How to Run the PacBio® RS II Instrument 

100‐338‐600‐01 

1. Run Instrument  

1.1 How to Run the RS II Instrument   

 

Notes: 

Welcome to Pacific Biosciences' How to Run the PacBio® RS II Instrument training module.

This module provides an introduction to the procedures required to successfully set up and run the PacBio RS II Instrument for a DNA sequencing analysis. 

 

 

 

1.2 Contents 

 

Notes: 

In this presentation we will present the following:

A general overview of the PacBio® software suite

Sequencing run parameter setup

Instrument and reagent setup

Starting a sequencing run

Monitoring a sequencing run

Show some helpful resources for more in-depth information on using PacBio instrumentation

 

 

 

 

1.3 PacBio® Software Suite 

 

Notes: 

PacBio® Software Suite 

 

 

 

1.4 Instrument Software 

 

Notes: 

The PacBio® RS II Instrument can be accessed from various interface software systems:

RS Remote:

RS Remote enables users to design and monitor a sequencing run

Is used remotely from the instrument

Provides customization through user-defined fields

Allows for fully-automated secondary analysis

Provides feedback on the time and consumables needed to complete the run

RS Touch:

RS Touch is used to load, run, monitor and look at primary analysis results, on the instrument

Has a user-friendly touch-screen interface

 

 

Is equipped with barcode tracking, which provides efficient management and traceability of samples and reagents

RS Dashboard

The RS Dashboard is comprehensive QC tool combining metrics from RS Touch, RS Remote and SMRT® Portal (Secondary analysis software)

RS Dashboard gives a quick performance view across single SMRT® Cells with comprehensive metrics of histograms of the cells within a run.

It helps a user quickly glance at trends, yield per cell, and reads of insert to determine library size.

The system performance display shows historical trends of runs, movies and uptime on a system for a given time period.

There is also an email link to provide feedback to PacBio directly within the interface. This is for customers to send ideas to PacBio developers. 

 

1.5 Designing a Sequencing Run 

 

Notes: 

Designing a Sequencing Run

 

 

 

1.6 RS Remote – Designing a Run 

 

Notes: 

RS Remote- Designing a Sequencing Run:

The RS Remote interface enables a user to design and monitor a sequencing run.

RS Remote provides customization through user-defined fields.

It allows for fully automated secondary analysis.

RS Remote provides feedback on the time and consumables needed to complete the run.

 

 

 

1.7 RS Remote Demonstration 

 

Notes: 

Shown here is a video demonstration using RS Remote to design a run.

 

 

 

1.8 RS Touch Setup Run 

 

Notes: 

RS Touch is the user interface on the instrument touch screen that enables a user to select a run, load the instrument, and then start the run.

RS Touch enables a user to load, run, monitor and view primary analysis results, on the instrument.

In addition, the user-friendly RS Touch screen interface is equipped with barcode tracking which provides efficient management and traceablity of samples and reagents.

 

 

 

1.9 RS Touch Demonstration 

 

Notes: 

Video demonstration using RS Touch to start a sequencing run:

From the Instrument Status window, press Select Run.

Runs available for the instrument are shown.

Select a run from the list.

The run displays on the main screen and shows the quantities of reagents, mixing plates, samples, SMRT® Cells and tips that you will need to load.

A run can be previewed to show the well positions, SMRT® Cells , movie time and collection protocol used.

 

 

 

 

1.10 Reagent & Sample Plate Preparation 

 

Notes: 

There are specific handling procedures for the OS Enzyme, SMRT® Cell Oil tube, reagent plate setup and sample plate setup.

Follow the procedures for reagent and sample plate preparation described in the PacBio® Template Preparation and Sequencing Guide.

Summary notes are provided for reference in the left side of your screen.

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Thaw the following kit components accordingly:

1. OS Enzyme: Remove the OS Enzyme from the reagent package. Keep at -20ºC at all times. When ready to use, spin down the tube for 5 seconds, replace cap with tube septa, and place on the reagent drawer of the instrument.

2. Reagent plate: The reagent plate can be thawed overnight at 4ºC. Thaw an additional 15 minutes, at room temperature, before using. Note the

 

 

following precautions which may impact reagent performance:

Do not thaw in hot water.

Do not remove plate seal until it is ready to be placed on instrument.

Do not keep at room temperature after thawing.

Do not keep the unsealed plate exposed to air. Place the septa mat on plate immediately.

3. Place the reagent plate on a VWR® Microplate Shaker and shake for 1 minute at 1200 rpm.

4. If undissolved precipitates are present in any of the filled wells, mix thoroughly by vigorously vortexing until the solution is clear.

5. Spin the plate in a centrifuge briefly at 2000 rpm.

6. Peel the foil seal off the plate

7. Place the supplied septa mat on the plate.

8. Using a roller, further press down the septa mat onto the plate.

Prepare Your Sample Plate

1. Load your samples into the appropriate wells in the sample plate.

2. Place the white septa mat on the plate and press down with a roller to ensure a tight fit.

3. Spin your sample plate down for 30 seconds at 2000 rpm.

4. Place the sample plate onto the instrument reagent loader

 

 

 

 

1.11 Samples Reagent Loading 

 

Notes: 

Samples, reagents and consumables are loaded into the PacBio® RS II Instrument.

Place the Sample Plate with septa onto the drawer.

Place the Reagent Plate with septa onto the drawer.

Insert the OS Enzyme tube with septa into the left well.

Insert the SMRT Cell Oil tube with septa into the right well. Note: SMRT Cell Oil tubes are single use only.

Place a 384-well Mixing plate onto the drawer.

Lower doors and press close button.

For more detailed information on sample, reagents and consumables preparation, please see the PacBio Template Preparation and Sequencing Guide.

