PCR amplification specificity
Sequences identified as the most likely partial barcodes by taxonomic
filtering were the maximally abundant sequence per specimen in 345 cases
for the FC amplicon and in 365 cases for the BR amplicon. Combined, the
selected FC and BR sequences were both the maximally abundant sequences
per specimen in only 286 cases. A further 29 selected FC sequences and
50 selected BR sequences each had abundance ranks between two and ten.
Two selected FC sequences had abundance ranks of 18 and 30,
respectively, and 10 selected BR sequences each had abundance ranks
between 11 and 101. In 37 cases where the maximally abundant FC sequence
was not selected, the maximally abundant sequences were identified as
deriving from insects (25), annelids (8), arachnids (2), algae (1), and
amoebae (1). Similarly, in 74 cases where the maximally abundant BR
sequence was not selected, these were identified as deriving from
insects (44), other hexapods (2), annelids (2), or gastropods (1); and
as Homo sapiens (5), and eukaryote (2) or prokaryote (18)
micro-organisms, including 10 cases of Wolbachia .
To investigate the origins of maximally abundant but non-target
sequences (i.e. those with unexpected taxonomic identifications), the
allpairs_global function in VSEARCH was used to identify any identical
sequences among the maximally abundant sequences per specimen, plus the
selected (presumed correct) sequences (if not maximally abundant) from
each specimen. Out of 37 specimens with maximally abundant but
non-target FC sequences, only three of those sequences were identical to
another maximally abundant but non-target FC sequence, in two cases from
adjacent PCR wells and in all three cases from within the same PCR
plates. Similarly, out of 74 specimens with maximally abundant but
non-target BR sequences, ten of those sequences were each identical to
one or more other maximally abundant but non-target BR sequence, in only
four cases from adjacent PCR wells but in all cases from within the same
PCR plates.