Development and Verification of an Economical Method of Custom Target Library Construction

Although
technological advances have greatly reduced the cost of DNA sequencing,
sample preparation time and reagent costs remain the limiting factors
for many studies. Based on low-cost targeted amplification, we developed
an economical method for custom target library construction based
on DNA nanoball (DNB) technology and two-step polymerase chain reaction
(PCR). Here, we refer to this method as the two-step PCR, which was
compared to traditional multiplex PCR methods in three aspects, data
quality, efficiency, and specificity to humans. The results confirmed
that two-step PCR reduces to finishing 128 sequencing libraries in only 2 h 24
min 59 s of the total PCR time and at a data utilization rate of 0.44
at a cost of approximately $1.70 per sample for targeted sequencing
via the two-step PCR. The replacement of traditional multiplex PCR
methods with this strategy makes the sample preparation process before
sequencing relatively more cost-effective and further reduces the
cost of next-generation sequencing (NGS). This method may also be
free from the interference of other species and the limitations of
sample type and DNA content. These findings reveal possibilities for
broad applications of this approach in forensic research.