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Dealing with Cross-Contamination in Fixed Barcode Protocols

Contributors

AvatarMehmet Tekman AvatarAlex Ostrovsky

Questions

Objectives

Requirements

last_modification Last modification: Mar 17, 2020

Fixed Barcode Protocols and Multiplexing

The main contending questions are:

  1. How large is each batch?

Speaker Notes How many slots in a batch that we need to barcode for

  1. How many batches on a plate?

  1. Should each batch use the same barcodes?

  1. What constraints are there on the plate?

Speaker Notes A technician always has to balance quality against cost, and this is illustrated in the following examples:


Example Setup

.pull-left[

]

.pull-right[

Speaker Notes Here we use N as an extra base just for example purposes, but you do sometimes see this in other barcodes. 2 batches at a time, only half


Example 1: Single Plate with a Single Lane

.bottom-info-box[ Available Barcodes

AAA ACC AGG TTT TAA TCC
ATT CCC CAA TGG NAA NCC
CGG CTT GGG NGG NTT ANN
GAA GCC GTT CNN GNN TNN

]

.image-75[slide36]

.pull-left[

– .pull-right[

Speaker Notes


Example 2: Single Plate with 2 Batches

.bottom-info-box[ Available Barcodes

AAA ACC AGG TTT TAA TCC
ATT CCC CAA TGG NAA NCC
CGG CTT GGG NGG NTT ANN
GAA GCC GTT CNN GNN TNN

]

.image-75[slide38]

Speaker Notes Here we use all barcodes since these batches will be sequenced at the same time Let’s look at one final example to see why using all our barcodes on a plate might not be optimal.


Example 3: Single Plate with 2 Batches, only 1 active

.bottom-info-box[ Available Barcodes

AAA ACC AGG TTT TAA TCC
ATT CCC CAA TGG NAA NCC
CGG CTT GGG NGG NTT ANN
GAA GCC GTT CNN GNN TNN

]

.image-75[slide40]

Speaker Notes All barcodes used, why leave one lane empty?


Example 3: Single Plate with 2 Batches, only 1 active

.bottom-info-box[ Available Barcodes

AAA ACC AGG TTT TAA TCC
ATT CCC CAA TGG NAA NCC
CGG CTT GGG NGG NTT ANN
GAA GCC GTT CNN GNN TNN

]

.image-75[slide40]

Speaker Notes If we see any reads in the Plate which contain barcodes {TTT,TAA,TCC, etc} then we know that some contamination has occurred because there should be no cells there. One reason is that the second lane was not completely cleaned before being used.


Example 4: 2 Plates with 2 Batches, only 1 active (alternate)

.bottom-info-box[ Available Barcodes

AAA ACC AGG TTT TAA TCC
ATT CCC CAA TGG NAA NCC
CGG CTT GGG NGG NTT ANN
GAA GCC GTT CNN GNN TNN

]

.image-50[slide44]

Speaker Notes


Example 5: 1 Plate with 2 Batches, both active

.bottom-info-box[ Available Barcodes

AAA ACC AGG TTT TAA TCC
ATT CCC CAA TGG NAA NCC
CGG CTT GGG NGG NTT ANN
GAA GCC GTT CNN GNN TNN

]

.image-100[slide46]

.pull-left[

]

.pull-right[

A1. This setup is actually the same as example 4, but with the two plates merged. Here we can check for cross-contamination in each lane by measuring the real cell labels against the false barcodes. If in lane 1, we detect a significant number of reads with cell barcodes of TAA or ANN, we can assume that some cross-contamination has occurred since we should not be able to detect these barcodes in that lane. The converse is also true of lane 2.

A2. We have the benefit of detecting cross-contamination with the same advantages as example 4, but with the cost advantage of sequencing two batches at the same time.


Summary


Key Points

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.