User manual

Next-Generation Sequencing Library Analysis
68 | Droplet Digital
™
PCR Applications Guide
Fig. 7.6. Plot of the reads vs. input library concentration. Impact of input library concentration on total usable
reads. Cluster density at 5 pM was approximately 800,000/mm
2
. PF, passing filter.
Total reads, millions
10
8
6
4
2
0
Concentration, pM
0 2 4 6 8 10 12 14 16
Total reads
PF reads
Q30 reads
As the cluster density and therefore number of reads is intimately tied to the loading
concentration of the prepared library, moderate differences can compromise read
capacity and quality of the MiSeq platform. NGS library quantification with ddPCR is
extremely accurate, providing accuracy better than 15% with a confidence level of 95%.
This method provides absolute quantification, eliminating the need to develop standards.
Use of ddPCR in NGS significantly increases reliability and quality, and optimizes use
of consumables, labor, and instrument time. Additional information, not available with
other methods, such as adapter-adapter dimers and improperly adapted species can be
seen. We have observed an inverse relationship between the size of the amplicons and
fluorescence intensity with our ddPCR library quantification kit for Illumina TruSeq.
The smaller the amplicon size, the higher the fluorescence attained, most likely due to
PCR efficiency within the droplets. This information-rich content provides you with a
digital quality check in the library construction before a sequencing run.
Library Balancing
When performing NGS on the MiSeq platform, it is important to aim for a cluster density of
approximately 800,000/mm
2
for optimal performance. ddPCR measurements can be used
to establish the functional relationship between input library concentration and the number
of usable reads on the MiSeq platform.
We examined the precision of ddPCR in balancing 12 TruSeq DNA libraries from human
genomic DNA using concentration measurements obtained from the QX100 system
using the ddPCR library quantification kit (Figure 7.7). Based on the ddPCR concentration
measurements, libraries with an average fragment length of 447 bp could be balanced
within less than 15% of each other with a confidence interval of 95%. Similar balancing
results were observed when RNA-Seq libraries with an average fragment length of 280 bp
were used.