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walkthrough
Original Walkthrough
Table of Contents
  1. Logging In
  2. Projects
  3. Incoming Samples
  4. Propagating Samples
  5. Using Boxes
  6. Libraries
  7. Library Aliquots, Pools, and Sequencing Orders
  8. Runs and Sequencing Containers
  9. Sequencers

1. Logging In

You need to log in to MISO LIMS in order to make changes to any LIMS entities. Logging in lets us record any changes you make and also allows us to set appropriate permissions.

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

  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 as they are often processed into Library Aliquots, which are then Pooled and sequenced. Projects also have Overviews, which hold information about a Project proposal. In this part of this workshop, we will create projects, set permissions, and familiarize ourselves with the project overview page.

Projects represent a sequencing effort toward a particular goal, usually led by a particular group or principal investigator. For example, the PCSI project is sequencing pancreatic tumors, references, cell lines and xenografts as part of the International Cancer Genome Consortium, and the FFPER project sequences samples from a bio-bank with a number of different preparations and treatments in order to determine the impact of each on data quality.

In this workshop, you will make your own project where you will create samples, libraries and other entities.

Most pages have a "Quick Help" tab at the top right under the Save button where you can find basic information about the current page.

2.1 Creating a new project

  1. After logging in, click the Projects link under Preparation in the menu on the left side of the screen.
  2. Select the Add Project button at the top right corner.

The Create Project page will display with a number of fields that you can fill out.

  1. Ignore Project ID and Name, since they are set by MISO once the Project is saved.
  2. Enter a unique Alias. The alias is a name, chosen by us, that is associated with a project. The aliases must be a unique and contain only letters or numbers. It cannot contain spaces or punctuation. e.g. DoIt4Science (be creative!)
  3. Enter a Short Name for your project. The short name should be 2-5 letters in all CAPS and related to the project alias. This short name will be used to automatically generate sample and library names. e.g. short name: DI4S
  4. In the Description field, enter MISO training workshop [Date]
  5. Choose the amount of Progress that your project has accomplished:
  • Unknown : I was told to enter this data and I does what they tells me.
  • Active : We are receiving/have received samples and are actively working on this project.
  • Inactive : Project is not yet done, but waiting for external collaborators, REB approval, papers to be published, etc.
  • Cancelled : Project was scrapped because we did not get funding/samples/REB approval.
  • Proposed : Project Initiation form has been received and is awaiting go-ahead by Genomics leadership.
  • Pending : We are waiting for the project to start.
  • Approved : Project has been approved by Genomics leadership.
  1. 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.
  2. In the Permissions section, select your name from the Owner drop-down.
  3. Make sure that Allow all internal users access? is selected.
Create Project DI4S
  1. Click the Save button at the upper right.

Upon save, you will be taken to the Edit Project page, where you can see the project you just created. Notice that the Project ID and Name now have values. The Project ID will be an integer, and the Name will begin with PRO. These are specific to this project and used by MISO to track the project internally.

2.1.1 Add a Study

In order to make sure MISO is SRA-compliant, you must add a Study to your Project. Studies are legacy objects that are not really used in the current system, but need to be added anyway.

  1. Click on the Studies section to expand the section.
  2. Hover over the Options menu at the top right of the Studies table, and click Add new Study.
  3. Much like creating a Project, enter:
  4. Alias (letters and numbers only): this can have any name, but make sure it is recognizable as belonging to your Project.
  5. Description: any free-text description
  6. Select a Study Type from the drop-down menu : Unless you are certain of the sequencing type, select Other.
  7. Click the Save button at the upper right.

2.2 Projects List

  1. Click again on the Projects link under Preparation in the menu on the left side of the screen. You will see a list of all of the projects you have access to in MISO.
  2. Find the project you just created in the list. You can sort any column in the table by clicking the column header. You can also search for your project by any of the displayed columns and the table will filter as you type.
  3. Click on the link in the Project Name, Short Name or Alias (they all go to the same page).

2.3 Edit Project page

The Edit Project page is used for viewing basic information about a project as well as all of the entities associated with that project, such as the samples, libraries, pools, and runs.

