2.3 Dietary DNA amplification and sequencing
PCR amplification was carried out using mitochondrial COΙ-targeting
primer (LCO-1490/Uni-MiniBar-R), which produced a COΙ (cytochrome
oxidase Ι) amplicon of 177 bp (Brown et al. 2014) for animal
identification. Existing COΙ-based approaches is widely preferred to
identify unknown arthropod sequences (Zeale et al. 2011). The used
primers were tested against Chinese mole shrew sequences to ensure no
significant amplification of host DNA. And the rbcL
(ribulose-bisphosphate carboxylase gene) primers (h1aF and h2aR primers)
were used to identify the plant species (Pierre et al. 2007). Sample
specific barcode sequences were added to the COΙ and rbcL primers.
PCR were performed with PCR Using Q5® High-Fidelity
DNA Polymerase (M0491, NEB) according to the manufacturer’s instruction.
And PCR protocols were conducted primarily following Bohmann et al.
(2018). Blank extraction controls were included on each PCR plate, and
for each different primer set. PCR products were then purified using a
PCR purification kit (AXYGEN). Taking the purified PCR product as the
template, quantitative real-time PCR was performed on a Microplate
reader (BioTek, FLx800) using Quant-iT PicoGreen dsDNA Assay Kit. The
amplicons for each sample were then mixed and purified according to the
next high throughput sequencing requirements. Libraries for sequencing
were constructed using the TruSeq Nano DNA LT Library Prep Kit
(Illumina, San Diego, CA, United States) as recommended by the
manufacturer. Libraries were sequenced on an Illumina Miseq platform (2
× 250 bp paired-end reads) by Personalbio Bioinformatics Technology
Corporation (Shanghai, China).