User manual
Next-Generation Sequencing Library Analysis
66 | Droplet Digital
™
PCR Applications Guide
Fig. 7.2. Design of the ddPCR library quantification kit for Illumina TruSeq assay.
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
P7
Index
Denature and amplify
for final product
Probe 1 Probe 2
FP
RP
H FQ Q
An example of the 2-D plot of ddPCR FAM versus HEX data observed for this assay is
shown in Figure 7.3.
Fig. 7.3. 2-D plot of ddPCR data from the ddPCR library quantification kit for Illumina TruSeq.
Channel 1 amplitude
0 1,000 2,000 3,000 4,000 5,000 6,000 7,000
Channel 2 amplitude
11,000
10,000
9,000
8,000
7,000
6,000
5,000
4,000
3,000
2,000
1,000
0
Library Quality Analysis
The ddPCR library quantification kit for Illumina TruSeq contains TaqMan assays designed
to detect and quantify both the P5 and P7 adapter arms. Each assay can be used to
independently confirm the formation of bona fide library fragments by exploiting the ability of
the QX100
™
or QX200
™
Droplet Digital PCR system to detect subpopulations of templates
with differing amplification efficiencies and with different combinations of P5 and P7 moieties.
A 2-D plot of the library can be used to provide information on the quality of the library
(Figure 7.4). Inserts of varying sizes within the library can be observed along a diagonal
path, with larger fragments near the negative droplets and smaller inserts in the upper-right
corner. This is because the smaller amplicons produce greater fluorescence during PCR
amplification. This characteristic pattern enables you to assess the quality of your library.
At the highest point in the upper-right corner is a population that appears separate from the
bulk of the library. This population is the adapter-dimer population with no inserts (red circle
in Figure 7.4).
Fig. 7.4. Additional library information produced by ddPCR.
Channel 1 amplitude
1,000 2,000
2,000
0
0
3,000
4,000
4,000
5,000 6,000
6,000
7,000 8,000
8,000
10,000
12,000
Channel 2 amplitude
Decreasing size of library
insert with adapters as
part of construct
Adapter dimers