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4. GS De Novo Assembler and GS Reference Mapper Appendices : 4.7 Multiplex Identifiers
4.7
Multiplex Identifiers
4.7.1 The Use of Multiplex Identifiers in the Data Analysis (MIDs)
4.7.2 The MIDConfig.parse File
4.7.1
The Use of Multiplex Identifiers in the Data Analysis (MIDs)
The path for any filename or data directory name can be pre-pended with a multiplexing information string, separated from the filename/directory-string by an ”@” sign. Multiplexing information strings should be used when multiple samples have been prepared using different initial “tag” sequences (such as those provided by the GS Multiplex Identifiers (MID) Kits), then mixed together for sequencing. The software uses these tag sequences to segregate the reads from each sample, by matching the initial bases of the reads to the known tag sequences used in the preparation of the libraries. If a multiplex string is given, sfffile will automatically match the 5’ bases of each read against the multiplex sequences, and will only use the reads that match the specified sequences (and reset the trim point of those reads so that the multiplex sequence is trimmed off the output read sequence).
Three examples of multiplex information strings are the following:
•
mid2@myreads.sff
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GSMIDs:mid1,mid4,mid8@/home/xxx/morereads.sff
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aattctc/1@1-3,4,6-8:D_2005_01_01_01_01_01_testuser_runImagePipe
The first example gives “myreads.sff” as the input SFF file and tells sfffile to only use reads whose initial 5’ sequence matches the “mid2” MID (as listed in the MID configuration file). The second example tells sfffile to use the file “/home/xxx/morereads.sff” and filter that file, using only the reads starting with the mid1, mid4 and mid8 sequences, as defined in the “GSMIDs” MID set. The third example tells sfffile to read regions 1, 2, 3, 4, 6, 7 and 8 of the “D_2005_...” sequencing Run, but only use the reads whose 5’ end matches the DNA sequence “AATTCTC” with 1 error or less. (Note that regions only apply to data produced by a GS FLX+ Instrument).
The format of the multiplexing information string is the following:
[setname:]mid[/#](,mid[/#]…)@
where
•
the “setname” is an optional name of an MID set found in the MID configuration file and can be given in uppercase or lowercase letters (the matching is case-insensitive)
•
each “mid” is either an MID name found in the configuration file or a DNA sequence
•
each “#” is an optional allowed number of errors
No spaces are allowed in the multiplexing information string, and no colons, slashes or ”@” signs are allowed in the MID set names or MID names.
A default MID configuration file can be found in /usr/local/rig/config/MIDConfig.parse. This file is read by sfffile and used to match MID set names and MID names with their multiplexing information. Users can edit this file to add their own MID sets (following the format and syntax described in the file), or can copy this file locally and create their own custom MID configuration file and then use the ‘-mcf filedesc’ option to specify the newly created MID configuration file.
The list of MID schemes provided in the Scheme drop down menu is read from the selected MID configuration file (by default, this is the MIDConfig.parse file). For pre-existing MID schemes, the names and sequences of the MIDs, and the number of errors allowed, cannot be changed in the Select GS Read Data window. To modify these values, or to add new user-specified MID schemes, edit the MIDConfig.parse file using any text editor. The standard location of this file is /opt/454/config/MIDConfig.parse; more generally, if the software is installed to the /install/directory folder, the configuration file will be found at /install/directory/config/MIDConfig.parse. See section 4.7.2 to view the default MIDConfig.parse file and for a description of how to edit it.
You can also create new MIDs directly in the Select GS Read Data window, by selecting the “Custom Multiplexing” option. In this case, a table will appear and new MID names, sequences, and error limits can be typed into the table (see Figure 94). However, these names, sequences and error limits will only apply to the sff files being imported.
Figure 94: GS Read Import Dialog Rapid Library MID Support
There is no formal limit to the number or length of custom MIDs that one can define in an Assembly or Mapping project. However, efficient de-multiplexing requires that the MID sequences be divergent enough that the software will be able to differentiate them from one another (within the “number of errors allowed” specified). The 454 Sequencing System software comes with 2 MID schemes, GSMIDs and RLMIDs. 14 MIDs (of which MIDs 1 to 12 match the “MID Adaptors” available in kit form) are listed for GSMIDs. 12 MIDs are available as RLMIDs.