Oxford Nanopore tutorial

1. Logging in

You need to log in to MISO LIMS in order to make changes to any LIMS entities. Logging in lets MISO record any changes you make on entities you have access to.

If you are a new user, you will need to contact helpdesk@oicr.on.ca so that they put you into the appropriate Active Directory group, MISO_ROLE_INTERNAL.

Try to log in now:

  1. Click on http://miso.gsi.oicr.on.ca.
  2. Enter your username (e.g. jdoe) and password and click the Login button.

MISO uses the same username and password as your OICR email account.

If all goes well, you should see the MISO Dashboard and see a message at the top right: "Logged in as: jdoe".

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2. Projects

A Project contains information about a set of Studies that may comprise many different Samples, Experiments and Runs. Samples are attached to Projects and they are often processed into Libraries and then Library Aliquots, which are then Pooled and sequenced. We will create a new project for our Oxford Nanopore Samples.

2.1 Creating a new project

To create a new project:

  1. After logging in, click the Projects link under Preparation in the menu on the left side of the screen.
  2. Click the Add button at the top left corner. The Create Project page will display with a number of fields that you can fill in.
  3. Ignore Project ID and Name, since they are set by MISO once the Project is saved.

  4. Enter a unique Alias. The alias is a name, chosen by us, that is associated with a project. e.g. Do It 4 Science (be creative!).

  5. Enter a Short Name for your project. The short name should be 2-5 letters and/or numbers in all CAPS and related to the project alias. e.g. DI4S. This short name will be used to automatically generate sample and library names.

  6. In the Description field, enter MISO training workshop [Date]
  7. For the Progress field, choose Active
  8. Select the Reference Genome Human hg19 random. This should be the primary species that will be sequenced in the course of the project. Xenografts count as human.
  9. Select the Pipeline Research. This value identifies the analysis pipeline to put sample data through after sequencing
  10. Click the Save button at the upper right.
  11. MISO will generate an ID and name for the project. The name will be 'PRO' followed by the ID (e.g. "PRO123").

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3. Receiving Libraries

Propagating libraries from samples is only possible if there are existing samples to propagate from, and only makes sense if the libraries were prepared in-house. Libraries may also be received without the samples. The sample information is still required, but it can all be entered in one step.

When a library is entered in this way, the automatically-created sample hierarchy will include an Identity, Tissue, Stock, and Aliquot. Existing Identities and Tissues may be included in the hierarchy if they match the data entered.

3.1 Entering received Libraries

  1. On the left hand menu under Preparation, click Libraries.

  2. Click the Receive button at the top of the table.

  3. In the dialog, select Aliquot Class: gDNA (aliquot) and Quantity: 1, and click Receive

  4. Select or enter the following data:

    • Project: Select your project from the drop-down.

    • Subproject: Select the subproject you created.

    • Sample Type: GENOMIC
    • Sci. Name: Homo sapiens
    • External Name: Enter PROJ_ID10, replacing the PROJ with your own project short name. The Identity Alias column should change to show First Receipt (PROJ).
    • Donor Sex: Select any.
    • Tissue Origin: Pa (Pancreas)
    • Tissue Type: P (Primary Tumour)
    • Times Received: 1
    • Tube Number: 1
    • Timepoint: Week 8
    • Date of receipt: Select today's date
    • Received From: Select any lab.
    • Received By: Select any group.

    • Matrix Barcode: Enter PROJ_lib5, replacing PROJ with your own project short name.

    • Design: Select any.

    • Platform: Oxford Nanopore

    • Type: 1D Genomic DNA by ligation

    • Index Kit: No indices
    • Kit: Select any.
    • QC Status: Ready
    • Size (bp): 430
  5. Click Save at the top right. Record the Library alias and barcode in your worksheet.

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4. Pooling Libraries

A library cannot be directly loaded into a flow cell. A library aliquot must be made, and then many aliquots (or just one) can be mixed into a pool for sequencing.

Seqeuncing Orders are requests for sequencing pools a certain number of times. They are used to keep track of sequencing progress for project management and book-keeping.

