2. Example Amplicon Project Design and Analysis
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2.6 Other Issues of Special Interest
: 2.6.4 When should MIDs be used?
2.6.4
When should MIDs be used?
The GS Amplicon Variant Analyzer (AVA) software provides a number of mechanisms for demultiplexing reads, allowing multiple Amplicons from the same or different Samples to be sequenced simultaneously within a PTP region. The simplest demultiplexing method, which has been available since the first release of the AVA software, exploits the template-specific primer regions of the Adaptors, used to prepare the library, to identify the Amplicons. The Amplicon library preparation method places these sequences at the beginning of the reads, just after the sequencing key (which is part of Primers A and B). If an experiment calls for measuring multiple distinct Amplicons from the same Sample, those Amplicons may be mixed together in a PTP region, and the Project can be set up such that reads of the various Amplicons are associated with the appropriate Sample by virtue of their known template-specific primer sequences.
But with the large number of sequencing reads that can be obtained in a single PicoTiterPlate Device region in the GS FLX+ System, the situation may be common whereby a single region would produce a vast excess of reads compared to what is necessary for any given Amplicon library (Sample). If the experiment includes multiple Samples, the obvious economical solution would be to load multiple Samples in each region such that each Sample will be covered at the appropriate depth, in a single sequencing Run. If different Amplicons were to be sequenced in each of the Samples, the standard demultiplexing method using the template-specific Primer 1 and Primer 2 sequences would be sufficient to assign each read to the proper Sample.
However, experiments where the same Amplicon, or set of Amplicons, are to be sequenced in several Samples are probably much more common. In such cases, one would face the restriction that an Amplicon can be associated with no more than one Sample, within a Read Data Set (equivalent to a PicoTiterPlate Device region, unless the data has been manipulated using the SFF Tools).
MIDs are short, recognizable sequence tags that can be added to the design of the Adaptors used for library preparation. Multiple Amplicon libraries (the Project’s Samples) can be prepared that include the same Amplicon target sequences (with the same template-specific primers), each labeled with different MID tags. The MID sequences provide extra context that is specific to each library and that, in concert with the template-specific primers, allows flexible demultiplexing options and, specifically enables the sequencing of the same Amplicon across multiple Samples within the same PTP region.
