The list of characters within the character class gives the set of characters matched by the class. "[abc]" matches a single "a" or "b" or "c". But if the first character after the "[" is "^", the class instead matches any character not in the list. Within a list, the "-" character specifies a range of characters, so that a-z represents all characters between "a" and "z", inclusive. If you want either "-" or "]" itself to be a member of a class, put it at the start of the list (possibly after a "^"), or escape it with a backslash. "-" is also taken literally when it is at the end of the list, just before the closing "]". (The following all specify the same class of three characters: [-az], [az-], and [a\-z]. All are different from [a-z], which specifies a class containing twenty-six characters, even on EBCDIC-based character sets.)
By default, the "^" character is guaranteed to match only the beginning of the string, the "$" character only the end (or before the newline at the end), and Perl does certain optimizations with the assumption that the string contains only one line. Embedded newlines will not be matched by "^" or "$". You may, however, wish to treat a string as a multi-line buffer, such that the "^" will match after any newline within the string (except if the newline is the last character in the string), and "$" will match before any newline. At the cost of a little more overhead, you can do this by using the "/m" modifier on the pattern match operator. (Older programs did this by setting $*, but this option was removed in perl 5.10.)
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Otherwise, use locale sets the default modifier to /l; and use feature 'unicode_strings, or use v5.12 (or higher) set the default to /u when not in the same scope as either use locale or use bytes. (use locale ':not_characters' also sets the default to /u, overriding any plain use locale.) Unlike the mechanisms mentioned above, these affect operations besides regular expressions pattern matching, and so give more consistent results with other operators, including using \U, \l, etc. in substitution replacements.
The rules used for matching decimal digits are slightly stricter. Many scripts have their own sets of digits equivalent to the Western 0 through 9 ones. A few, such as Arabic, have more than one set. For a string to be considered a script run, all digits in it must come from the same set of ten, as determined by the first digit encountered. As an example,
Any pattern containing a special backtracking verb that allows an argument has the special behaviour that when executed it sets the current package's $REGERROR and $REGMARK variables. When doing so the following rules apply:
Zhang et al. (2020) recently compiled a list of 22 primer sets for fish eDNA metabarcoding based on a literature survey, which performed more comprehensive in silico and in vitro comparisons of the primer performance. Those primer sets targeted the mitochondrial 12S rRNA, 16S rRNA, cyt b, and COI genes (7, 6, 7, and 2 primers, respectively), exhibiting considerable differences in the amplified taxonomic ranges and proportions, richness, species discriminatory power, and community compositions (Zhang et al. 2020). For example, the number of fish species detected from eDNA from freshwaters in Beijing, China ranged from 0 to 66, with the 12S primers consistently detecting greater fish diversity than the other primers. Specifically, the top six primer sets that recovered the greatest number of fish taxa were all 12S primers, of which four primers, including MiFish, showed outstanding detection of fish diversity. Zhang et al. (2020) also found that the results from in silico PCR and in vitro tests did not always agree, leading these authors to argue that primer choice for biodiversity monitoring should not be based solely on in silico evaluation. We favor their argument, as in silico evaluation of PCR primers only considers primer/template mismatches and does not accommodate the technical tips described above (Ficetola et al. 2010; Elbrecht and Leese 2017; Taberlet et al. 2018), which enhance primer annealing to the template.
These two examples strongly suggest that it is essential to perform preliminary experiments using MiFish primers based on a few water samples from the target study area before starting the actual survey. By comparing the preliminary results with direct observations through capture-based sampling, underwater visual censuses, and/or historical records through literature survey, apparent false-negative species (or groups of species) can be found among common species that are lacking in the preliminary results. In freshwater fishes of Japan, some species of lampreys (Lethenteron and Lampetra) and some osmerid fishes (Plecoglossus altivelis and Hypomesus spp.) are underrepresented in the MiFish eDNA metabarcoding data in comparisons with their known distributions and abundances, possibly owing to primer/template mismatches (Fig. 7b, c), and modified primers to accommodate their unique variations effectively increase their detections (M. Miya, unpublished data). More recently, a similar example was reported for cryptic seahorse taxa (family Syngnathidae) from Perth, Australia, in which false negatives were apparent with the existing primer sets, including MiFish, although the newly developed primers could detect these sea horse taxa (Nester et al. 2020). This finding is, however, in contrast with the consistent detections of two species of seahorse in the UK using MiFish primers (Tang et al. 2018). 2ff7e9595c
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