4.1 Creating a Library Aliquot

  1. If you are still on the Create Libraries page, click the Make aliquots button in the toolbar. Otherwise, go to the Libraries page, select the library you created in the previous exercise, and click the Make aliquots button.
  2. Click the Create button in the dialog that pops up.
  3. Select QC Status Ready
  4. Leave the default values for everything else and click Save.

4.2 Creating a Pool

Here we will create a pool that contains the single library aliquot that we just created.

  1. If you are still on the Create Library Aliquots from Libraries page, click the Pool separately button in the toolbar. Otherwise, go to the Library Aliquots page, select the aliquot you created in the previous exercise, and click the Pool separately button.
  2. Click the Create button in the dialog that pops up.
  3. Enter the pool information:
    • Volume: Enter a volume.
  4. Leave the remaining fields at their default values and click Save.
  5. A dialog will pop up, warning you about saving without a barcode. Click Save again.

4.3 Creating a Sequencing Order

  1. If you are still on the Create Pools from Library Aliquots page, click the Create Orders button in the toolbar. Otherwise, go to the Pools page, select the pool you created in the previous exercise, and click the Create Orders button.
  2. Enter the order information:
    • Purpose: Production
    • Instrument Model: MinION
    • Container Model: FLO-MIN107
    • Sequencing Parameters: Sequencing Run
    • Partitions: 2
  3. Click Save.
  4. Go to the Outstanding Sequencing Orders page and select the Oxford Nanopore tab. The order you just created should show up here.

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5. Creating runs from scratch

MISO supports runs from Illumina, PacBio, Oxford Nanopore, and other sequencers, so the terms used for instrument runs and associated libraries are intentionally different from those used by the vendor. A MISO Run represents a sequencing run where the sequencer has been loaded and sequencing has begun. The Run Scanner can be configured to detect new Illumina, PacBio, and Oxford Nanopore runs and automatically create corresponding runs in MISO, while runs for other platforms must be created manually. A Sequencing Container is the link between the library information and the instrument Run, and contains one or more lanes. Each Illumina lane, PacBio SMRT cell, or Oxford Nanopore flow cell is loaded with exactly one Pool. Runs and Containers can be associated as soon as the Run and Container have both been created.

5.1 Create a Container

  1. On the Sequencing Containers page, select the Oxford Nanopore platform and click the Add … button near the top left of the table. The text on this button will vary depending on the platform
  2. Select MinION from the dialog.
  3. Select FLO-MIN107 from the dialog. Only active (non-archived) models are available.

  4. Enter the name of your project as the serial number. Record the serial number in your worksheet.

  5. Select a Pore Version.

  6. Select a Received Date. The Returned Date is the date that the flow cell was returned to be recycled. This should be left blank for now, and may be filled out later.

  7. Click Save in the upper right corner of the page.

5.2 Create a Run

  1. On the Sequencing Runs page, select the Oxford Nanopore platform and click the Add Oxford Nanopore Run button near the top left corner of the table.
  2. Select MN16303 (MinION) from the sequencers list. Only active (non-retired) sequencers are available.
  3. Enter a unique and memorable Alias for your run. Record the alias in your worksheet.
  4. Select Sequencing Parameters Sequencing Run

  5. In the Run Path field, enter path as the file path to the sequencer output.

  6. Enter a MinKNOW version.

  7. Enter a Protocol version.

  8. Select Status Running. Note that if MISO does not automatically detect runs from this sequencer, all status updates will have to be entered manually.
  9. Enter a date into Start Date.
  10. Click Save in the upper right corner of the page.
  11. In the Flow Cell section, click the Add Flow Cell button.
  12. Enter the flow cell serial number of the container you created in exercise 5.1 and click the Add button.

5.3 Adding pools to runs

The Run (representing an instrument run) is associated with Pools using a Sequencing Container.

  1. On the Edit Run page, scroll down to the Flow Cells section.
  2. Select the only flow cell (Number 1).
  3. Click Assign Pool from the toolbar.
  4. Choose Outstanding Orders (Matched Chemistry) to see the Pools which have sequencing orders matching the run parameters.
  5. Click the pool that you created in exercise 4.2.

Now check on the sequencing order.

  1. Click on the Outstanding Sequencing Orders page and verify that the Remaining column now shows 1 for the pool you added to the run.
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