“In silico” testing
Before this test, it was verified through real-time PCR that the three
new mini-barcodes showed the capacity to amplify the DNA of the 7
different tissue samples belonging to 6 different phyla.
In the “in silico” PCR, all the barcodes tested showed high taxonomic
coverage of eukaryotic organisms (Figure 1), along with a null capacity
to amplify organisms of the taxa Bacteria and Archaea (Figure 1). Of the
three mini-barcodes designed in this study, MiniB18S_81 showed the
higher taxonomic coverage of eukaryotic organisms. This barcode served
to amplify 91.1% of eukaryotic sequences present in the SILVA reference
database, which is more than the other 28S mini-barcodes tested in this
study. Only the large barcode 566F/1200R showed a slightly better
amplification capacity for eukaryotic organisms (92.6%; Figure 1). In
addition, all barcodes showed a good capacity to amplify eukaryotic
phyla sequences: MiniB18S_41 = 94.9% (131/138); MiniB18S_43 = 93.5%
(129/138); MiniB18S_81 = 95.7% (132/138); 566F/1200R = 98.6%
(136/138); nucLSUDf1/nucLSUDr1 = 96.6% (85/88); and Short28SF/Short28SR
= 96.6% (85/88).
The six barcodes were able to amplify large numbers of organisms of the
10 target phyla (Supplemental information: Table S3). However,
MiniB18S_81 amplified more than 90% of the sequences of 7 of these 10
phyla, and showed the best taxonomic coverage among the mini-barcodes
tested, as good as the amplification capacity of the large barcode
566F/1200R (Supplemental information: Table S2). Of the previously
designed 28S mini-barcodes, nucLSUDf1/nucLSUDr1 showed poor taxonomic
coverage of several phyla, amplifying less than 80% of the sequences of
5 of the 10 phyla examined (Supplemental information: Table S3).
MiniB18S_43 and the reference nucLSUDf1/nucLSUDr1 mini-barcodes showed
the worst capacity to detect sequences of the phyla Phragmoplastophyta,
Arthropoda, Nematoda, Platyhelminthes and Apicomplexa (Figure 2). The
former did not feature a high amplification capacity for Nematodes and
the latter behaved poorly when used on all taxa, especially Nematoda and
Apicomplexa (Figure 2). The MiniB18S_81 mini-barcode provided the best
results, showing an excellent amplification capacity on Arthropoda,
Nematoda, Platyhelminthes, despite a slightly lower amplification
capacity for the Apicomplexa phylum than MiniB18S_41, MiniB18S_43 and
Short28SF/Short28SR (Figure 2). It should also be mentioned that all
five mini-barcodes and 566F/1200R displayed a high amplification
capacity for the Fungi kingdom and its phyla Ascomycota and
Basidiomycota (Supplemental information: Table S3).
All tested barcodes showed short mean amplicon lengths (Table 1), as
well as a quite constant amplicon length (Supplemental information:
Figure S1). Although the five mini-barcodes generated amplicons of 30 to
900 bp, percentages of sequences longer than 300 bp were reduced (0.06%
MiniB18S_41; 0.05% MiniB18S_43; 1.16% MiniB18S_81, 0.34%
nucLSUDf1/nucLSUDr1 and 0.32% Short28SF/Short28SR). In the case of
MiniB18S_81, cestodes showed larger amplicons with a mean length of 299
bp including some longer than 300 bp.
In contrast, our taxonomic resolution revealed a higher resolution
capacity of the large barcode 566F/1200R than any of the mini-barcodes
(Table 2; Supplemental information: Table S3). The resolution capacity
of the five mini-barcodes was not poor (Supplemental information: Table
S3), yet MiniB18S_81 seemed to have the best resolution capacity for
the phyla Phragmoplastophyta, Arthropoda, Nematoda, Platyhelminthes and
Apicomplexa (Table 2). In effect, this mini-barcode offered high
taxonomic resolution at the order level within the phyla Arthropoda,
Nematoda, Platyhelminthes and Apicomplexa, at the family level in
Platyhelminthes and Apicomplexa, at the genus level in Apicomplexa, and
at the species level in Arthropoda, Nematoda and Platyhelminthes (Table
2). However, no results were obtained at the family and genus levels for
various phyla as this information was lacking in the reference dataset
(Table 2).