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Droplet Digital
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PCR Applications Guide | 65
Next-Generation Sequencing Library Analysis
ddPCR Quantification on Illumina TruSeq v2 Chemistry
For the Illumina MiSeq and HiSeq platforms, the total possible reads is directly related
to the concentration of prepared library loaded. These platforms have a narrow loading
concentration range requirement for successful runs. To maximize the sequencing
information from a given sequencing run, accurate measurements of library concentration
must be made.
Measuring concentration by ddPCR before amplification may help determine the number of
additional PCR cycles needed, if any, to obtain enough library for loading.
Measuring concentration by ddPCR before the library is loaded for sequencing determines
concentration and helps identify any library construction quality anomalies accumulated
during the process.
The TruSeq v2 library preparation protocol is shown in Figure 7.1. TruSeq Y-adapters,
containing both P5 and P7 sequences, are ligated to library DNA inserts. Following PCR
amplification, the resulting amplicons contain P5 and P7 sequences directionally oriented on
either strand of the fragment library.
Fig. 7.1. TruSeq v2 library preparation.
DNA insert
Rd1 SP
Index
Rd2 SP
P5
P7
P
A
A
P
P
T
DNA insert
Ligate index adapter
Rd1 SP
Rd1 SP
Index
Rd2 SP
Rd2 SP
P5
P5P7
P7
Index
DNA insertRd1 SP
Rd2 SP
P5
5'
P7
Index
Denature and amplify
for final product
Probe 1 Probe 2
FP
RP
H FQ Q
3'
Our duplex assays target the flanks of the library fragments with two probes targeting both
the P5 and P7 moieties. We can directly measure linkage between probes 1 and 2 because
they are co-localized in the same droplet (Figure 7.2). Targeting both flanking segments
ensures testing for both well-formed and poorly formed fragments, allowing quantification
of species possessing both adapter arms.