User manual

Droplet Digital
™
PCR Applications Guide | 69
Next-Generation Sequencing Library Analysis
Fig. 7.7. ddPCR library balancing results. PF, passing filter.
A
B
PF reads (x 1,000)
Average
Library 1
Library 2
Library 3
Library 4
Library 5
Library 6
Library 7
Library 8
Library 9
Library 10
Li b ra r y 11
Librar y 12
800
700
600
500
400
300
200
0
Index number
100
0
0
2
4
6
8
10
2 4 6 8 10 12 14
PF reads identified, %
Channel 2 amplitude
Library
1 2 3 4 5 6 7 8 9 10 11 12
Input
total
brain
RNA
4,000 1,000 100 10
8.3% expected
Amplicon Recovery from Droplets
Bio-Rad’s ddPCR supermix for probes (no dUTP) has been optimized for PCR amplification
of rare target DNA sequences and for NGS library preparations. The droplet PCR supermix
provides unbiased amplification and greater template coverage of sequencing template.
Generate droplets by following the ddPCR standard workflow and protocols. If your goal is
to read droplets as well as recover material from droplets, make the desired number of wells
to be read on the QX100 or QX200 system (nonrecoverable), and also make replicates to be
broken open (not to be read on the QX100 or QX200 system).
For example, a column of wells could be generated (eight wells), four of which are read
after PCR and four of which are not read. In QuantaSoft software, set up the plate where
only four of the eight wells are read. After the QX100 or QX200 system has finished the run,
remove the plate and pierce the foil of the four remaining unread wells and proceed with
breaking the droplets from those wells.
If your goal is to generate droplets and break them open only after PCR, without reading
them on the QX100 or QX200 system, then proceed directly with the following protocol
for recovery of DNA from droplets after PCR:
1. Pipet out the entire volume of droplets and oil from a well into a 1.5 ml tube
(combine up to ten replicates if desired).
2. Pipet and discard the bottom oil phase after droplets float to the top.