Titration Runs Calculations for Older Chemistries

7.2	Titration Runs Calculations for Older Chemistries

For sequencing Runs carried out using the GS 20 or GS FLX standard chemistries, it was recommended to titrate the libraries to determine the optimal amount of library DNA to add to the emPCR Amplification reactions. The titration included a set of amplification reactions carried out with different amounts of input DNA, followed by a sequencing Run with the beads produced. For details on how to carry out a titration experiment in the GS FLX standard chemistry, see the GS FLX Shotgun DNA Library Preparation Method Manual (December 2007).

When the data set being viewed in the GS Run Browser is identified as having been generated using one of the older chemistries, the Filters tab can compute an “Expected bead recovery” figure (also known as “Predicted emPCR bead yield”) for each region of a multi-lane gasket (”small regions” format). This figure, assuming these data belong to a titration experiment, can help determine the optimal amount of input DNA to use for future emPCR amplifications / sequencing Runs with this library.

It is important to note that this feature DOES NOT APPLY to sequencing Runs carried out using the GS Junior Titanium or the GS FLX Titanium chemistry. Titration Runs still exist in the newer chemistry but they are no longer mainstream and the method to evaluate them has been greatly simplified (see the emPCR Amplification Method Manual – Lib-L SV or – Lib-A SV).

To support these calculations, the Filter tab displays the following additional feature:

•

Regions for Titration: Only for sequencing Runs carried out with the GS 20 or the GS FLX standard chemistry kits, choice of whether to evaluate the data as a titration and if so, choice of which regions to include in such evaluation (Figure 47). This is not applicable (grayed out) if the Control DNA reads are selected; and is not displayed at all for sequencing Runs carried out with a GS Junior Titanium or a GS FLX Titanium series sequencing kit.

Figure 47: Regions for Titration selector, available only for sequencing Runs carried out with the GS 20 or the GS FLX standard chemistry kits, when data for the library wells category is selected.

If any region(s) are selected to be evaluated as a titration, the Expected bead recovery line is added at the bottom of the Summary (Figure 48). An “Expected bead recovery” value (also known as “Predicted emPCR bead yield”) is calculated for each selected lane and provides the number of enriched beads (that will produce key pass reads) one might expect to get from a full emPCR kit (16 reactions) with this library, assuming the performance observed in this lane. See the GS FLX Shotgun DNA Library Preparation Method Manual, Section 3.7.3.3, for a description of how it is calculated and how to use it to determine the optimal emPCR amplification conditions (DNA concentration) for your library.

Figure 48: Summary area from the Filters tab, showing lanes 1 through 4 evaluated as a titration Run, with corresponding entries in the “Expected bead recovery” row. In this example, lane 3 provides the best conditions. Region 2 might appear to be best based on quality criteria (more % Passed Filter and less % Dot + Mixed and % Short), but the Expected bead recovery data shows that it would provide far too few beads. Conversely, the conditions of lane 4 would generate a lot more beads, but their quality would be too low (less than 50% Passed filter and much higher % Dot + Mixed and % Short). This type of assessment DOES NOT APPLY to sequencing Runs carried out using the GS Junior Titanium or the GS FLX Titanium chemistry.