3. Results
3.1. Summary for the data
analysis results of the shotgun sequencing
Through DNA barcoding technology,
except for Poria, the other nine herbal materials and the positive
control are all identified to be authentic medicinal materials
(Supplementary Table 1 ). The ITS2 sequence of Poria was not
successfully obtained and the herbal material was identified as
authentic by macroscopical and morphological identification. The GenBank
accession numbers of each DNA barcode sequence are listed inTable 1 . Certain DNA barcode regions of some herbal materials
were not successfully obtained due to the fail of PCR amplification.
Their relevant reference sequences of these species were downloaded from
GenBank for subsequent assembly accuracy analysis (Table 1 ).
A
total of 39.52 Gb of raw data were obtained from the six samplesvia shotgun sequencing, including 131,786,682 paired-end
sequencing reads. An average of 6.59 Gb raw data was obtained for each
sample. A total of 3,391,155 paired-end sequencing reads were enriched
for the ITS2, psbA-trnH , matK , and rbcL regions
after removing low-quality sequences. The detailed sequencing results
from all samples were shown in Supplementary Table 3 . There
were 460, 10158, 75, and 127 contigs belonging to ITS2,psbA-trnH , matK , and rbcL were obtained by combing
the assemble results of MEGAHIT and MetaSPAdes. Then, there were 139,
49, 44, and 45 sequences of ITS2, psbA-trnH , matK , andrbcL DNA barcoding regions were yielded after annotation and
removal of the chimeras. After clustering the obtained DNA barcode
sequences according to 100% sequence similarity, the ITS2 region
yielded a total of 53 OTUs, with an average length of 209 bp and an
average GC content of 55.8%. The number of OTUs obtained for the
chloroplast region was significantly lower than that of the ITS2
sequences, with 14, 13 and, 11 for the psbA-trnH , matK ,
and rbcL regions, respectively. In addition, the GC content ofpsbA-trnH , matK , and rbcL sequences was also lower
than that of the ITS2 sequences (Table 2 ).