select

Sequencing Primer Sequencing Primer v4 Enable 20:1 Polymerase:Template

30:1 Polymerase:Template Binding Kit Sequel Binding Kit 3.0

Sequencing Mode CLR

• In Run Design, select sequencing mode = CCS.

• For detailed recommendations for sequencing specific amplicon library insert size ranges, refer to the Quick Reference Card – Diffusion Loading and Pre-Extension Time Recommendations for the Sequel System here.

Sequel II System:

• For primer annealing and polymerase binding, follow the instruction in SMRT Link Sample Setup (SMRT Link 8.0). Select the following options:

Sample Setup Select Sequencing Primer Sequencing Primer v4 Enable 20:1

Polymerase:Template 30:1 Polymerase:Template

Binding Kit Sequel II Binding Kit 2.1 Sequencing Mode CLR

• In Run Design, select sequencing mode = CCS

• Recommended Run parameters:

On-Plate Loading Conc (pM) 50 pM

Movie Time (hours) >10

Pre-Extension Time >0.5 Hours

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Sequencing Data Analysis on the Sequel II System

The SMRT Link v8.0 user interface can support a maximum number of 384 barcodes. To analyze more than 384 barcoded samples containing different barcodes on either end of the template, follow the instructions below. Alternatively, data can be analyzed on the command line using SMRT Tools or PacBio Developers tools in Bioconda. If you need additional information, please contact [email protected].

CCS Analysis:

• When you are designing your sequencing run in the Run Design module of SMRT Link v8.0 or higher use the Auto Analysis option to set up CCS analysis. Data will be automatically analyzed with the CCS analysis application following the sequence acquisition.

• If CCS analysis was not performed automatically, manually run CCS analysis in SMRT Link with default parameters

Demultiplexing Analysis:

• Use the Demultiplex Barcodes analysis application in SMRT Link v8.0 or higher to demultiplex the CCS reads.

• Use PacBio Barcode Set “Sequel_96_barcodes_v1” for demultiplexing For details on the Demultiplex Barcodes analysis application, see pg. 62 in the SMRT Link User Guide

Analysis steps: At the SMRT Analysis module in SMRT Link, click on Create New Analysis:

1. Enter an analysis name 2. Select Data Type “CCS Data” 3. Select your dataset for analysis and click Next 4. Select Analysis Application “Demultiplex Barcodes” 5. Select Barcode Set “Sequel_96_barcodes_v1” 6. Provide the output a New Dataset Name 7. Set Same Barcodes on Both Ends of Sequence to “Off” 8. Set Infer Barcodes Used to “Off”* (Note: Analysis will fail if turned on.) 9. Optionally, set Minimum Barcode Score to 70

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https://www.pacb.com/wp-content/uploads/SMRT-Tools-Reference-Guide-v8.0.pdf

https://github.com/PacificBiosciences/pbbioconda

https://github.com/PacificBiosciences/pbbioconda

mailto:[email protected]

https://www.pacb.com/wp-content/uploads/SMRT-Link-User-Guide-v8.0.pdf

https://www.pacb.com/wp-content/uploads/SMRT-Link-User-Guide-v8.0.pdf


For Research Use Only. Not for use in diagnostic procedures. © Copyright 2020, Pacific Biosciences of California, Inc. All rights reserved. Information in this document is subject to change without notice. Pacific Biosciences assumes no responsibility for any errors or omissions in this document. Certain notices, terms, conditions and/o r use restrictions may pertain to your use of Pacific Biosciences products and/or third p arty products. Please refer to the applicable Pacific Biosciences Terms and Conditions of S ale and to the applicable license terms at http://www.pacificbiosciences.com/licenses.html. Pacific Biosciences, the Pacific Biosciences logo, PacBio, S MRT, SMRTbell, Iso-Seq and Sequel are trademarks of Pacific Biosciences. BluePippin and SageELF are trademarks of Sage Science, Inc. NGS-go and NGSengine are trademarks of GenDx. FEMTO Pulse and Fragment Analyzer are trademarks of Agilent Technologies. All other trademarks are the sole property of their respective owners.

Revision History (Description) Version Date Customer Collaboration. DRAFT May 20, 2020 Customer Collaboration. Step 9 on page 9, 23 cycles changed to 20. Some links also updated.

DRAFT May 22, 2020

Updated volumes in step 3, page 6. Added link to demultiplexing FASTA file on page 8. Various updates to Sequencing instructions on page 15.

DRAFT June 8, 2020

Updated table volumes in step 3 on page 6 (Multiplex 1st-Round PCR Reaction with Primer Pool Set B Working Solution)

DRAFT June 11, 2020

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Documents

Procedure & Checklist – Multiplexing 2.5 kb Amplicons for ... · 4. For higher multiplexing (e.g. ≥96-plex), purifying and quantifying each individual PCR product may be difficult