What Does ‘Sample’ Mean?

2. Example Amplicon Project Design and Analysis : 2.6 Other Issues of Special Interest : 2.6.1 What Does ‘Sample’ Mean?

2.6.1

What Does ‘Sample’ Mean?

One of the major decisions to make is what the term ‘Sample’ means to you within the context of your Project. The software recognizes a ‘Sample’ as a generic grouping unit of data. It is at its essence, merely a label with some optional annotation. It is up to you to decide how to best group your experimental data into Samples.

A typical Project might use a Sample to represent DNA from a distinct source, such as a tube of genomic DNA from a particular subject. Another Project might have a more specific definition of Sample and might split out different classes of DNA from a single individual and call them separate Samples such as control and experimental Samples or pre-treatment and post-treatment research Samples. Yet another project might define Samples as distinct replicates of a DNA source to allow for statistical comparison between them.

You are free to get more granular with Sample definitions (such as assigning each amplicon for an individual to its own Sample), but you should keep in mind that you can only view a single Sample-Reference Sequence alignment/difference plot at a time. You can examine cross-Sample Variant frequency statistics in the main ‘Variant’ tab, but reads from different Samples cannot be viewed in the same alignment. You can, however, navigate from Sample to Sample for a particular Reference Sequence.

There is a limit on the granularity of your Sample definitions. The fundamental unit of computation is the individual Read Data Set. If you intend to divide an individual Read Data Set into multiple Samples, it must be feasible for the software to assign the individual reads from that Set to Samples. If MIDs are not used, this Sample assignment will be performed based solely on the primer content of the Amplicons being measured for the Samples. If MIDs are used, then a read’s primer content will first be used to determine the Amplicon it represents, and then that Amplicon’s association with a Multiplexer, within the read’s Read Data Set of origin, will be used to assign the read to the appropriate Sample. It is important to remember that for a given Read Data Set, each Amplicon may be associated with at most one Sample, unless MIDs are used. With MIDs, a Multiplexer can associate an Amplicon with multiple Samples within a Read Data Set, but each Amplicon may still be associated with at most one Multiplexer.