Specifying Multiplex Identifiers (MIDs)

1.6.3

When the “Use Multiplex Filtering” checkbox is selected, special controls for MID filtering are displayed in the bottom half of the ”Set GS Read Data Attributes” window (Figure 9). Multiplex Identifiers (MIDs) allow you to design an experiment whereby multiple libraries are prepared using distinct MID tags and sequenced together, on the same PTP Device. On the GS FLX+ System, multiple libraries can be sequenced together in the same region of a PTP Device. (Use of MIDs can greatly improve the workflow and cost effectiveness of your experiment.)

Figure 9: Set GS Read Data Attributes dialog with the MID Scheme options expanded

The MID controls on this window allow you to filter the reads in the selected Read Data files for inclusion in the Assembly project. Note, this filtering operation does not produce a different Assembly for each specified MID. Rather, the reads of the SFF files are scanned for the presence of MIDs, and the reads containing the selected MIDs are made eligible for assembly in the project. The selected MID filtering criteria are associated with the sff files when the OK button is clicked (Figure 9). Any reads from these file(s) without the specified MIDs will be ignored. To produce independent Assemblies for different MIDs (or sets of MIDs), you must create independent Assembly projects for each of the desired MID subsets of the data.

MID filtering information is contained in ‘MID schemes’. Available MID schemes may, in turn, be found in MID configuration files. The default MID configuration file supplied with the GS De Novo Assembler software is MIDConfig.parse; the MID schemes provided in this file are ‘No Multiplexing’, ‘GSMIDs’ for use of the original Genome Sequencer MID sets, and ‘RLMIDs’ for the Rapid Library Preparation Kit MIDs. An alternate configuration file can be specified by pressing the “browse for MID configuration file” button to the right of the “MID Config File” textbox (Figure 9). The “Select MID Config file” dialog (Figure 10) will then appear and can be used to navigate to the desired location. Choose the file to be used as the configuration file for the sff file(s) being imported and click the Select button. The MID schemes specified in the file selected will then populate the Scheme dropdown. Press the “Reset” button to set MIDConfig.parse (the default MID configuration file) as the configuration file to be used in the file import. ‘Custom Multiplexing’ can be selected if custom MIDs were used.

Figure 10: Select MID Config file browser

By default, no MID/Multiplexing scheme is associated with Read Data files. To use MIDs, first, specify the MID Config File, then select the desired MID Scheme (Figure 9) using the Scheme drop down menu. When an MID scheme is selected, a table of the MIDs present in that scheme is displayed, as shown in Figure 11. Use the checkboxes on the left to select or deselect the MIDs to be included in the assembly. Shift-clicking selects or deselects all MIDs in the scheme at once. The names, sequences and error limits of the MIDs selected will be used to filter the reads (from the Read Data sets selected in the top part of the window). The result is that only the reads that contain the selected MIDs, within the number of errors specified in the scheme, will be included in the project.

For more information about MIDs, see Section 4.7.

Figure 11: Set GS Read Data Attributes dialog with the RLMIDs Scheme active

Once the MID scheme has been specified, click the OK button to add the selected data files (with MID filtration information) to the list of GS read data files (see Figure 9).

In certain circumstances, you may want to apply a different MID selection to different Read Data sets, within the same project. For example, you may have multiple libraries of the same organism, made with different MIDs (or with/without MIDs), that you want to assemble together. In this case, select the Read Data set(s) that need to be filtered with any given MID (or combination of MIDs), and click OK to add the MID-filtered data file(s) to the project. Back on the GS Reads sub-tab of the GS De Novo Assembler window (with the GS Reads sub-tab active) (Figure 5), click the Add button (

) again, and select other Read Data sets, to be filtered with different MIDs.

Incorrect MIDs may be assigned: In some cases, the MID(s) you select may be within the allowed number of errors of another MID, resulting in incorrect MID assignment. If this occurs, you should use the sfffile command (with the -s option) to split the reads with different MIDs into different files. The sffffile command, described in section 3.1, considers all MIDs in the MIDConfig.parse file, and will assign the MID(s) more accurately. The default MIDs and RLMIDs present in the MIDConfig.parse differ from each other by more than the allowed number of errors so this should occur very infrequently