User's Manual

12
Chapter 2: Droplet PCR Reaction Design
for Target Selection
The Xdrop™ technology requires a simple assay design with the following components:
• A high molecular weight DNA sample ( > 30 kb, depending on the assay) of high purity. Calculate the
required amount of input DNA needed based on the desired enrichment and desired amount of
output DNA using the online DNA input calculator at samplix.com
• One droplet PCR (dPCR) primer pair for enrichment. Please see the design guidelines below and
use the online primer design tool at samplix.com
• One quantitative PCR (qPCR) QC primer pair for validation of Xdrop™ DNA enrichment. Please see
the design guidelines below and use the online primer design tool at samplix.com
Target sequence
The Xdrop™ technology allows targeted enrichment by selection and amplification of a genomic region of
interest without the need for long-range PCR. The target DNA of interest can contain repeat regions, GC-rich
regions or other regions that are otherwise difficult to amplify. Specific primers amplifying a short fragment
of 120-160 bp (ID sequence), within the target or in flanking regions, are used for capturing the kilobases-
long region of interest. The Xdrop™ technology compartmentalizes the amplification reaction in small
droplets and makes use of a highly processive DNA polymerase to enable amplification of almost all regions
of the genome.
The length of the enriched target DNA will depend on the length of the input DNA. Consider using high
molecular weight DNA as input with a DNA fragment size > 30 kb and of high purity. Calculate the optimal
amount of input template DNA using the online sample input calculation tool at samplix.com
DNA sample preparation
When purifying the DNA sample, use a method that maintains the integrity and the purity of the DNA. Take
all required measures to avoid contaminations. The Xdrop™ enrichment technology can be affected by
contamination of the DNA sample by RNA, proteins, carbohydrates, salt and phenol among others. Purify the
DNA to the same quality as required for long read sequencing.
Calculate the optimal amount of input template DNA using the online enrichment calculation tool at
samplix.com