_________________________________________________________
JUMP TO TOPIC:
- Introduction
- Standard Illumina sequencing
- Barcode Index Reads vs Inline Barcodes
- Mixing Barcode Index Reads with Inline Barcodes
- PacBio Sequencing
- 10x Genomics sequencing (Single cell sequencing applications)
- Pools
__________________________________________________________
Introduction
If you've multiplexed your samples, you'll need to enter your barcodes into iLab. This page tells you how to do so!
You'll notice that there's a column "Pools" in our new Illumina/PacBio iLab service request forms. Please read about Pools below before using them.
__________________________________________________________
Standard Illumina sequencing
Under construction!
Note: If you're doing 10x sequencing of any type, please ignore this section and instead jump to 10x Genomics sequencing below.
__________________________________________________________
PacBio sequencing
Coming soon!
__________________________________________________________
10x Genomics sequencing
Under construction!
If you've requested 10x Genomics sequencing of any kind, this section is for you!
Instead of entering in barcode nucleotide sequences, you'll be entering the 10x barcode names (e.g., "SI-TT-E7"). This applies to all 10x Genomics applications (e.g., scRNA-seq, scATAC-seq, etc).
10x Genomics offers both dual and single indexes (we encourage the use of dual indexes if possible). Below are examples of how to enter each into iLab. NOTE: This is the current interface, but we will likely be modifying it in the near future to be simpler. Stay tuned!
iLab 10x dual index example
iLab 10x single index example
__________________________________________________________
Pools
Note: Apologies, we use "barcodes" and "indexes" interchangably below.
You may have noticed a new column in the barcodes tables: "Pool # (if submitting >1 pool)". What is that, and how do you use it?
Explicit Pools
The "Pools" column was introduced mainly to allow customers to submit one iLab service request that can be applied to multiple separate lanes / cells of sequencing.
In the past, users would often be forced to submit multiple iLab service requests to accomplish the same thing, i.e., one iLab service request per lane or cell of sequencing. For example:
iLab Service Request 1:
iLab Service Request 2:
With the addition of the "Pools" column, you can now submit a single iLab service request with both pools and two lanes of sequencing, like so:
The one and only iLab Service Request:
Because you the customer are explicitly "naming" your pools, we refer to these as "Explicit Pools".
Implicit Pools
Additionally, GC3F supports the notion of "Implicit Pools". Why are implicit pools helpful?
Sometimes customers mix either:
- Single and Dual indexed samples into the same lane, or
- Barcodes of different lengths into the same lane.
The tools we use to demultiplex your sequencing data don't really like this arrangment; they require that you have only one type (i.e., either single indexed, or dual indexed, but not both), and that the barcodes are all the same length.
To get around this, we automatically detect when you have a mix, and then perform multiple demultiplexings as needed. They'll be lableled "pool-1", "pool-2", "pool-3", and so forth.
For the most part, you don't need to worry much about how this works. You only need to be aware of this behavior, and that if you upload a mix of barcodes as described above, that multiple pools will be published to you.
In this first example below, the customer has uploaded a mix of dual and single indexed pools, all to be sequenced on one lane. The resulting pools will be "pool-1" and "pool-2".
Notice that the user did not enter anything in the "Pool" column, yet they will still receive "pool-1" and "pool-2". Hence, we refer to these as "Implicit Pools".
In this second example below, the customer has uploaded barcodes with two different lengths, all to be sequenced on one lane. The resulting pools will be "pool-1" and "pool-2".
