DNA barcoding for Nee Soon flora and fauna 
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species in which taxonomically important parts were not readily accessible, we also 
developed a NGS-based technique for identifying trees to genus based on sapwood 
samples. This was challenging because such samples contain little DNA and large 
amounts of PCR-inhibitors. 
Methods 
Faunal barcoding 
Barcodes for insects were generated through COI amplification using direct-PCR 
(dPCR) (Wong et al., 2014), where a small amount of tissue is dissected from each 
individual specimen and serves as a template for amplification without prior DNA 
extraction. In addition, DNA from many specimens was also extracted using a 
novel reagent known as QuickExtract™ DNA Extraction Solution (EPICENTRE 
Biotechnologies); DNA extracts obtained with QuickExtract were used directly as 
input template for amplification of barcoding genes. A short fragment (313 bp) of COI 
was used as the general faunal barcoding gene. Subsequent downstream sequencing 
was conducted using a combination of traditional Sanger sequencing and high- 
throughput, paired-end NGS on Illumina™ platforms. Most barcodes were generated 
with NGS (Srivathsan et al., 2015). For NGS barcodes, each specimen’s amplicons 
were tagged with a uniquely labelled primer pair in the PCR step; the use of indexed 
primers allowed for barcodes to be traced accurately to their specimen of origin in the 
downstream bioinformatic process. Sequences generated from either Sanger or NGS 
methods were aligned using MAFFT ver.7 (Katoh & Standley, 2013), before being 
grouped into molecular operational taxonomic units (mOTUs) based on objective 
clustering, whereby sequences are grouped by similarity based on uncorrected 
pairwise (p) distances at specific uncorrected percentage thresholds (Meier et al., 2006; 
Srivathsan & Meier, 2012). Ideally, the threshold value set for clustering into mOTUs 
should be within a numerical range where the number of clusters remains stable; this 
is because stable clusters are likely to represent species. When used appropriately, 
intraspecific and interspecific variability can also be compared to further assess the 
stability of the species boundaries (Meier et al., 2008). 
Floral barcoding 
DNA extraction for all floral specimens were carried out on leaf and sapwood samples 
using a modified CTAB-chloroform extraction protocol (Doyle & Doyle, 1987). 
DNA barcodes for plant species were generated using both Sanger sequencing and 
NGS platforms. Initially, sequences for four selected barcode genes were amplified 
and sequenced: maturase K ( matK ), ribulose-bisphosphate carboxylase (rbcL ), a 
chloroplast tRNA gene ( trnL ), and the internal transcribed spacer 2 ( ITS2 ) region of 
nuclear ribosomal DNA. The trnH-psbA intergenic spacer region was also explored. 
With the advent and availability of high throughput sequencing, Illumina™ 
platforms were used for both amplicon sequencing and chloroplast genome skimming. 
