Editing BioMicroCenter:Multiplex (section)

== MULTIPLEX BARCODING ==

Multiplexing methods allow for running multiple samples in a single Illumina lane. There are multiple strategies for multiplexing Illumina samples but they can generally be broken down into two classes: 5' barcodes and 3' barcodes. 

[[IMAGE:BioMicroCenter_Barcode.png|1200px]]

The main advantage of 5' barcodes is the fact that they are read as the first part of read 1, eliminating the need for a separate barcode reprime. A disadvantage is that sample prep will require two different primers (both halves of the Y-shaped adapter). Also, because the cluster-calling algorithms require a balanced base composition over the first few nucleotides, you must use a large number of barcodes in each run (16+). In addition, because the barcodes are present during the ligation reaction, care must be taken to prevent them from introducing biases in library construction. <br><br> 3' Barcodes are more flexible as they are added during the PCR step, can have a number of different compositions, and can be run in any number. They do require a separate barcode read to sequence, which can add extra cost to the flowcell.