Notes are provided for reference on the left side of your screen.

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At the instrument, open the reagents/samples drawer. You can either press

 

 

the illuminated Open button on the instrument Reagents/Samples drawer or the Open button on the touchscreen user interface.

Load your Reagents on to the Instrument:

1. When the drawer opens, lift up the metal door covering the reagents slots.

2. Place your reagent plate in the reagent slot. Note that there are two slots, if you have only one plate, you can place it in either slot. The barcode scanner will locate the plate.

3. Remove the OS Enzyme from the refrigerator or ice.

4. Replace the cap with a tube septa. Be careful not to remove the barcode collar, the Machine Vision System will read the barcode.

5. Place the tube in the left slot encircled by your reagent plate location.

6. Remove the blue cap from the SMRT® Cell Oil tube.

7. Replace the cap with a tube septa. Place the SMRT Cell Oil tube in the right hand reagent slot. Note that the SMRT Cell Oil tube is slightly larger and will fit only in that slot.

8. Gently close the metal door.

Load your samples and mixing plate on to the Instrument:

9. Lift up the metal door covering the sample and mixing slots.

10. Place your sample plate in the slot labeled Sample.

11. Place a 384-well mixing plate (Eppendorf® LoBind Deepwell plates) in the slot labeled Mixing.

12. Gently close the metal door.

13. Press the Close button on the touchscreen user interface (optionally, press the Close button on the instrument).

 

 

 

 

1.12 SMRT Cells ‐ Tips Loading 

 

Notes: 

Load your SMRT® Cells/tips onto the instrument:

Note: Open the SMRT® Cells/tips drawer by either pressing the illuminated Open button on the instrument SMRT Cells/tips drawer or the Open button on the touchscreen user interface.

When the drawer opens, place the appropriate number of SMRT Cell 8Pacs (determined previously during project planning) into the SMRT Cells tray.

Place tip boxes in the slots. Note that, at a minimum, there should be a tip box in slot 1 and slot 6. Be sure to remove the lid and tape from the tip boxes.

Gently close the drawer door.

Press the Close button on the touchscreen user interface (optionally, press the Close button on the instrument).

WARNING! Pinch Point. Do not place fingers inside drawers as they are closing. 

 

 

 

1.13 RS Touch – Start Run 

 

Notes: 

RS Touch - Start Run Demonstration

Before starting the run, from the RS Touch Instrument Status screen: press the Scan Contents button.

The system will scan the contents of the drawers to verify that there are enough consumables loaded to complete the sequencing run.

Once the contents have been scanned and verification passes, the green Start button will be activated.

Press the Start button to begin the run.

Note: After pressing Start, the user interface will prompt you to verify that certain steps have been performed prior to sequencing:

Waste has been emptied. Verify that the waste bin is less than half full.

Nitrogen supply is within range. Locate your facility's N2 tank supply gauge and verify that the supply is > 80 psi (preferably > 100 ~ 120 psi).

 

 

 

 

1.14 Monitor Sequencing  Run 

 

Notes: 

Monitoring a Sequencing Run

 

 

 

1.15 RS Touch – Monitoring Run 

 

Notes: 

Once a run begins, the run can be monitored at the instrument or from a remote workstation.

The system displays real-time base incorporations.

View the remaining time for all SMRT® Cells.

The cell-by-cell status is displayed from cell prep through QVs.

Once QVs are available, view primary analysis on the RS Touch screen.

 

 

 

1.16 RS Dashboard – Monitor and QC Runs in a Web Browser 

 

Notes: 

The PacBio® RS II Instrument utilizes the RS Dashboard, a web-based tool that will help monitor performance trends and perform run QC remotely.

Within individual run reports are summaries of the run parameters for each sample with QC statistics updated as the run progresses.

Instrument performance trending includes statistics for each SMRT® Cell to ensure that the resulting throughput and quality are as expected.

At the bottom of the report are charts for each SMRT Cell showing distributions of read lengths and accuracy, as well as graphs helpful for troubleshooting. 

 

 

 

1.17 Getting to RS Dashboard 

 

Notes: 

There are two ways to get to the RS Dashboard:

The easiest way to access RS Dashboard is via a web browser.

Just type the URL into your web browser including the name of your system.

You can also access RS Dashboard via RS Remote.

 

 

 

1.18 Completed Run 

 

Notes: 

Here is an example of a completed run.

The finished status indicates the run is complete and corresponding read lengths are displayed.

 

 

 

1.19 Help Resources 

 

Notes: 

Help Resources 

 

 

 

1.20 Resources 

 

Notes: 

Additional help resources include the following:

PacBio Software Getting Started Guide

RS Remote Online Help

Official PacBio documents are available from the Pacbio website ( www.pacificbiosciences.com) within the Services & Support / Documentation & Training pages.

Another resource is the SMRT® Community page called SampleNet that provides protocols and recommendations outside of our standard sequencing workflow that were suggested by our customers.

SampleNet is the central resource for information and discussion on sample preparation and sequencing with the PacBio® System.

In addition to downloading the latest PacBio sample preparation documents, you can find out what other users are thinking and doing, share your protocols, ask questions and join in the discussion. 

 

 

 

1.21 Summary 

 

Notes: 

In summary, we have reviewed the following concepts in running the PacBio® RS II Instrument:

A general overview of the PacBio® software suite

Sequencing run parameter setup

Instrument and reagent setup

Starting a sequencing run

Monitoring a sequencing run

Helpful resources for more in-depth information on using PacBio instrumentation.

 

 

 

1.22 Thank You 

 

Notes: 

Thank you for your participation. For more information, please contact your local PacBio 

Field Applications Scientist (FAS) or PacBio Account Representative. 

 

www.pacificbiosciences.com