Plates, Batches, and Barcodes

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# Plates, Batches, and Barcodes

<div class="contributors-line">
		<a href="/training-material/hall-of-fame/nomadscientist/" class="contributor-badge contributor-nomadscientist"><img src="/training-material/assets/images/orcid.png" alt="orcid logo" width="36" height="36"/><img src="https://avatars.githubusercontent.com/nomadscientist?s=36" alt="Wendi Bacon avatar" width="36" class="avatar" />
    Wendi Bacon</a><a href="/training-material/hall-of-fame/mtekman/" class="contributor-badge contributor-mtekman"><img src="https://avatars.githubusercontent.com/mtekman?s=36" alt="Mehmet Tekman avatar" width="36" class="avatar" />
    Mehmet Tekman</a><a href="/training-material/hall-of-fame/astrovsky01/" class="contributor-badge contributor-astrovsky01"><img src="/training-material/assets/images/orcid.png" alt="orcid logo" width="36" height="36"/><img src="https://avatars.githubusercontent.com/astrovsky01?s=36" alt="Alex Ostrovsky avatar" width="36" class="avatar" />
    Alex Ostrovsky</a>
	</div>

</div>

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## Requirements

Before diving into this slide deck, we recommend you to have a look at:

- [Introduction to Galaxy Analyses](/training-material/topics/introduction)

---

### <i class="far fa-question-circle" aria-hidden="true"></i><span class="visually-hidden">question</span> Questions

- What is a batch when it comes to single cells?

- What’s the difference between a barcode and an index?

- What is batch effect and how do you prevent it?

- What is a lane?

---

### <i class="fas fa-bullseye" aria-hidden="true"></i><span class="visually-hidden">objectives</span> Objectives

- How to set up plates to prevent batch effect

- Proper naming conventions when dealing with scRNA-seq samples

---

### Sorting Plates

.image-100[![slide5](../../images/scrna-intro/wab96wellplate.svg)]

.left[Plates are *N x M* arrays of wells that cells are sorted, to then be individually amplified and sequenced.]

???
- Sorting plates are 2 dimensional arrays of wells that individual cells are placed into.
- Each cell in each well is tagged with a unique barcode and then primed to then be amplified and sequenced
- The method in which this is performed can yield significant pros and cons as we will see.

---

### Sorting Plates

.image-80[![slide6](../../images/scrna-intro/wabexampleplates.png)]

???
- Here we see 3 different sorting strategies with different advantages for same treatment sample.
- In the top image: We see all wells are filled, to maximise the number of cells on a plate
- In the middle image: We see only the inner wells are filled, to maintain a protective border during library preparation
- In the bottom image: We see a striping pattern, which mitigates contamination of material from adjoining wells

---

### Setting up Plates

.image-75[![slide7](../../images/scrna-intro/wabbatchproblems.png)]

.center[What is the problem with this plate setup?]

???
- However, if we consider different treatment samples, then we must consider different challenges.
- What is the advantage of the top example, i.e. Several different treatments on one plate?
- What is the advantage of the bottom example, i.e. One treatment on one plate?

---

### Setting up Plates

.image-50[![slide7](../../images/scrna-intro/wabbatchproblems.png)]

.center[Batch effect (plate vs plate) cannot be separated from treatment effect in either scenario.]

???
- Trick question!
- In both examples, the treatment effect cannot be separated from any technical effect from the plate itself.
- For example, the biological effect of the yellow cells might just be the technical confounding effect from Batch 1.
- What can we do instead to reduce the effect of these technical confounding effects across treatments?

---
### Setting up Plates

.image-50[![slide7](../../images/scrna-intro/wabbalancedbatches.png)]

.left[Either of these are better set-ups. Mixing columns is good, but not required. Ultimately, batch effect can now be separated from variable effect.]

???
- The answer is that we can balance the treatments across our plates better.
- By ensuring the same treatment occurs in comparable amounts in different plates, we can reduce the effect that each plate would have on the treatment.

---

### Setting up Plates

.image-90[![slide7](../../images/scrna-intro/wabreplicates.png)]

.center[Can't mix samples on plates? Separate replicates evenly and process together.]

???
- If putting multiple treatments on the same plate is not an option, then having enough separated replicates will also allow for batch correction.
- Here, you can assess the variation between replicates, and thus batches, as well as between treatments.

