Process tagged sequences

Multiplexing based on the name of the sequence is of course only possible if proper sequence names could be assigned from the sequencing process. With many of the new high-throughput technologies, this is not possible.

However, there is a need for being able to input several different samples to the same sequencing run, so multiplexing is still relevant - it just has to be based on another way of identifying the sequences. A method has been proposed to tag the sequences with a unique identifier during the preparation of the sample for sequencing [Meyer et al., 2007].

With this technique, each sequence will have a sample-specific tag - a special sequence of nucleotides before and after the sequence of interest. This principle is shown in figure 23.14 (please refer to [Meyer et al., 2007] for more detailed information).

Image barcode_concept_web
Figure 23.14: Tagging the target sequence. Figure from [Meyer et al., 2007].

The sample-specific tag - also called the barcode - can then be used to distinguish between the different samples when analyzing the sequence data. This post-processing of the sequencing data has been made easy by the multiplexing functionality of the CLC Genomics Workbench which simply divides the data into separate groups prior to analysis. Note that there is also an example using Illumina data here.

Before processing the data, you need to import it as described in Import high-throughput sequencing data.

The first step is to separate the imported sequence list into sublists based on the barcode of the sequences:

        Toolbox | NGS Core Tools (Image ngsfolder) | Multiplexing (Image multiplex_group) | Process Tagged Sequences (Image multiplex)

This opens a dialog where you can add the sequences you wish to sort. You can also add sequence lists.

When you click Next, you will be able to specify the details of how the de-multiplexing 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 23.15.

Image add_barcode_element
Figure 23.15: Defining an element of the barcode system.

At the top of the dialog, you can choose which kind 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. For the example from figure 23.14, the dialog should include a linker for the SrfI site, a barcode, a sequence, a barcode (now reversed) and finally a linker again as shown in figure 23.16.

Image process_tagged_sequences_step2
Figure 23.16: Processing the tags as shown in the example of figure 23.14.

If you have paired data, the dialog shown in figure 23.16 will be displayed twice - one for each part of the pair.

Clicking Next will display a dialog as shown in figure 23.17.

Image process_tagged_sequences_step3
Figure 23.17: Specifying the barcodes as shown in the example of figure 23.14.

The barcodes can be entered manually by clicking the Add (Image add) button. You can edit the barcodes and the names by clicking the cells in the table. The name is used for naming the results.

In addition to adding barcodes manually, you can also Import (Image import) barcode definitions from an Excel or CSV file. The input format consists of two columns: the first contains the barcode sequence, the second contains the name of the barcode. An acceptable csv format file would contain columns of information that looks like:


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

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).

Click Next to specify the output options. First, you can choose to create a list of the reads that could not be grouped. Second, you can create a summary report showing how many reads were found for each barcode (see figure 23.18).

Image process_tagged_sequences_report
Figure 23.18: An example of a report showing the number of reads in each group.

There is also an option to create subfolders for each sequence list. This can be handy when the results need to be processed in batch mode.

A new sequence list will be generated for each barcode containing all the sequences where this barcode is identified. Both the linker and barcode sequences are removed from each of the sequences in the list, so that only the target sequence remains. This means that you can continue the analysis by doing trimming or mapping. Note that you have to perform separate mappings for each sequence list.