Once you create different entities for your project like Samples, Libraries, Library Aliquots, Pools and Runs, you can view and search them on this page. Each section can be expanded by clicking on its name or the arrow next to it. The tables under each section can be sorted by the column header or searched using the field in the upper right. The Options menu above the search bar contains actions you can perform on the entities in the project.

2.3.1 Add a project overview

Project Overviews specify the origin of a specific set of samples that are part of the project. For example, if you are receiving some samples from a clinician and other samples from a hospital, you can enter the expected numbers of samples from each and indicate how many of them passed quality control and what stage of preparation has been completed. Project overviews are optional and only used to indicate overall progress on the My Projects page.

  1. Click Add Overview at the right side under the Project Information.
  2. In the pop-up, enter a principal investigator (You Yourname!)
  3. In No. proposed samples, enter 5.
  4. Click the Add Overview button.

The Edit Project page will reload and now there will be a table with your name under the Project Information section. You can fill in other values if you wish, or leave them blank for now. You can also select the checkboxes below the table to show that certain stages have been completed. Remember to press the Save button after you are finished editing.

2.3.2 Add Project Files

In the Project Files section, you can add attach any necessary documents to the project (e.g. Project Information form, REB).

  1. Click the Project Files section on the Edit Project page.
  2. Find a document or image that is appropriate for your study

Now that your project has been created, continue to make your first samples.

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3. Incoming samples

A Sample contains information about the material upon which the sequencing experiments are to be based. Samples can be used in any number of sequencing Experiments in the form of a Library that is often processed further into pooled Library ALiquots.

Every received Sample must have an Identity. The Identity corresponds to the individual or organism with whom the sample originated, i.e. the donor. MISO requires you to assign an external name, which is usually an identifier from another institution like a Donor ID.

When material is received for sequencing, it can be in many different forms, called Sample Classes in MISO. Here are the classes of Sample that can be received:

  • Cell line
  • Tumour tissue (Primary or Metastatic)
  • Reference tissue
  • Xenograft tissue
  • gDNA (untreated or whole genome amplified)
  • cDNA
  • whole RNA

Depending on which Sample Class is chosen, more or less fields appear on the Create Sample page.

In this workshop, we will create six Samples with four different Identities in the Project you created in the last session.

3.1 Entering a single Sample

There are two ways of entering Samples into MISO: Single and Bulk. We will start by entering a single Sample for reference tissue from the Identity ID1.

  1. On the left hand menu under Tracking, click Samples.
  2. Click the Add Sample button on the right hand side. There are two tabs across the top. Ensure that Single is selected.
  3. In the Sample Information section, enter or select the following:
  4. Project: Select the project you created in the last exercise.
  5. Alias: leave blank. This will be auto-generated based on other information in this form.
  6. Description: Reference 1.
  7. Date of Receipt: Select a date
  8. Scientific Name: Homo sapiens.
  9. Sample Type: select GENOMIC from the drop down.
  10. QC Status: select Ready from the drop down.
  11. In the Identity section:
  12. External names : 1. Click Find or Create Identity. 1. In the pop-up window, enter external name: project short name _ID1 (e.g., PROJ_ID2). This is the name given to the donor by the external institute that the tissue came from. 1. Click Validate External Name(s). 1. From the dropdown, select First Receipt. 1. Click Select.
  13. Sex: Select any item from the dropdown.
  14. In the Details section, select the Sample Class Reference Tissue.
  15. In the Tissue section, select or enter the following to create a reference Sample.
  16. Tissue Origin: Ly (Lymphocyte)
  17. Tissue Type: R (Reference or non-tumour, non-diseased tissue sample)
  18. Tissue Material: Select any from the drop-down.
  19. External Institute Identifier: BioBankID 1. This is the Biobank ID or Tube ID. It may also be left blank.
  20. Lab: Select BioBank (University Health Network) from the drop-down.
  21. Times Received: 1
  22. Tube Number: 1
  23. At the upper right hand side, click Save.

Upon saving, a number of fields will be filled in, including the Alias. The Tissue Alias will be in the form PROJ_0001_Ly_R_nn_1-1: (Project Short Name)_(Individual ID)_(Tissue Origin)_(Tissue Type)_(Passage number)_(Times Received)_(Tube Number). Passage number is only required for Xenografts and Cell lines. For more information about Sample nomenclature, see Sample Nomenclature.

