3.2. The evaluation of the shotgun metabarcoding assembly
accuracy and species detected in mock samples
There were 27, 10 12, and 10 reliable OTUs of ITS2, psbA-trnH ,matK , and rbcL were obtained in mock samples (HSZY159 and
HSZY171). All the ingredients mixed in HSZY159 can be successfully
re-detected, whereas nine ingredients (except for C.
lacryma-jobi ) and the positive control, the roots of P.
quinquefolius , could be detected in HSZY171 (Table 3 ).
To confirm the reliability of OTUs obtained from shotgun sequencing,
this study compared the sequence differences between these OTUs and the
corresponding reference barcodes obtained from Sanger sequencing(Table 1) . For ITS2, the OTUs of seven species were exactly the
same with their reference barcodes; two, two, and three types of ITS2
OTUs have been obtained for W. villosa , L. purpureus , andN. nucifera . Except for OTUs consistent with the three species’
reference barcodes, there 1-6 different bases between other OTUs and
reference barcodes. Furthermore, the proportions of reads belonging to
the OTUs same as the reference barcodes of W. villosa , L.
purpureus , and N. nucifera were 37.3%, 47.9%, and 50.0%,
respectively. For thepsbA-trnH region, OTUs of N. nucifera , W. villosa ,C. lacryma-jobi , G. uralensis , D. oppositifolia ,
and Panax ginseng were identical to their reference barcodes,psbA-trnH OTUs of L. purpureus and Platycodon
grandiflorus showed eight and three bases difference with their
reference barcodes. For the matK region, OTUs of N.
nucifera , G. uralensis , and Panax ginseng were completely
consistent with their reference barcodes. Three types of OTUs had been
obtained for both Platycodon grandiflorus and W. villosa ,
and there were 0-3 different bases comparing to their reference
barcodes. Finally, rbcL OTUs of seven species were identical to
their reference barcodes. Similar to the situation in matK two
additional OTUs with two and three different bases has been observed forW. villosa in addition to the OTU same as its reference barcode.
In addition, all the OTUs of ITS2, psbA-trnH , matK , andrbcL for the positive control, Panax quiquefolium , were
exactly the same as its reference barcodes.