---

### What about sequencing lanes?

.center[So now it's time to sequence our samples! How do we combine samples into sequencing lanes?]

.image-90[![slide7](../../images/scrna-intro/wabseqlane.png)]

.center[This works well, but what if you have too many samples for one lane?]

???
- So now it's time to sequence our samples! How do we combine samples into sequencing lanes?
- In many cases, it is enough to sequence 1 or 2 plates together in a single sequencing lane.
- But what if we wish to sequence 8 plates?

---

### What about sequencing lanes?

.center[So now it's time to sequence our samples! How do we combine samples into sequencing lanes?]

.image-90[![slide7](../../images/scrna-intro/wablanesbad.png)]

.center[Does this look ok?]

???
- In the example show, Treatment A would go to the first sequencing lane.
- Treatment B would go to the second sequencing lane.
- Is it okay to sequence different treatments across different sequencing lanes?

.center[No! You've turned each treatment (A & B) into a batch!]

???
- No, because you've turned Treatment A and Treatment B into separate batches.
- This gives us the same unbalanced design we saw with the sorting plates.

---

### What about sequencing lanes?

.image-75[![slide7](../../images/scrna-intro/wablanesgood.png)]

.center[This is the way to balance your batches at the lane-level.]

???
- Instead we should balance the availability of each treatment across different sequencing lanes
- This way, each treatment is sequenced at the same time as another treatment, reducing the technical effect from the sequencing on the treatment.

---

### Distinguishing cells in a plate

.image-60[![slide11](../../images/scrna-intro/wabplate.png)]

* Cells are selected from a plate by their *barcodes*
* Barcodes must be unique
  + e.g. 96 wells in a plate, need 96 barcodes to sequence them together

???
- How do we distinguish each cell in each well?
- Do we remember the physical location of that cell on that well, or do we tag it somehow?
- The answer is that each cell is given a unique barcode that distinguishes from other cells which have different cell barcodes.

---

### What are Cell Barcodes?

.image-100[![Add Barcodes](../../images/scrna-intro/scrna_pbb_barcodes_add.svg)]

* Barcodes are added to *all* transcripts of a *specific* cell
* Transcripts with different cell barcodes originate from different cells

???
- What exactly are cell barcodes?
- These are usually short nucleotide barcodes that are added to all transcripts of a specific cell
- The idea is that transcripts with different cell barcodes originate from different cells.

---

### What are Cell Barcodes?

.image-100[![Barcodes in Plate](../../images/scrna-intro/scrna_pbb_barcodes_overview.svg)]

* Many different cell barcodes are used across many different cells
* Each well in a plate contains a cell, indexed by its cell barcode

???
- Within a plate, many different barcodes are used, and each cell is indexed by the cell barcode assigned to that well.

---

### Questions about Cell Barcodes

.image-50[![slide14](../../images/scrna-intro/wabplate.png)]

.left[Assuming you sequence one 96-well plate:]

1. How many cell barcodes are needed for a single lane?

1. How many cell barcodes are needed if you combine 10 plates into a single sequencing lane?

1. What would be the minimum length of the barcodes for each of the previous questions?

???
- Consider a 12 by 8 plate with 96 wells.
- How many cell barcodes are needed if you sequence a single plate?
- How many cell barcodes are needed if you sequence 10 plates into a single sequencing lane?
- What would be the minimum length of the barcodes for each of the previous questions?

.footnote[
1. *96 unique barcodes per lane*
1. *96 x 10 = 960 unique barcodes per plate*
]

???
- For one plate you would just need 96 unique barcodes
- For 10 plates you would need 960 unique barcodes

---

### Questions about Cell Barcodes

.left[<small>3) What would be the minimum length of the barcodes for each of the previous questions?</small>]

.pull-left[

A single lane?

* 96 barcodes, 4 nucleotides for each base of a barcode

| Barcodes | Result |
|---------|------|
| $$4^2 =  16$$ | <small>No, 2 bases is not enough |
| $$4^3  = 64$$ | <small>No, 3 bases is not enough |
| $$4^4 = 256$$ | <small>Yes, 4 bases is enough to cover 96 barcodes (and more!) |

]

???
- To answer the third question, we need to make some assumptions about the barcodes.
- If we assume a barcode is made up of a chain of 4 possible nucleotides, then we can solve the problem as a power of 4.
- For a single lane with only 96 barcodes, we require our barcodes to be of at least 4 base pairs in length.