3.1.1 Enter a matrix tube barcode

After saving the Sample, you will be able to enter the barcode for the tube.

  1. On the Edit Sample page for the sample you just created, click the arrow next to the blue ID box at the top right hand corner.
  2. Select Assign New Barcode from the menu.
  3. Use the hand-scanner or type a barcode into the pop-up. For this exercise, enter your Project Short Name and _R1. e.g. PROJ_R1. We will use this barcode later in the Box section.
  4. Click Save on the pop-up.

The page will re-load with the 2D barcode at the top right.

3.2 Automatically created Samples

  1. Click the My Projects tab at the top and select your project from the list.
  2. Open the Samples section on the Edit Project page to see your newly created samples.

(You can also find your samples by searching on the Samples page or by using the widget on the MISO front page. At the moment, searching on the Samples page is quite slow but you are welcome to try using that page to view your samples. The widget is fast but does not show enough information for the following exercises.)

You only created a single Sample but at least two are in this list: the Reference tissue as well as the Identity. The Identity sample was automatically created because you provided an External name that had not been previously used in this Project, and has a name in the format (Project short name)_(Individual number), e.g. PROJ_0001. Other types of Samples are created automatically depending on how you propagate them through to libraries. Some of them will be addressed in the following tutorials.

3.3 Bulk create Samples

Next, we will create four more Samples using the much faster bulk method. The four samples will be the Primary Tumour Tissue for individuals 1-5.

  1. On the left hand menu under Tracking, click Samples.
  2. Click the Add Sample button on the right hand side. There are two tabs across the top. Ensure that Bulk is selected.
  3. Select project dropdown: select your project.
  4. Select class dropdown: Primary Tumor Tissue.
  5. Number of samples text box: 4.
  6. Click Make Table.

A table will appear with the requested number of samples in table format. We will fill in the first row and use the quick-fill option to fill in the rest of the table.

Enter the following values into the first row only.

  1. Sample Alias: leave blank. Again, this will be automatically generated from the rest of the table.
  2. Select or enter the following fields:
  3. Description: Primary.
  4. Date of Receipt: select a date
  5. Sample Type: select GENOMIC from the drop-down.
  6. Sex: select any item from the drop-down.
  7. Tissue Origin: select Br (Breast) from the drop-down.
  8. Tissue Type: select P (Primary Tumour) from the drop-down.
  9. Times Received: 1
  10. Tube Number: 1
  11. Lab: select BioBank (University Health Network) from the drop-down.
  12. Ext. Inst. Identifier: BioBankID
  13. Material: Select any item from the drop down.
  14. QC Status: Select Ready from the drop down.

Now we will fill in the rest of the table. Like in Excel, you can fill down a column by double-clicking the square at the lower right hand side of a selected cell. You can also click and drag to only fill in a certain number of cells.

  1. Click the Sample Type cell in the first row. A blue square will appear at the lower right hand side. Double click it to fill in the rest of the table with the word "Primary".
  2. Fill in the columns in the same way for: Sex, Tissue Origin, Tissue Type, Times Received, Tube Number, and Material.

Some fields cannot be filled down, so enter each of those separately.

  1. Matrix Barcode: you would normally use a hand-scanner or copy and paste a list of barcodes from a spreadsheet. In this case, enter the project short name followed by P and a number. The fill down functionality does not auto-increment, so these need to be typed. For example:PROJ_P1, PROJ_P2, PROJ_P3, PROJ_P4
  2. External Name: replace PROJ with your own project name.
  • PROJ_ID1
  • PROJ_ID2
  • PROJ_ID3
  • PROJ_ID4

  1. Click Look up Identities (above the top left corner of the table).

  2. Select First Receipt from the dropdown menu in each cell of the Identity Alias column. Since these are all first receipt, you may fill them down after selecting it in the top row. If the identities already existed, you would have to select them individually.

  3. Ext. Inst. Identifier: add a number to each row starting from 2, i.e.. BioBankID 2, BioBankID 3, BioBankID 4, BioBankID 5.

  4. Description:

    Copying and pasting from Excel and Word is supported.

