Creating and Computing an MID Project with the AVA-CLI

3.6	Creating and Computing an MID Project with the AVA-CLI

3.6.1 Example MID Project Script

The EGFR example Project shown in detail in sections 2 (GUI version) and 3.5 (CLI version) does not display the usage of MIDs and Multiplexers. The example below briefly shows many of the special features of the AVA software that come into play when MIDs are used, and how they would be set up in a Project using the CLI.

A few things to remember are that in a Project that contains multiple Read Data sets, a given Multiplexer can be used for more than one Read Data set, or distinct Read Data sets can each have specific Multiplexer(s). It is also possible to associate more than one Multiplexer with the same Read Data set and to mix regular Samples with Multiplexers on a Read Data set. However, for a given Read Data set, an Amplicon can only be assigned to one entity (a regular Sample or a Multiplexer). Figure 3‑1 shows the Read Data Tree and Multiplexers Definition Table for an example Project that is atypically complex for tutorial purposes, as it was intentionally constructed to illustrate a wide variety of Multiplexer features, whereby:

•	4 Multiplexers are used, showing all 4 encoding types (Both, Either, Primer 1 MID, and Primer2 MID)

•	“MultiPlexerBoth” and “MultiplexerP1” are associated with Read Data ESS716O01 (as seen in the Tree), and “MultiplexerEither” and “MultiplexerP2” are associated with Read Data set ESS716O02

•	the Tree nodes are partially expanded to reveal which Amplicons are being demultiplexed by which Multiplexer:

◦	“MultiplexerBoth” is being used to assign a single amplicon “amp1” to 16 Samples

◦	“MultiplexerP1” is being used to assign “amp4” to 3 different Samples

◦	“MultiplexerEither” is being used to assign two different Amplicons (“amp2” and “amp3”) to 4 different Samples

◦	“MultiplexerP2” is being used to assign “amp5” to 3 different Samples

◦	MIDs are not being used for “amp6” and it is being assigned to Sample “A001” on the basis of its template-specific primers without the benefit of a Multiplexer

Figure 3‑1: The Multiplexer Definition Table and Read Data Tree for the MID example Project described in this section. Note that this Project is atypically complex, as it serves to illustrate a wide variety of MID / Multiplexer features.

he usual CLI commands can be used to set up an MID Project, using the appropriate options. For example, the ‘associate’ command supports the definition of both MID-based and non-MID-based multiplexing relationships. Read section 3.4.1 (or run ‘help associate’) for more details on how to create these multiplexing relationships. For more information on creating Multiplexers and their associated constituents using the CLI, run ‘help create multiplexer’ (section 3.4.4.4), ‘help create mid’ (section 3.4.4.2), and ‘help create midGroup’ (section 3.4.4.3).

To display some of these commands in context, an example CLI script is provided below. This script would produce a Project setup that would match the one shown in Figure 3‑1. Just as in non-MID project setup CLI scripts (as shown in the example in section 3.5), the standard objects such as References, Amplicons, and Samples must be created, and Read Data must be imported. The script shows these entities being loaded via the ‘-file’ option to keep the script more succinct.

The “here” file contents that are shown are tab-delimited. The double-quoting of the “here” file contents isn’t a requirement, but it is used here to help make it clear visually when particular fields have been deleted. These empty fields are generally interpreted as an intent to set an appropriate empty value for a field (such as an empty string for an annotation). However, the “associate” command is unique in this regard, as empty fields are interpreted as “ignore this field for this line”. This allows different types of associations to be specified within a single table (e.g. in the script below, the associations between Multiplexers, MIDs, and Samples for all four different types of Multiplexers are shown in the same association “here” file).

In the example script, the associations of the Multiplexers, MIDs, and Samples are split logically from the association of the Multiplexers, Read Data, and Amplicons, but they could have been combined into a single association table. This would have resulted in a larger, more complicated table with more repetition of fields across lines, which would be more difficult to create error-free by manual typing. However, the large association table would be convenient when creating the setup script via programmatic means (such as using Perl scripts to construct the commands by consulting a database or spreadsheet of the experimental design).

This script also illustrates the use of the new “utility execute” command to load the “454Standard” MID Group via an existing default script called “create454StandardMIDs.ava”, which is also used as part of the automatic project initialization functionality. Documentation of the “utility execute” command is available in section 3.4.17.5 and more information on automatic project initialization can be found in sections 4.4 and 4.5.

Due to the limitations of this printed document, certain lines of the script below appear on multiple lines. This occurs for certain tab-separated entries in the tables given as arguments to certain commands. Be aware that these should actually be single lines in the script.