Demultiplexing single reads

To demultiplex your data, please go to:

        Toolbox | Prepare Sequencing Data (Image sequencedataprep_closed_16_n_p) | Demultiplex Reads (Image multiplex)

This opens a dialog where you can specify the sequences to process (figure 25.17).

Image demultiplex_single_reads_step2
Figure 25.17: Specify the sequences to demultiplex.

When you click on the button labeled Next, you can then specify the details of how the demultiplexing should be performed. At the bottom of the dialog, there are three buttons, which are used to Add, Edit, and Delete the elements that describe how the barcode is embedded in the sequences.

First, click Add to define the first element. This will bring up the dialog shown in 25.18.

Image add_barcode_element
Figure 25.18: Defining an element of the barcode system.

At the top of the dialog, you can choose the type of element you wish to define:

The concept when adding elements is that you add e.g. a linker, a barcode, and a sequence in the desired sequential order to describe the structure of each sequencing read. You can of course edit and delete elements by selecting them and clicking the buttons below. In the example shown in figure 25.16, the dialog should include a linker, a barcode, and a sequence as shown in figure 25.19.

Image demultiplex_single_reads_step3
Figure 25.19: Processing the tags as shown in the example of figure 25.16.

Click Next to set the barcode options (figure 25.20). At the top, you can choose to search on both strands for the barcodes; this is needed for some 454 protocols where the MID is located at either end of the read. You can also choose to allow mismatches: only one per barcode will be allowed, regardless of whether the barcodes are on the same read, or distributed on both R1 and R2.

Note: If a sequence is one mismatch away from two barcodes, it will not be assigned to any of them.

In the table below, the Preview column will show a preview of the results by running through the first 10,000 reads.

Image demultiplex_reads_barcode_preview
Figure 25.20: A preview of the results.

If you would like to change the name of the sequence(s), this can be done at this step by double-clicking on the specific name that you would like to change. This is shown in figure 25.21.

Image demultiplex_remame
Figure 25.21: The name of the sequence can be renamed by double-clicking on the existing name.