    Go to http://pastebin.com/uQXhafqJ and copy the list of descriptions by selecting it with your mouse, right clicking and selecting Copy. Then click on the first cell in the top row of Description and press Ctrl+V on your keyboard to paste.

  5. Click Save at the upper right hand corner.

If everything is correct, the Alias will be auto-generated for each row and the samples will be saved. If you navigate back to your Edit Project page, there should be nine Samples:

  • 4 Identity Samples
  • 1 Reference Sample
  • 4 Primary Samples

Notice also that because you used the same External Name, ending in ID1, for two samples, reference and primary, they have the same Identity.

3.4 Receiving Stock DNA/RNA

The process for receiving Stock DNA is very similar to receiving tissue. Every stock derives from a Tissue, which originated from an Identity. MISO will create the tissue for you when you enter a Stock. These samples are known as ghost samples, which do not exist at OICR but are in MISO for sample tracking purposes.

In this section, we will 'receive' a single Stock DNA tube from individual 2 reference tumour.

  1. On the left hand menu under Tracking, click Samples.
  2. Click the Add Sample button on the right hand side. There are two tabs across the top. Ensure that Single is selected.
  3. In the Sample Information section, enter or select the following:
  4. Project: Select the project you created in the last exercise.
  5. Alias: leave blank. This will be auto-generated based on other information in this form.
  6. Description: Stock 1.
  7. Date of receipt: select a date.
  8. Scientific Name: Homo sapiens.
  9. Sample Type: select GENOMIC from the drop-down.
  10. QC Status: select Ready from the drop-down.
  11. Volume (µl): 300
  12. In the Identity section, enter the
  13. External name : project name _ID2.
  14. Sex: Select any item from the dropdown.
  15. In the Details section, select the Sample Class: gDNA (stock).
  16. In the Tissue section, select or enter the following to create a reference Sample.
  17. Tissue Class: Reference Tissue
  18. Tissue Origin: nn (Unknown)
  19. Tissue Type: R (Reference or non-tumour, non-diseased tissue sample)
  20. Tissue Material: Select any from the drop-down.
  21. External Institute Identifier: BioBankID 6.
  22. Lab: BioBank (University Health Network).
  23. Times Received: 1
  24. Tube Number: 1
  25. At the upper right hand side, click Save.

Stock aliases are created from their tissue alias by appending _D_S# or _R_S#. For example, the first DNA stock that derives from a tissue PROJ_0002_Ly_R_nn_1-1 has the name PROJ_0002_Ly_R_nn_1-1_D_S1.

After saving, go back to your project page and look at the samples that were automatically created. Although you received Stock DNA, it has created a Tissue for you as well.

  1. Click on the Tissue with the alias similar to PROJ_0002_nn_R_nn_1-1. It should have a Sample Description that says only "Tissue".

At the top, you will see a grey section with the warning: "This entity does not exist except for sample tracking purposes!". This message means that the Tissue does not exist in a freezer at OICR. Eventually these ghost samples will be hidden from the MISO interface.

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4. Propagating Samples

Samples in MISO exist for each step in the tissue preparation: from identity, to tissue, optionally though tissue preparation, to stock, to aliquot. At each step, the possible options are limited based on the established workflows. Group IDs may be assigned at any time and are copied when propagating. Different QC information is available at each step. For instance, STR status is attached to the stock.

For the tissue samples created previously (by bulk and single entry), we will create stocks for library preparation.

4.1 Bulk Propagate Samples

For three of the tissues you created in the previous section, create a stock.

  1. On the Samples page, enter your project name in the search box.
  2. Check the boxes for the tissue samples (not the received stock). They will have names that end in two hyphenated numbers, like PROJ_0001_Ly_R_nn_1-1:
  • PROJ_0001_Br_P_nn_1-1
  • PROJ_0001_Ly_R_nn_1-1
  • PROJ_0002_Br_P_nn_1-1
  1. From the Bulk actions dropdown at the bottom, select Propagate (sample) selected.
  2. A new dropdown will appear. Select gDNA (stock) and click Go.
  3. Fill out the table:
  • Description: Free text description. In this case, use "Stock (Tissue Type)(Individual)". (e.g. Stock P2 for PROJ_0002_Br_P_nn_1-1)
  • Matrix Barcode: (Project short name)_(Tissue Type)(Individual)_St,
    • PROJ_P1_St
    • PROJ_R1_St
    • PROJ_P2_St
  • Vol.: 300
  1. Click Save.

