«Worfklows» has been renamed «protocol lists»

This section is outdated. We have renamed "workflows" (meaning an applied sequence of protocols) to "protocol lists". LabID now uses the term "workflow" to refer to data analysis workflows.

Hands-on: Sample creation and lineage

• 25 min
• Easy

Overview

The goal of this tutorial is to understand the basics of the LabID Biomaterial & Specimen sections i.e. what is the difference between specimen & samples and why there are different sample sub-types. We will see how specimen & samples are connected with parent-child relationships to form a lineage graph that precisely reflects the transformations executed at the bench.

• step 1: Creating a Specimen
• step 2: Deriving samples into a lineage
• step 3: Using the lineage viewer
• step 4: Adding protocols
• step 5: Constraints on parents

Walkthrough

Step 1. Creating a Specimen

In LabID, a Specimen represents the living source of samples. It can be a patient, a mouse, a fly line, bacteria, viruses, a cell line... There should always be a specimen at the origin of a sampl e.g. a blood sample comes from a patient; and we encourage you to maintain the list of your biological sources as specimen.

Let's create our first Fish specimen.

• Navigate to the Specimen menu and click the Fish sub-menu.

• Bring up the new Fish creation form using the New fish
  available at the usual place (top left of the list page).

• Fill in all the properties you like with at least:

  • Name : Trainee XX Fish (use your own trainee number in place of the XX)
  • Organism : Zebrafish (Danio rerio)

• Click Save Item

We now have a Fish specimen representing a particular fish line i.e. not a single individual. These fishes are maintained in their tank and happily reproduce.

Step 2. Deriving samples into a lineage

Let's imagine that you are a developmental biologist using this particular fish line (that we just created) to study embryonic development. Regularly, you collect fish embryos at a particular developmental stage to study gene expression by sequencing approaches.

We now learn how to reflect this in LabID.

• Navigate to the Biomaterials menu and click the Sample sub-menu.

• Bring up the new Sample creation form using the New sample available at the usual place (top left of the list page).

• Fill in all the properties you like with at least:

  • Name : TXX_embryos_09-25-22_E25 (use your own trainee number in place of the XX)
  • Organism : Zebrafish (Danio rerio)
  • Project : Tea Project
  • Material type : whole organism
  • Under the Sample Lineage section:
    • Derives from : Select Specimen
    • Specimen : Select Fish and then your Trainee XX Fish
  A sample deriving from a Fish specimen

  

  A new sample with a fish specimen set as parent (green)

• Click Save Item button at the top right and then immediately exit the edit mode with the Exit edit

This sample represents the collection of fish embryos at embryonic stage E25 that were collected on 09-25-22. Next, we use some of it to extract polyA-RNA and prepare a library for Illumina sequencing.

• Click on the Create Child button at the top right of the detail page and select the Sequencinglibrary

  Deriving a sequencing library from a sample

  

• When creating a child sample, the following properties are pre-filled automatically (red arrows):

  • The Organism and Project are automatically propagated from the parent
  • The parent sample is automatically set in the lineage section
  Automated property propagation upon child creation

  

  LabID propagate properties and set the parent (red arrows)

• Fill in missing properties as visible in above picture, importantly:

  • Name: TXX_embryos_09-25-22_E25_RNA-lib with XX your trainee number

  • The new Sequencing Library Preparation section holds properties that become mandatory upon data submission. Although these are not mandatory, you should always provide these at creation.

    • This library is not multiplexed so we use NA for the mandatory Barcode field.

• Click Save Item button at the top right and then immediately exit the edit mode with the Exit edit

Learn more...

You can learn more about the different sample & specimen sections/properties in the documentation

Step 3. Using the lineage viewer

We now have a chain of three steps with:

Specimen (fish line) -> Sample (an embryo collection) -> Sequencing Library (RNA ready for sequencing)

This is a simple lineage but sometimes they are quite complex with samples being pooled (e.g. when one sample does not contain enough material) or samples giving rise to many children (e.g. different treatment or protocols to extract DNA, RNA and proteins from the same sample/cell).

To help you navigate between the different layers and have an ensemble view on sample relationships, LabID offers a lineage viewer available on every sample detail page.

• Scroll down to the Lineage Graph section. The embedded graph displays the lineage of the object on focus (circled in black), here the sequencing library. The graph displays the routes up to the sample origin (here the Specimen). The graph display can be customised using the different controls. Item types can be hidden using the (red arrow) and the graph can be rearranged, re-layered ... using the top left controls (boxed in purple). Explore these options in a few clicks

  The lineage viewer offers many options to display and navigate lineages

  

  The graph can be displayed in full-page mode using the top rigth button (boxed in red). Objects have different colors and shapes reflecting their type (orange squarres for Specimen, green circle for samples)

• Put the viewer in full screen mode ( at top-right) and enter embr in the search box to find all lineage items whose name contains embr (case insensitive). Matching nodes can easily be identified :

  • they have a dashed red or black border (black for the node under focus)
  • a search result summary together with controls (boxed in red below) allowing to navigate across results are displayed directly below the search box
    • note the red cross that erases the search
  • use the left/right arrow to navigate between matching nodes
  • Click on the Matched Nodes (boxed in purple) to bring the list of matched nodes as a table. Take a few seconds to explore the Action columns.
  Searching the lineage

  

  The Matched Nodes button (boxed in purple) brings up the matched nodes as a table.

