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PacBio Onso Operation Manual

Cluster generator and short-read sequencer
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Onso™ system
cluster
generator and
short-read
sequencer
Operations Guide

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  • Page 1 Onso™ system cluster generator and short-read sequencer Operations Guide...
  • Page 2 PACIFIC BIOSCIENCES IS ADVISED OF THE POSSIBILITY OF SUCH DAMAGES. Certain notices, terms, conditions and/or use restrictions may pertain to your use of PacBio products and/or third party products. Refer to the applicable PacBio terms and conditions of sale and to the applicable license terms at pacb.com/license.

  • Page 3: Table Of Contents

    Required materials and equipment ........................... 30 Sequencing procedure .............................. 31 1. Thawing sequencing reagent pack thaw .......................... 31 2. Optional custom primer addition ............................31 © 2024 PacBio. All rights reserved. For research use only. Not for use in diagnostic procedures. PN 103-140-400 REV05 JUN2024...
  • Page 4 6. Instrument washes ................................43 7. Long-term shutdown ................................46 8. Run management .................................. 46 9. System checks ..................................47 Troubleshooting ................................ 49 © 2024 PacBio. All rights reserved. For research use only. Not for use in diagnostic procedures. PN 103-140-400 REV05 JUN2024...

  • Page 5: Before You Begin

    This guide describes the workflow for preparing Sequencing By Binding (SBB™) libraries for cluster generation on ® the PacBio Onso™ cluster generator system, as well as preparation for and sequencing of clustered flow cells on the Onso system. Below are descriptions of the main consumables required for both clustering and sequencing procedures.

  • Page 6: Mica Configuration

    Flip the power switch to turn the instruments on. For the sequencer, wait approximately 30 seconds to then power on the computer by pressing the power button on the PC. © 2024 PacBio. All rights reserved. For research use only. Not for use in diagnostic procedures. PN 103-140-400 REV05 JUN2024...

  • Page 7: Safety Guidelines

    Library QC (LQC) spike-in. Using fewer than 4 samples in the absence of an LQC spike-in will result in too low sample diversity during sequencing. If multiplexing samples in the absence of LQC spike-in, a minimum of 8 Onso indexed adapters are required to supply sufficient index diversity.

  • Page 8: Reagent And Sample Handling

    This section describes the workflow for flow cell clustering of Onso compatible libraries in preparation for sequencing on the Onso sequencing system. After completing this section of the guide, a clustered flow cell can either be sequenced immediately, by following the...

  • Page 9: Cluster Generator

    The (H) magnetic flow cell clamp and metal guide pins are seated within the workspace door, allowing the user to properly place and secure the flow cell. The flow cell clamp lifts upward, hinging away from the user. © 2024 PacBio. All rights reserved. For research use only. Not for use in diagnostic procedures. PN 103-140-400 REV05 JUN2024...

  • Page 10: Clustering Workflow

    Denature and dilute DNA library 10 minutes Cluster reagent plate preparation 5.5 hours Cluster generator setup and run Safe stop 30 minutes Post-run wash © 2024 PacBio. All rights reserved. For research use only. Not for use in diagnostic procedures. PN 103-140-400 REV05 JUN2024...

  • Page 11: Required Materials And Equipment

    Any MLS Vortexer Any MLS Microcentrifuge Any MLS Pipettes, 2-1000 µL pipetting range Any MLS Pipette tips, 2-1000 µL Any MLS © 2024 PacBio. All rights reserved. For research use only. Not for use in diagnostic procedures. PN 103-140-400 REV05 JUN2024...

  • Page 12: Clustering Procedure

    Each clustering reagent plate supports clustering of up to 2 lanes on a single flow cell.  Step Instructions Remove the Onso clustering reagent kit from -20°C storage, unbox and remove clustering reagent plate from mylar bag. Discard mylar bag. Thaw contents of the Onso clustering reagent kit: •...
  • Page 13 If clustering a flow cell with an empty lane, simply add 380 µL of DIL buffer to a DNA low-bind tube designated for the empty lane. Immediately proceed to section 4 titled “Onso Clustering reagent plate preparation” OR (see next page) © 2024 PacBio. All rights reserved. For research use only. Not for use in diagnostic procedures. PN 103-140-400 REV05 JUN2024...
  • Page 14 = a - c User input 3b.4 Desired total loading concentration (pM) LQC spike-in concentration (pM) Final sample library concentration (pM) © 2024 PacBio. All rights reserved. For research use only. Not for use in diagnostic procedures. PN 103-140-400 REV05 JUN2024...

  • Page 15: Onso Clustering Reagent Plate Preparation

    Leave the oval trough in column 2 empty until after the clustering run has completed (Refer to section 5 “Post-run wash”). © 2024 PacBio. All rights reserved. For research use only. Not for use in diagnostic procedures. PN 103-140-400 REV05 JUN2024...