Upon successful save, a green status will show at the top that says "Saved 3 items". The Sample Alias will have been filled in with aliases that end in D_S1 (for each first DNA stock of that tissue).

4.2 Bulk Editing

Samples can be edited in bulk. Assume that we have done some quality control and wish to update the QC status of the samples.

In this case we will update several fields of 4 stock samples. We will use the stocks we entered in the previous step as well as the reference stock entered in part 3 of this tutorial.

  1. On the Samples page, enter your project name in the search box.
  2. Check the boxes for the stock samples (propagated and received). These are the samples that end in D_S1:
  • PROJ_0001_Br_P_nn_1-1_D_S1
  • PROJ_0001_Ly_R_nn_1-1_D_S1
  • PROJ_0002_Br_P_nn_1-1_D_S1
  • PROJ_0002_Ly_R_nn_1-1_D_S1
  1. From the Bulk actions dropdown at the bottom, select Update selected and click Go.
  2. Change the QC Status column to Ready for all rows.
  3. Enter the missing Matrix Barcode, e.g. PROJ_R2_St
  4. Click Save.

Upon successful save, a green status will show at the top that says "Saved 4 items.".

4.3 Creating Aliquots

Propagate again from the 4 gDNA (stock) samples to gDNA (aliquot).

  1. On the Samples page, enter your project name in the search box.
  2. Check the boxes for the stock samples. They will have names that end in D_S1:
  • PROJ_0001_Br_P_nn_1-1_D_S1
  • PROJ_0001_Ly_R_nn_1-1_D_S1
  • PROJ_0002_Br_P_nn_1-1_D_S1
  • PROJ_0002_Ly_R_nn_1-1_D_S1
  1. From the Bulk actions dropdown at the bottom, select Propagate (sample) selected.
  2. A new dropdown will appear. Select gDNA (aliquot) and click Go.
  3. Fill out the table:
  • Sample Alias: Skip this field. It will be automatically filled in upon save.
  • Description: Free text description. In this case, use "Aliquot (Tissue Type)(Individual)". (e.g. Aliquot P2 for PROJ_0002_Br_P_nn_1-1_D_S1)
  • Matrix Barcode: (Project short name)_(Tissue Type)(Individual)_Al
    • PROJ_P1_Al
    • PROJ_R1_Al
    • PROJ_P2_Al
    • PROJ_R2_Al
  • Purpose: Select Library
  1. Click Save.

Upon successful save, a green status will show at the top that says "Saved 4 items". The Sample Alias will have been filled in with aliases that end in D_1 (for each first aliquot of that stock).

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5. Using Boxes

Boxes hold samples, libraries, and pools. They do not track reagents or primers. Boxes are separated into uses for different kinds of storage (e.g., tissue samples versus extracted DNA), but there is no check that items in a box match. Each box also has a size that includes the physical dimensions of the box as well as whether the box is compatible with the VisionMate scanner. Every position in the box is identified by a standard row letter + column number format (e.g., C05).

The goal is to create a box for the samples created in the previous step.

  1. From the navigation panel, choose Boxes and then Add Box.
  2. On the Create Box page, enter the information:
  3. Alias: A short name for the box. Enter your short name followed by _OUTBOX (e.g. PROJ_OUTBOX)
  4. Description: a human description of the box purpose “gDNA ready for library prep for ”.
  5. Use: The contents of the box. Select DNA.
  6. Size: Select 8 x 12 scannable.
  7. Click Save.

Upon clicking save, a graphic of the box will appear. You can now load your samples into the box. All types of samples can be entered in the box, including the tissues, stocks and aliquots you made in the previous steps.

  1. Click on a position in the displayed Contents grid.
  2. Enter a matrix barcode into the box on the right and click Lookup. Normally a hand scanner would be used. For this exercise, use this handy reference list:
  • PROJ_P1_St
  • PROJ_P2_St
  • PROJ_R1_St
  • PROJ_R2_St
  • PROJ_P1_Al
  • PROJ_P2_Al
  • PROJ_R1_Al
  • PROJ_R2_Al
  1. Click Update Position.
  • ⚠ The Save button does not work for individual positions, only for Box Information.
  1. Repeat for all the samples listed above.