• Cancel the search with the and right-click on the central node to bring up the contextual menu.

  • The menu offers more control to customize the graph display: (un)grouping nodes, hiding children or parents. Many of these become really useful as the graph gets more complex.

  • Note how the node border turned red, indicating that the node is selected. Clicking around on the white background unselects the node

  Navigating the lineage

  

  Right click on a node to bring the contextual menu

• Click on the open detail menu option. What happened?

Step 4. Adding protocols

Let's now see a very important property of samples: Workflow. A Workflow in LabID represents an ordered list of protocols_i.e._it is made of 1 or more protocols. It is ordered because it reflects the order in which you executed the protocols to obtain the sample under focus.

• Create a protocol of type Growth named Zebrafish Embryo Production By In Vitro Fertilization.

  • Summary: paste this URL https://zfin.atlassian.net/wiki/spaces/prot/pages/352157844/Embryo+Production+By+In+Vitro+Fertilization
  • Description: copy content from here
  • Save with & exit

• Create a protocol of type Extraction named Poly(A) mRNA isolation.

  • Summary: paste embryos were homogenised in TRI Reagent and total RNA extraction was performed according to the manufacturer's instructions followed by NEBNext Poly(A) mRNA Magnetic Isolation Module
  • Description: also paste the text above
  • Save with & exit

• Create a protocol of type Library Preparation named Illumina TruSeq for mRNA-Seq

  • Summary: paste standard Illumina TruSeq protocol.
  • Description: paste mRNA-Seq libraries (from polyA+ RNA) were created according to the standard Illumina TruSeq protocol.
  • Save with & exit

• Navigate to the Biomaterial menu and click the Sequencing Library sub-menu; open your TXX_embryos_09-25-22_E25_RNA-lib

• Edit the library using Edit, click in the "...or add an individual protocol" and select the Illumina TruSeq ... library construction protocol. Finally, save the page Save

  Adding a protocol

  

• In the lineage editor, right-click on the intermediate sample (should be named TXX_embryos_09-25-22_E25) and click open details to navigate to the sample page. Let's assume that this sample represents the RNA-seq extracted from collected fish embryos. We therefore want to link 2 protocols as a workflow.

  Warning

  in a sample lineage, each sample expects to have a workflow with a minimum of one protocol. The workflow attached to a particular sample reflects the steps executed from the parent sample only. In our example:

  TraineeXX Fish => TXX_embryos_09-25-22_E25 => TXX_embryos_09-25-22_E25_RNA-lib

  • The protocol(s) attached to TXX_embryos_09-25-22_E25 should only reflect how this sample was obtained from the specimen TraineeXX Fish.
  • Similarly, the protocol(s) attached to TXX_embryos_09-25-22_E25_RNA-lib should only reflect how this library was prepared from the sample TXX_embryos_09-25-22_E25_i.e._the protocols on how TXX_embryos_09-25-22_E25 was obtained from the specimen TraineeXX Fish should not be repeated.

• Edit the sample using Edit :

  • click in the "...or add an individual protocol" and select the Zebrafish Embryo Production By In Vitro... growth protocol
  • click again in the "Add an individual protocol" and select the Poly(A) mRNA isolation extraction protocol

• Finally, save the page Save .

  A workflow with two protocols

  

  An unlimited number of protocols can be added in the workflow (red arrow), steps can be easily deleted or re-ordered using the buttons boxed in red.

Step 5. Constraints on parents

We'll finish up this hands-on by checking a few constraints imposed on the lineage.

• Navigate to the Biomaterial menu and click the Sequencing Library sub-menu; open your T10_embryos_09-25-22_E25_RNA-lib

• Edit the library using Edit and set the organism to Fruit Fly.

• Save the page Save .

  What happens?

  LabID does not let you save and complain about incompatible organisms between the parent sample and this sample.

  Handson

  

• Set the organism back to Zebrafish

Handling mixed organisms samples?

This situation is so rare that LabID does not yet have an advanced way to deal with it. Still a few tricks can help:

• There is a special Mixed Organisms option in the organism list. You can specify the list of organisms in the annotations using the Applicable Organisms (might not be available on the training platform)

• LabID checks organism compatibility between the sample you are editing and its parents.

Assuming you have a "Mixed Organisms" sample C resulting from merging sample A (e.g. fly) and sample B (mouse).

You could create your connection A -> C & A -> B with A, B & C saved as "Mixed Organisms".

Then edit the organism of A & B to fly & mouse respectively. The only drawback is that you cannot edit sample C anymore.

Lastly, you should not mix up a Mixed Organisms situation (very rare) with a sample with spike-ins from another organisms (for quantitation and/or normalization purpose). Spike-ins should be described using the spike-ins section and the sample's organism should remain that without considering the spike-ins.

Congrats! You now have completed this exercise. You can now efficiently create your sample lineages reflecting your work.