  • Page 16: Cluster Generator Setup And Run

    5.2 below. Load Consumables In the screen, select “Open workspaces”. © 2024 PacBio. All rights reserved. For research use only. Not for use in diagnostic procedures. PN 103-140-400 REV05 JUN2024...
  • Page 17 This will cause the base of the flow cell kit to sit against the flow cell clamp hinge. © 2024 PacBio. All rights reserved. For research use only. Not for use in diagnostic procedures. PN 103-140-400 REV05 JUN2024...
  • Page 18 © 2024 PacBio. All rights reserved. For research use only. Not for use in diagnostic procedures. PN 103-140-400 REV05 JUN2024...
  • Page 19 If placed properly, the clustering reagent plate QR code should be visible from the right side of the instrument to scan into the software. © 2024 PacBio. All rights reserved. For research use only. Not for use in diagnostic procedures. PN 103-140-400 REV05 JUN2024...
  • Page 20 Verify that the consumables entered are correct and select “Continue” to advance. If changes need to be 5.14 made, click the back arrow at the top left to make the necessary changes. © 2024 PacBio. All rights reserved. For research use only. Not for use in diagnostic procedures. PN 103-140-400 REV05 JUN2024...
  • Page 21 If an error is encountered, refer to the Troubleshooting section at the end of the Operations guide. 5.15 © 2024 PacBio. All rights reserved. For research use only. Not for use in diagnostic procedures. PN 103-140-400 REV05 JUN2024...
  • Page 22 4°C the following morning, or user can proceed to section 6 below (“Post-run wash”) as soon as clustering is complete. 5.16 SAFE STOPPING POINT © 2024 PacBio. All rights reserved. For research use only. Not for use in diagnostic procedures. PN 103-140-400 REV05 JUN2024...

  • Page 23: Post-Run Wash

    Repeat step 4 above with the left MICA instrument connector side to disengage from the instrument and seat within the flow cell kit base. © 2024 PacBio. All rights reserved. For research use only. Not for use in diagnostic procedures. PN 103-140-400 REV05 JUN2024...
  • Page 24 Lower the magnetic flow cell clamp and close the workspace door. Prepare at least 30 mL of fresh 0.1% hypochlorite (bleach) solution. © 2024 PacBio. All rights reserved. For research use only. Not for use in diagnostic procedures. PN 103-140-400 REV05 JUN2024...
  • Page 25 When the post-run wash is complete, the user will be allowed to access and dispose of the clustering reagent 6.13 plate, if necessary. © 2024 PacBio. All rights reserved. For research use only. Not for use in diagnostic procedures. PN 103-140-400 REV05 JUN2024...

  • Page 26: Instrument Washes

    Load the four empty troughs onto the plate and ensure they are pressed firmly down until the tip of each trough meets the plate. On the clustering instrument screen, click on the top right hamburger menu and select “Washes”. © 2024 PacBio. All rights reserved. For research use only. Not for use in diagnostic procedures. PN 103-140-400 REV05 JUN2024...
  • Page 27 Add 28 mL of user prepared 0.1% NaOCl (Bleach) solution to the first trough. © 2024 PacBio. All rights reserved. For research use only. Not for use in diagnostic procedures. PN 103-140-400 REV05 JUN2024...

  • Page 28: Long-Term Shutdown

    If the clustering instrument was powered off for long-term shutdown, power on instrument. See “Before you begin” section titled “Powering ON instrument” to identify power switch location. © 2024 PacBio. All rights reserved. For research use only. Not for use in diagnostic procedures. PN 103-140-400 REV05 JUN2024...

  • Page 29: Sequencing

    Once the instrument maintenance wash is complete, the instrument is ready to use. Sequencing This section describes the workflow for sequencing an Onso clustered flow cell on the Onso sequencing system. Each clustered flow cell and each sequencing reagent pack only support a single sequencing run. The number of cycles supported per reagent pack is indicated by the specific reagent pack being used (e.g., 200-cycle reagent...

  • Page 30: Sequencing Workflow

    Kimtech or other MLS Serological Pipettor Any MLS Serological Pipettes (10 mL, 25 mL, 50 mL, or 100 mL) Any MLS © 2024 PacBio. All rights reserved. For research use only. Not for use in diagnostic procedures. PN 103-140-400 REV05 JUN2024...

  • Page 31: Sequencing Procedure

    Page 31 Sequencing procedure 1. Thawing sequencing reagent pack thaw An Onso sequencing reagent pack can be removed from the box and mylar bag prior to thaw, as described below.  Step Instructions Remove the sequencing reagent pack from -20°C storage. Remove the reagent pack from packaging and the mylar bag.
  • Page 32 Note: if setting up in this configuration DO NOT select custom primers during run setup. © 2024 PacBio. All rights reserved. For research use only. Not for use in diagnostic procedures. PN 103-140-400 REV05 JUN2024...
  • Page 33 Visually inspect the side of the custom primer tubes during mixing to ensure the pipette tip is submerged in the solution during mixing. © 2024 PacBio. All rights reserved. For research use only. Not for use in diagnostic procedures. PN 103-140-400 REV05 JUN2024...

