User manual

Droplet Digital
™
PCR Applications Guide | 67
Next-Generation Sequencing Library Analysis
Droplets that appear above and below the insert population (large, diagonal cluster of
increasing fluorescence) represent rare species with three or more adapters ligated to
the insert (Figure 7.5). These populations can be selected in QuantaSoft
™
software using
the lasso function. By selecting the desirable bulk population that lies along the diagonal
(green circle, Figure 7.5), excluding the adapter-adapter population in one color channel
(blue circle, Figure 7.5), and excluding the undesirable side populations (adapter-adapter
and extra adapter populations in red circles, Figure 7.5), you can then use the Ratio tab in
QuantaSoft software to select Fractional Abundance (a/a+b), and get a readout of the
percentage of your library with inserts. This can be a quick and easy readout of the quality
of your libraries. As anything with two adapters can still contribute to your cluster densities,
you must include the adapter-adapter populations in your quantification and balancing
of your libraries for sequencing. This quality measurement can be used to enable you
to appropriately load and balance your sequencing runs, thus compensating for poorly
formed library fragments and improving greater reading depth.
Fig. 7.5. Various species visualized by the ddPCR library quantification kit assay.
FAM amplitude
1,0000
–1,000
2,000 3,000 4,000 5,000 6,000 7,000
10,000
8,000
6,000
4,000
2,000
0
HEX amplitude
The library quality information obtained from 2-D plots will fuel further investigations
into improvements of NGS sequencing, possibly by determining library fragment PCR
efficiencies. Improvements in the NGS workflows will likely result from extremely accurate
quantification by ddPCR. For example, you may be able to eliminate secondary amplification
steps if enough library material is generated for sequencing directly after library construction,
thus avoiding unnecessary steps and further skewing of fragment representation.
Next-Generation Sequencing Reads
In Figure 7.6, we demonstrate the relationship between the ddPCR-determined library
concentration loaded and the number of total reads from sequencing on a MiSeq platform.
By measuring library concentrations, the amount of input can be balanced across samples
before loading the NGS instrument.