Effectiveness of blocking primers and a peptide nucleic acid (PNA) clamp for 18S metabarcoding dietary analysis of herbivorous fish

Abstract

The structure of food webs and carbon flow in aquatic ecosystems can be better understood by studying contributing factors such as the diets of herbivorous fish. Metabarcoding using a high-throughput sequencer has recently been used to clarify prey organisms of various fish except herbivorous fish. Since sequences of predator fish have dominated in sequences obtained by metabarcoding, we investigated a method for suppressing the amplification of fish DNA by using a blocking primer or peptide nucleic acid (PNA) clamp to determine the prey organisms of herbivorous fish. We designed three blocking primers and one PNA clamp that anneal to fish-specific sequences and examined how efficient they were in suppressing DNA amplification in various herbivorous fish. The results showed that the PNA clamp completely suppressed fish DNA amplification, and one of the blocking primers suppressed fish DNA amplification but less efficiently than the PNA clamp. Finally, we conducted metabarcoding using mock community samples as templates to determine whether the blocking primer or the PNA clamp was effective in suppressing fish DNA amplification. The results showed that the PNA clamp suppressed 99.3%–99.9% of fish DNA amplification, whereas the blocking primer suppressed 3.3%–32.9%. Therefore, we propose the application of the PNA clamp for clarifying the prey organisms and food preferences of various herbivorous fish.

Using DNA metabarcoding and a novel canid-specific blocking oligonucleotide to investigate the composition of animal diets of raccoon dogs (Nyctereutes procyonoides) inhabiting the waterside area in Korea

“…The lower reduction efficiency may be due to the shorter length of RacBlk (27 nt) than other blocking oligonucleotides (40–50 nt). In fact, the fact that longer blocking oligonucleotides are associated with higher blocking efficiencies has been reported by previous studies [ 69 , 70 ]. We do not know the mechanism behind, but one possible explanation is that a short-length blocking oligonucleotide allows annealing and extension of the universal primer to the target region of the blocking oligonucleotide before the blocking oligonucleotide is annealed to it, resulting in amplification of the target organism (which should be blocked) by the universal primers.…”

Contaminants reach everywhere: Fish dietary samples should be surface decontaminated prior to molecular diet analysis

“…It is also a fact that the predator DNA is generally present in both good quality and quantity in the gut samples (Drake et al, 2022 ; Leray, Yang, et al, 2013 ), and very low amounts of template DNA of whitefish might have been outcompeted during the PCR by the dominant predator DNA (sensu Cuff et al, 2022 ; Kebschull & Zador, 2015 ; Paula et al, 2015 ). Although there was a positive association between host and prey DNA reads, and we retained a usable amount of prey reads for further analyses, the proportion of reads could be increased using host‐specific blocking primers (Homma et al, 2022 ; Leray, Agudelo, et al, 2013 ).…”

Improved 18S rDNA profiling of parasite communities in salmonid tissues using a host blocking primer

“…Commonly, primers are modified at 3’ ends by addition of short 3-carbon chains (C3 spacers), phosphate groups, inverted dT bases or amine groups, to prevent polymerase extension (Lee et al 2011 ; Wang et al 2013 ; Leroux et al 2022 ; Liu et al 2019 ). Other methods use peptide nucleic acid (PNA) clamps, which are synthetic DNA molecules with modified peptide backbones that anneal to target sequences and prevent amplification from upstream primers (Belda et al 2017 ; Homma et al 2022 ). The net effect is lowered PCR efficiency of host DNA amplification, allowing target DNA to be more readily detected by amplicon sequencing with low sequencing depth (Liu et al 2019 ; Leroux et al 2022 ; Belda et al 2017 ; Homma et al 2022 ).…”

“…Other methods use peptide nucleic acid (PNA) clamps, which are synthetic DNA molecules with modified peptide backbones that anneal to target sequences and prevent amplification from upstream primers (Belda et al 2017 ; Homma et al 2022 ). The net effect is lowered PCR efficiency of host DNA amplification, allowing target DNA to be more readily detected by amplicon sequencing with low sequencing depth (Liu et al 2019 ; Leroux et al 2022 ; Belda et al 2017 ; Homma et al 2022 ).…”