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Plates, Batches, and Barcodes

Contributors

AvatarWendi Bacon AvatarMehmet Tekman AvatarAlex Ostrovsky

Questions

Objectives

Requirements

last_modification Last modification: Apr 12, 2021

Sorting Plates

.image-100[slide5]

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

Speaker Notes


Sorting Plates

.image-80[slide6]

Speaker Notes


Setting up Plates

.image-75[slide7]

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

Speaker Notes


Setting up Plates

.image-50[slide7]

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

Speaker Notes


Setting up Plates

.image-50[slide7]

.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.]

Speaker Notes


Setting up Plates

.image-90[slide7]

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

Speaker Notes


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]

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

Speaker Notes


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]

.center[Does this look ok?]

Speaker Notes

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

Speaker Notes


What about sequencing lanes?

.image-75[slide7]

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

Speaker Notes


Distinguishing cells in a plate

.image-60[slide11]

Speaker Notes


What are Cell Barcodes?

.image-100[Add Barcodes]

Speaker Notes


What are Cell Barcodes?

.image-100[Barcodes in Plate]

Speaker Notes


Questions about Cell Barcodes

.image-50[slide14]

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

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

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

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

Speaker Notes

.footnote[

  1. 96 unique barcodes per lane
  2. 96 x 10 = 960 unique barcodes per plate ]

Speaker Notes


Questions about Cell Barcodes

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

.pull-left[

A single lane?

Barcodes Result
\(4^2 = 16\) No, 2 bases is not enough
\(4^3 = 64\) No, 3 bases is not enough
\(4^4 = 256\) Yes, 4 bases is enough to cover 96 barcodes (and more!)

]

Speaker Notes

– .pull-right[

10 plates in a single lane?

Barcodes Result
\(4^4 = 256\) No, 4 bases is not enough
\(4^5 = 1024\) Yes, 5 bases is enough to cover 960 barcodes (just barely!)

]

Speaker Notes


Barcode Safeguarding

.pull-left[

.pull-right[ Edit distance = 1bp

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

Speaker Notes


Guarding against Sequencing Errors

.pull-left[

]

Speaker Notes

\[4^{5-1} = 512\]

Speaker Notes


Edit Distance : General Principle

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

Speaker Notes

– .pull-right[ Number of Barcodes :

\[4^{N-(E-1)}\]

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

]

Speaker Notes


How many available barcodes are there?

.pull-left[

]

Speaker Notes

– .pull-right[

Speaker Notes


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

Speaker Notes


Cell Barcodes: Summary

.pull-left[

]

Speaker Notes

.pull-right[#### Need to balance: | | | | |-|-|-| | Edit Distance | vs. | Barcode Length | | Number of Barcodes | vs. | Plate and Lane Size | ] Speaker Notes - 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 ] Speaker Notes - 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 ] Speaker Notes - Cell barcodes indicate what cell a transcript came from - They reduce sequencing errors when spaced appropriately

Key Points

curriculum Do you want to extend your knowledge?

Follow one of our recommended follow-up trainings: - [Transcriptomics](/training-material/topics/transcriptomics) - Single-cell quality control with scater: [tutorial hands-on](/training-material/topics/transcriptomics/tutorials/scrna-scater-qc/tutorial.html)

Thank you!

This material is the result of a collaborative work. Thanks to the Galaxy Training Network and all the contributors! Galaxy Training Network This material is licensed under the Creative Commons Attribution 4.0 International License.