The table below the box diagram shows the position and information for the currently selected sample. If you would like to see all of the samples in the table, click the List all Box Contents button at the top right of the table.

In the lab, it is possible to use Options > Scan Box to use the plate scanner and update all positions at once, but that will not be covered in this tutorial.

Using Boxes

Boxes can be found either from the Sample or Library page or the Boxes page.

  1. On the Sample page, enter your project name.
  2. Click on the alias for individual 2 primary stock, i.e. PROJ_0002_Br_P_nn_1-1_D_S1.
  3. The Box and position is listed under Location near the top of the Sample Information section. Click on the link to go to the Box (e.g. PROJ_OUTBOX, A03).

Boxes can be used to store Samples, Libraries and Pools and one box can store all three types.

Removing and trashing tubes

When you have a position selected, you can also either remove the tube from the box (setting its location to "Unknown") or trash the tube, meaning it has been used up. Trashing the tube sets the volume of the sample to 0 and marks it as "emptied".

  1. From the list below the box, find the location of the PROJ_0002_Br_P_nn_1-1_D_S1 sample.
  2. Open the sample page in a new tab by clicking on the Sample link (Right click on the link and select "Open in new tab").
  3. Go back to the Box page if necessary. Select the sample you just opened in the Box and click Trash Tube. Click "OK" in the pop-up.
  4. Go to the other tab with the Edit Sample page and click the browser refresh button. The Location field will show as blank, Volume will be set to 0.0, and the Emptied box will be ticked.

Moving samples around in boxes

An item can only exist in one box. If assigned to a new box, it will disappear from the original.

  1. In the current tab, go to the Boxes page, find the TUTORIAL box. This box was previously created by the MISO developers for the tutorial..
  2. Choose an empty position and enter your barcode ending in PROJ_P1_Al (e.g., PROJ_P1_Al).
  3. Click Lookup and Update Position.
  4. Go back to the first tab with your PROJ_OUTBOX Box and refresh the browser, or return to the Boxes page and find your project outbox. The sample was removed from the outbox.

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6. Libraries

A library is made from one sample for a single target platform and has a specific design associated with it that decides the selection and strategies used to make the library. A library may also have indices (primers) and QC information.

MISO stores two important pieces of information about how a library was generated: the selection (e.g., PCR, cDNA) and the strategy (e.g., WGS, WXS, amplicon). A design captures both a selection and strategy and the list of allowed designs is limited based on the sample type (e.g., a cDNA sample can only have SM, WT, or MR library designs and these lock the selection and strategy type accordingly).

6.1 Bulk propagate aliquots into libraries

In this section, you will use the aliquots you created already and create libraries.

  1. On the Samples page, enter your project name into the search box.
  2. Check the gDNA aliquot samples to turn into libraries. These samples are the ones that end in _D_1:
  • PROJ_0001_Br_P_nn_1-1_D_1
  • PROJ_0001_Ly_R_nn_1-1_D_1
  • PROJ_0002_Br_P_nn_1-1_D_1
  • PROJ_0002_Ly_R_nn_1-1_D_1
  1. From the Bulk actions drop down, select Propagate (library) selected.
  2. A table will appear. Enter the library information:
  • Library Alias: The sample alias up to the tissue type (R or P), library type, insert size, library design (e.g., DI4S_0001_Br_P_PE_318_WG). For more information about Library nomenclature, see Library Nomenclature. ⚠ This alias does not automatically fill in yet, so it must be entered:
    • PROJ_0001_Br_P_PE_300_EX
    • PROJ_0001_Ly_R_PE_300_EX
    • PROJ_0002_Br_P_PE_300_EX
    • PROJ_0002_Ly_R_PE_300_EX
  • Description: Library (Tissue type)(individual), e.g. Library P1
  • Matrix Barcode: As before, usually this would be scanned by the hand scanner. In this tutorial, enter matrix barcodes in the form (Short name)_(Tissue type)(Individual)_Li, e.g. PROJ_P1_Li.
  • Platform: Illumina
  • Type: Paired End
  • Selection: PCR
  • Strategy: AMPLICON
  • Index Kit: Nextera Dual Index
  • Index 1 and Index 2: Select any combination of indices you wish. Select different indices for each library. Selecting the same index for two different libraries will make you unable to pool those two libraries together later.
  • Volume: 100
  • Kit: KAPA Hyper Prep
  1. Choose Save.