  • Page 34: Sequencing Set Up

    Discard the flow cell and attached MICA in the appropriate waste stream. © 2024 PacBio. All rights reserved. For research use only. Not for use in diagnostic procedures. PN 103-140-400 REV05 JUN2024...
  • Page 35 The cutout in the flow cell kit base should wrap around the preload cover. © 2024 PacBio. All rights reserved. For research use only. Not for use in diagnostic procedures. PN 103-140-400 REV05 JUN2024...
  • Page 36 Users can either retain the flow cell kit lid and base or dispose in appropriate waste streams. © 2024 PacBio. All rights reserved. For research use only. Not for use in diagnostic procedures. PN 103-140-400 REV05 JUN2024...
  • Page 37 • “Reagent pack” – use the barcode scanner to scan in the reagent pack. © 2024 PacBio. All rights reserved. For research use only. Not for use in diagnostic procedures. PN 103-140-400 REV05 JUN2024...
  • Page 38 100 base pair insert read 1, enter “102” in the “Insert read 1” field. • “Index 1” & “Index 2” – input the number of index reads up to 14 base pairs each. © 2024 PacBio. All rights reserved. For research use only. Not for use in diagnostic procedures. PN 103-140-400 REV05 JUN2024...
  • Page 39 “Onso sequencing” this guide. The example shown below is for a 2x100 sequencing run for native Onso libraries. Native Onso libraries contain 8 base pair index sequences, and do not require the use of custom primers. Confirm field entries are correct. Select “Continue” to advance.
  • Page 40 When all sequencing cycles have completed, the software will automatically begin a post-run wash to prepare 3.17 the instrument for the next run. Once completed, click “Continue” in the top corner of the user interface. © 2024 PacBio. All rights reserved. For research use only. Not for use in diagnostic procedures. PN 103-140-400 REV05 JUN2024...

  • Page 41: Unloading The Sequencing Instrument

    Close the reagent door and workspace area. Home Select the back arrow at the top left of the screen to return to the screen. © 2024 PacBio. All rights reserved. For research use only. Not for use in diagnostic procedures. PN 103-140-400 REV05 JUN2024...

  • Page 42: Kitting And Discarding The Reagent Pack

    Used flow cells can be discarded in a properly labelled sharps or broken glass disposal. For disposal of used MICA, check with local EH&S for appropriate disposal recommendations. © 2024 PacBio. All rights reserved. For research use only. Not for use in diagnostic procedures. PN 103-140-400 REV05 JUN2024...

  • Page 43: Instrument Washes

    The Onso sequencing instrument will require a standby wash in one or more of the following scenarios: 1) if pre-run checks fail and 2) if the instrument is idle for ≥5 days. For a standby wash, the first wash step using diluted bleach lasts approximately 20 minutes, and the second wash step using instrument wash reagent lasts approximately 20 minutes.
  • Page 44 6.10 of the guide. Once pre-wash checks complete successfully, the wash will automatically begin and will last approximately 20 minutes. © 2024 PacBio. All rights reserved. For research use only. Not for use in diagnostic procedures. PN 103-140-400 REV05 JUN2024...
  • Page 45 Once the wash is complete, consumables can remain on the instrument until next use. It is recommended the wash cartridges stay installed onto the system until next use. © 2024 PacBio. All rights reserved. For research use only. Not for use in diagnostic procedures. PN 103-140-400 REV05 JUN2024...

  • Page 46: Long-Term Shutdown

    “Complete” to clear space for the next run. Do not delete runs that do not have a Copy status as “Complete”, or data will be lost. © 2024 PacBio. All rights reserved. For research use only. Not for use in diagnostic procedures. PN 103-140-400 REV05 JUN2024...

  • Page 47: System Checks

    System checks should only be performed with guidance from a PacBio Technical Support representative. Navigate to the hamburger menu and select System checks to open the System checks menu to select the desired system check tests. Once system checks complete, test results can be found in D:\PacBio\Metrics\PreRun to be shared with PacBio Technical Support.
  • Page 48 Once the test is complete, results can be found in D:\PacBio\Metrics\PreRun Home 9.12 When finished, select “Close” to go back to the screen. © 2024 PacBio. All rights reserved. For research use only. Not for use in diagnostic procedures. PN 103-140-400 REV05 JUN2024...

  • Page 49: Troubleshooting

    Research use only. Not for use in diagnostic procedures. © 2024 Pacific Biosciences of California, Inc. (“PacBio”). All rights reserved. Information in this document is subject to change without notice. PacBio assumes no responsibility for any errors or omissions in this document.

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