Running Demultiplex Reads

Demultiplex Reads is in the Toolbox at:

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

Select one or more sequence lists as input. When you click on the button labeled Next, you can then specify the details of how the demultiplexing should be performed.

Defining the read structure

The 'Define tags' wizard contains three buttons, which are used to Add, Edit, and Delete the tags that describe the structure of the reads and how the barcode is embedded in the sequences.

Click on Add to define the first tag. This will bring up the 'Define tag' dialog (figure 28.18).

Image add_barcode_element
Figure 28.18: Defining a tag.

At the top of the 'Define tag' dialog, you can choose the type of tag you wish to define:

The concept when adding tags 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 edit and delete elements by selecting them and clicking the buttons below. For the example shown in figure 28.17, the structure should include a linker, a barcode, and a sequence as shown in figure 28.19.

Image demultiplex_single_reads_step3
Figure 28.19: Processing the tags as shown in the example of figure 28.17.

If the input contains paired reads, there are two wizards for defining the read structure: 'Define tags' for R1 and 'Define tags (mate)' for R2. If the two reads in the read pair have different barcodes such as illustrated in figure 28.20, the read structure would look like this:

R1 : -Linker1-Barcode1-Sequence

R2 : -Linker2-Barcode2-Sequence

Image barcode_concept_paired_reads
Figure 28.20: Paired reads with linkers and barcodes originating from two different samples.

Defining the barcodes

Click Next to set the barcode options (figure 28.21).

Image demultiplex_reads_barcode_preview
Figure 28.21: Defining the barcodes.

At the top, you can choose to search on both strands for the barcodes.

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 that if a sequence is one mismatch away from two barcodes, it will not be assigned to either of them.

Barcodes can be provided in several ways:

A preview of results (figure 28.21) based on 10,000 reads is presented. With a single input, the preview is based on the first 10,000 reads. When multiple inputs are provided, the 10,000 reads are take from across the inputs, with the contribution from each input being proportional to the relative size of that input.

If you would like to change the name of the barcode(s), this can be done by double-clicking on the specific name that you would like to change, see figure 28.23.

Image demultiplex_remame
Figure 28.23: The sequence can be renamed by double-clicking on the name.