Associating Read Data Sets with Samples

3. GS Amplicon Variant Analyzer Command Line Interface : 3.5 Creating and Computing a Project with the AVA-CLI : 3.5.9 Associating Read Data Sets with Samples

3.5.9

Associating Read Data Sets with Samples

With the Sample-Amplicon associations already made and the Read Data Sets loaded into the Project, the Samples with their associated Amplicons can now be associated to the proper Read Data Sets. This uses the “associate” command, described in section 3.5.7 where it was used to associate Samples with Amplicons (the usage statement is in section 3.4.1). In this case, the command must supply a Sample and a Read Data Set (or a Read Group).

assoc -file - << HERE_TERMINATOR

"readData"      "sample"

"DGVS90J01"     "Sample1"

"DGVS90J02"     "Sample2"

"DGVS90J03"     "Sample6"

"DGVS90J03"     "Sample7"

"DGVS90J03"     "Sample3"

"DGVS90J03"     "Sample5"

"DGVS90J03"     "Sample4"

HERE_TERMINATOR

When you make the association between a Sample and a Read Data Set, all the Amplicons associated with that Sample at that time are used to form three-way Read Data Set - Sample - Amplicon associations. This is equivalent to dragging a Sample, in the GUI, onto a Read Data Set in the Read Data Tree. These associations are used during Demultiplexing to determine which reads of a given Read Data Set belong to which Samples. As with dragging a Sample onto a Read Data Set in the GUI, when a Sample is associated with a Read Data Set, the Sample itself must already have an association with at least one Amplicon. An error is generated in the CLI if such a Sample-Amplicon association does not exist at the time you attempt to associate the Sample with the Read Data Set.

Rather than first creating Sample-Amplicon associations and using the Samples to then indirectly create Read Data Set – Sample – Amplicon associations, you can directly create these three-way associations by specifying all three entities in a single command. For example:

assoc -readData DGVS90J04 -sample Sample8 -amplicon EGFR_20_1

As this command only forms the association for the triad, the three individual entities must have been previously defined. It must be remembered that for a given Read Data Set, an Amplicon can only be assigned to one Sample; an “associate” command that would violate this constraint would return a warning message and the association will not be made. If the Sample and Amplicon specified in the “three-way association” form of the command, above, were not already associated with each other, that two-way association would simultaneously be made when the command executes.

In a Project where the same Sample (or list of Samples) is being measured against many Read Data Sets, the “associate” command can be simplified by the judicious use of Read Groups: associating a Sample (with its already associated Amplicons) to a Read Group forms the Read Data Set - Sample - Amplicon association triads with each Read Data Set present in the Read Group. For example:

assoc -readGroup ReadGrp_1 -sample Sample1

would associate Sample1 and all its currently associated Amplicons with each of the Read Data Sets of Read Group ReadGrp_1.

In a different context, where you may be measuring all the Amplicons defined in a Project for a Sample (or the same set of Amplicons for every Sample of the Project), you can more succinctly specify the triad association using the asterisk (“*”) notation for the Amplicons. For example:

assoc -readGroup ReadGrp_1 -sample Sample8 -amplicon *

The command above associates every Amplicon currently defined in the Project with the Sample specified and then associates the Sample and these Amplicons with every Read Data Set in the Read Group. You can include an “ofRef” parameter in the association command to restrict the Amplicons being associated to those derived from a single particular Reference Sequence.

Note that the command usage above is appropriate for establishing the Read Data Set – Sample – Amplicon associations in projects in which MIDs are not used. For an example of a CLI script that creates such associations for an MID-based experiment, see section 3.6