Note that for dual-index libraries, only the first index needs to be specified. The second is optional.

6.1 Quality control

There are three way to indicate library quality in MISO: 1) Enter quantitative QC values under the Library QC section; 2) The overall pre-sequencing quality flag QC passed; and 3) The post-sequencing quality control Low quality library.

6.2.1 Library QC

After measuring the insert size or concentration, this information can be entered into each library. There is no bulk entry for this information yet, it must be entered for each library.

  1. From the Libraries page, find the PROJ_0001_Br_P_PE_300_EX library using the search box and click the sample link.
  2. On the right side of the QCs heading, select Options → Add Library QC.
  3. Enter the information in the row:
  4. QC Date: Select a date.
  5. Method: Choose a QC instrument.
  6. Results: Enter the measurement.
  7. Insert Size: Enter the measured insert size.
  8. Click Add.

6.2.2 QC passed

QC Passed is a simple pass/fail flag for a library to decide if it is good enough for sequencing. If not measured, this can be left as "Unknown".

  1. From the Libraries page, find the PROJ_0001_Br_P_PE_300_EX library using the search box and click the sample link.
  2. Change QC passed from Unknown to True.
  3. Click Save.

6.2.3 Low Quality Sequencing

Libraries can be marked as having low sequencing quality, which will be shown after the Run exercises.

6.3 Boxes

Libraries can also be placed in boxes.

  1. From the Boxes page, find the project-specific box you created before, e.g. PROJ_OUTBOX.
  2. Select an unused position and enter a library matrix barcode PROJ_P1_Li, e.g. PROJ_P1_Li.
  3. Click Lookup and then Update position.
  4. Repeat for the remaining libraries:
  • PROJ_R1_Li
  • PROJ_P2_Li
  • PROJ_R2_Li

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7. Library Aliquots, Pools, and Sequencing Orders

A library cannot be directly loaded into a lane in a sequencing container (flowcell/SMRTcell) in MISO. A library aliquot must be made and then many aliquots (or just one) can be mixed into a pool for sequencing.

Sequencing 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.

7.1 Bulk creating Library Aliquots

Library Aliquots can be made in bulk from libraries.

In this exercise, we will create 4 library aliquots from the libraries we made previously.

  1. On the Libraries page, check all the libraries just created.
  • PROJ_0001_Br_P_PE_300_EX
  • PROJ_0001_Ly_R_PE_300_EX
  • PROJ_0002_Br_P_PE_300_EX
  • PROJ_0002_Ly_R_PE_300_EX
  1. Click the Make aliquots button at the top of the table. In the dialog that appears, click Create.
  2. Enter the concentrations of the aliquots (use any number you wish).
  3. Click Save.

7.2 Pools

Pools are the last step for libraries before sequencing and represent the entity that is loaded onto the flowcell lane or SMRTcell. A "pool" can have one or more library aliquots in it.

Here we will use all of the library aliquots we added previously to make a single pool of 4.

  1. On the Pools page, under the Illumina tab, click the Add button at the top of the table.
  2. Enter the Pool Information:
  3. Alias: A short description of the pool contents. Enter the project name followed by _POOL (e.g. PROJ_POOL)
  4. Description: A longer free-text description of the pool.
  5. Platform Type: Leave Illumina selected.
  6. Desired Concentration: Enter a concentration.
  7. Creation Date: Select a date.
  8. Ready to Run: Whether or not the pool is ready for sequencing. This flag is used together with the Sequencing Order to show the pool is ready to be sequenced. Tick this box.
  9. Volume: Enter a pool volume.
  10. Click Save. The pool must be saved before you can add library aliquots to it.
  11. In the Available Library Aliquots section, use the search box to find the aliquots created previously. Select each aliquot and click the Add button at the top of the table to add it to the pool.

7.3 Creating a Sequencing Order

Sequencing orders are created on the pool to be sequenced, and include the quantity of sequencing required (counted in lanes/SMRT cells), and the sequencing chemistry required (on Illumina).