--
.pull-right[

10 plates in a single lane?

* 960 barcodes, 4 nucleotides for each base of a barcode

| Barcodes | Result |
|---------|------|
|$$4^4 = 256$$ | <small>No, 4 bases is not enough |
|$$4^5 = 1024$$ | <small>Yes, 5 bases is enough to cover 960 barcodes (just barely!) |

]

???
- For 10 lanes with 960 barcodes, we require our barcodes to be of at least 5 base pairs in length.
- However these calculations also make one serious assumption about the barcodes.

---

### Barcode Safeguarding

.pull-left[
* Is 5 nucleotides really enough to capture 960 cells?

* What could go wrong if all barcodes are separated by 1 bp, as shown?
]

.pull-right[
<small>*Edit distance = 1bp*

AAAAA AAAAC AAAAG AAAAT AAACA AAAGA AAATA ····
    CCCCC CCCCA CCCCG CCCCT CCCAC CCCGC CCCTC ····
                        ·
                        ·
                        ·
]

???
- For example, we assume that all our barcodes are at minimum separated by a single base pair.
- This is known as an edit distance of 1.
- If we assume that every barcode is separated from every other barcode by 1 base pair, then 5 nucleotides is indeed enough to delineate 960 cells.
- However if there was even just one small sequencing error of 1 base pair on any barcode, it would immediately change that erronous barcode into another real barcode.
- That is to say, we have no way of detecting sequencing errors if we use an edit distance of 1.

---

### Guarding against Sequencing Errors

.pull-left[
* Sequencing errors can change the cell barcode

* To guard against a 1 bp sequencing error, need to space barcodes by 2 bp

* How many barcodes for a cell barcode of length 5 bp (ED = 2)?
]
.pull-right[
<small>*Edit Distance = 2 bp*

AAAAA AAACC AAAGG AAATT AACCA AAGGA AATTA ····
    CCCCC CCCAA CCCGG CCCTT CCCAA CCGGC CCTTC ····
                        ·
                        ·
                        ·

]

???
- Let us try to rectify this by increasing the edit distance to 2.
- In the example barcodes shown we see barcodes of length 5, separated by 2 base pairs.
- How many unique cell barcodes can we make given these restrictions?

$$ 4^{5-1} = 512 $$

???
- The answer is half the amount that we need.

---

### Edit Distance : General Principle

.pull-left[
e.g. For barcodes of length N=3:

* Edit Distance of E=1:
.center[`AAA AAC AAG AAT ACA ACC` ...]
.center[<small>64 barcodes</small>]

* Edit Distance of E=2:
.center[`AAA ACC AGG ATT CAA CCC` ...]
.center[<small>16 barcodes</small>]

* Edit Distance of E=3:
.center[`AAA CCC GGG TTT`]
.center[<small>4 barcodes</small>]
]

???
- Let us explore this more explicitly.
- Here we will be using barcodes only of length 3, and we will explore different edit distances of 1, 2, and 3.
- These yield 64, 16, and 4 barcodes respectively.

--
.pull-right[
Number of Barcodes :

$$ 4^{N-(E-1)} $$

.center[For barcodes of length *N*,
and Edit Distance of *E*.]

]

???
- This can be summarized by the formula given, where the length of the barcodes, minus one minus the edit distance, gives the number of available barcodes.

---

### How many available barcodes are there?

.pull-left[
* Barcodes typically limited to 4 main bases {A,C,T,G}.

* Number of available barcodes depends on:

1. The length of the barcode

2. The edit distance between adjacent barcodes

* Must take sequencing errors into account

]

???
- To summarize, barcodes are typically limited to 4 nucleotides, and the number of available barcodes depends on the length and edit distance.
- This is to ensure that sequencing errors are taken into account.