  1. On the Edit Pool page for the pool you just created, scroll down to the Requested Sequencing Orders section and click the "Create" button.
  2. Fill in the order details:
  • Instrument Model: Select the instrument for sequencing. Illumina - Illumina HiSeq 2500
  • Sequencing Parameters: Select v4 2×126 chemistry.
  • Partitions: the number of lanes/cells that should run for this pool. Enter 2
  1. Click Add. The sequencing order will now be visible in the Requested Sequencing Orders section.

7.3.1 Checking for outstanding orders

The Sequencing Orders page is used to decide what needs to be sequenced.

  1. From the navigation menu, choose Sequencing Orders - Outstanding.
  2. Verify that the pool you just created is listed in the Illumina tab.

Columns on this page will disappear if there are no entries (e.g., the Failed column will not be shown if there are no failed runs). When enough lanes have been sequenced, the row will disappear from the Unfulfilled tab, but remain in the All tab. Lanes currently being sequenced will be marked as in-progress and remain on the Unfulfilled tab until the run transitions to Completed.

A pool can have many sequencing orders. Sequencing orders for the same platform and chemistry are summed when displayed on this page.

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8. Runs and Sequencing Containers

MISO supports runs from both Illumina and PacBio sequencers (and others that are no longer used at OICR), so the terms used for instrument runs and associated libraries are intentionally different from those used by the vendor. Every time a sequencer is loaded and sequencing begins, a Run is created. Runs are picked up automatically from the instrument. A Sequencing Container is the link between the library information and the instrument Run and contains one or more lanes. Each lane (e.g. Illumina lane, PacBio SMRT cell) in the container is loaded with exactly one Pool. Runs and Containers can be associated as soon as the sequencer has started.

8.1 Viewing Run statistics

About five minutes after an instrument begins sequencing, MISO will detect it and create a Run. As sequencing continues, MISO will pull back information about the quality of the run similar to the on-instrument applications like SAV. This includes statistics like percent pass filter, the percent of bases with Qscores over 30, and cluster density.

  1. From the Runs page, find the run assigned to you for this tutorial.

Under the InterOp Metrics section, you will see a loading animation. After a few seconds to load, this page will show information from the sequencer about the run progress. However, this function does not work reliably under load (as when ~20 people look at runs at the same time during this tutorial), so it may not load. This functionality will be redesigned in the near future.

8.2 Adding a sequencing container to a run

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

  1. On the Run assigned to you, scroll down to the Containers section.
  2. Search for the pool you previously created and double click to assign it to the lane. If you did not tick the Ready to Run box when creating the Pool, you may need to untick this option next to the search field.
  3. Click the Select Study button on the Lane to accept the default Study.
  4. If the flowcell is being stored, enter the location.
  5. Click Save.

The validation field is used only by cBot; leave it blank.

Now check on the sequencing order.

  1. Click on the Sequencing Orders - Outstanding page and verify that the Remaining column now shows 1 for the pool.

8.3 Low Quality Sequencing

Not every library realises its full potential. After sequencing, specific libraries can be flagged as having low sequencing quality. The "Low Quality Sequencing" indicator causes any pool containing this library to be flagged, so that it can be checked before it is sequenced again.

  1. From the Libraries page, find the PROJ_0002_Ly_R_PE_300_EX library and click the link.
  2. Check Low Quality Sequencing.
  3. Click Save.

Now if the Pool containing that Library is added to a Sequencing Container, it will be flagged red.

  1. Go back to your Run page.
  2. Find your pool using the search box. It will be flagged red.

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9. Sequencers

Each sequencer has a service record.

  1. Select Sequencers.
  2. Choose an individual sequencer.
  3. Use the blue arrow to expand the Service Records section.
  4. From Options, choose Add Sequencer Record.
  5. Fill in the form as follows:
  • Title: a short description of the work (e.g., Remove gremlin from sequencer)
  • Description: a long description of the work (e.g., A gremlin was found outside of the terrible 80s movie. It had developed a taste for polymerase.)
  • Serviced By: your name
  • Service Date: the current date
  1. Click Save.

After saving, it is also possible to attach files to the record. Look under the Attachments heading.

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