--
.pull-right[
* Availability is balance between *barcode size* vs. *sequencing errors*

* Typically between 4-10 bases, range of cells that can be labelled:

$$[4^4, 4^{10}] = [256, 1048576]$$

* True range is lower due to edit distance.
]

???
- The availability of barcodes and the number of cells they can label is a design choice, where the technician must balance two opposing forces: barcode size vs sequencing errors.
- For the size, this means that for a barcode N bases long, there will 4 to the N barcodes available.
- Typically barcodes tend to span 4 to 10 bases, since longer barcodes tend to be more subjectable to sequencing errors.
- The true number of barcodes used is smaller than 4 to the N, due to the countermeasures used to space barcodes apart from one another, in order to reduce sequencing errors.

---

### Design Factors in Barcodes

Is it better to use Longer or Shorter Barcodes?

| Longer | Shorter |
  |--------|---------|
  | Offer larger range of barcodes | Offer smaller range of barcodes |
  | Prone to more sequencing errors | More robust against sequencing errors |
  | Must increase edit distance significantly | A smaller edit distance is more acceptable |
  | Can accommodate large edit distances | *Cannot* accommodate large edit distances |

???
- To weigh the pros and cons of longer and shorter barcodes, we need to take into account the likelihood of sequencing errors for a given size, and the resulting barcode address space given by the edit distance.

---

### Cell Barcodes: Summary

.pull-left[

* A single barcode sequence indexes a single cell

* Every transcript in a specific cell has the same cell barcode

* Barcodes are *designed* for smaller plate-based protocols, while for split-pool and similar techniques they are randomised.

* Barcode use is *limited* by length and read depth

]

???
- To summarize cell barcodes, we should note the following
- A single barcode sequence indexes a single cell
- Every transcript in a specific cell has the same cell barcode
- Barcodes are designed for smaller plate-based protocols, while for split-pool and similar techniques they are randomised
- Barcode use is limited by length and read depth

--
<hline>
.pull-right[#### Need to balance:

<small>

| | | |
|-|-|-|
| Edit Distance | vs. | Barcode Length |
| Number of Barcodes | vs. | Plate and Lane Size |

</small>
]

???
- The number of reads wanted per cell determines how many cells you run in a sequencing lane, which in turn tells you how many barcodes you need.

---

# Summary

.pull-left[

* Cell Barcodes are sequences attached to transcripts to indicate what cell a transcript came from

* Cell Barcodes are *designed* to the Plate/Lane setup

]

???
- From the content shown here, you have learned the following.
- Cell Barcodes are sequences attached to transcripts to indicate what cell a transcript came from
- Cell Barcodes are designed for the Plate and Lane setup.

--
.pull-right[

* Indicates what cell a transcript came from.

* Reduce sequencing errors

]

???
- Cell barcodes indicate what cell a transcript came from
- They reduce sequencing errors when spaced appropriately

---
### <i class="fas fa-key" aria-hidden="true"></i><span class="visually-hidden">keypoints</span> Key points

- Balanced batches and replicates allow bioinformatic batch correction

- A sequencing lane often contains multiple batches, and is itself a batch effect!

---

## Thank You!

This material is the result of a collaborative work. Thanks to the [Galaxy Training Network](https://training.galaxyproject.org) and all the contributors!

<div class="contributors-line">
		Authors: <a href="/training-material/hall-of-fame/nomadscientist/" class="contributor-badge contributor-nomadscientist"><img src="/training-material/assets/images/orcid.png" alt="orcid logo" width="36" height="36"/><img src="https://avatars.githubusercontent.com/nomadscientist?s=36" alt="Wendi Bacon avatar" width="36" class="avatar" />
    Wendi Bacon</a><a href="/training-material/hall-of-fame/mtekman/" class="contributor-badge contributor-mtekman"><img src="https://avatars.githubusercontent.com/mtekman?s=36" alt="Mehmet Tekman avatar" width="36" class="avatar" />
    Mehmet Tekman</a><a href="/training-material/hall-of-fame/astrovsky01/" class="contributor-badge contributor-astrovsky01"><img src="/training-material/assets/images/orcid.png" alt="orcid logo" width="36" height="36"/><img src="https://avatars.githubusercontent.com/astrovsky01?s=36" alt="Alex Ostrovsky avatar" width="36" class="avatar" />
    Alex Ostrovsky</a>
	</div>

</div>

</div>

Tutorial Content is licensed under <a rel="license" href="http://creativecommons.org/licenses/by/4.0/">Creative Commons Attribution 4.0 International License</a>.<br/>