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Parallel identification of novel antimicrobial peptide sequences from multiple anuran species by targeted DNA sequencing.

ABSTRACT: BACKGROUND:Antimicrobial peptides (AMPs) are multifunctional effector molecules that often combine direct antimicrobial activities with signaling or immunomodulatory functions. The skin secretions of anurans contain a variety of such bioactive peptides. The identification of AMPs from frog species often requires sacrificing several specimens to obtain small quantities of crude peptides, followed by activity based fractionation to identify the active principles. RESULTS:We report an efficient alternative approach to selectively amplify AMP-coding transcripts from very small amounts of tissue samples, based on RNA extraction and cDNA synthesis, followed by PCR amplification and high-throughput sequencing of size-selected amplicons. This protocol exploits the highly conserved signal peptide region of the AMP precursors from Ranidae, Hylidae and Bombinatoridae for the design of family-specific, forward degenerate primers, coupled with a reverse primer targeting the mRNA poly-A tail. CONCLUSIONS:Analysis of the assembled sequencing output allowed to identify more than a hundred full-length mature peptides, mostly from Ranidae species, including several novel potential AMPs for functional characterization. This (i) confirms the effectiveness of the experimental approach and indicates points for protocol optimization to account for particular cases, and (ii) encourages the application of the same methodology to other multigenic AMP families, also from other genera, sharing common features as in anuran AMPs.

SUBMITTER: Roncevic T

PROVIDER: S-EPMC6245896 | biostudies-literature | 2018 Nov

REPOSITORIES: biostudies-literature

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Parallel identification of novel antimicrobial peptide sequences from multiple anuran species by targeted DNA sequencing.

Rončević Tomislav T Gerdol Marco M Spazzali Francesca F Florian Fiorella F Mekinić Stjepan S Tossi Alessandro A Pallavicini Alberto A

BMC genomics 20181120 1

<h4>Background</h4>Antimicrobial peptides (AMPs) are multifunctional effector molecules that often combine direct antimicrobial activities with signaling or immunomodulatory functions. The skin secretions of anurans contain a variety of such bioactive peptides. The identification of AMPs from frog species often requires sacrificing several specimens to obtain small quantities of crude peptides, followed by activity based fractionation to identify the active principles.<h4>Results</h4>We report a ...[more]

PMID: 30458708

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Project description:BackgroundEffective innate responses against potential pathogens are essential in the living world and possibly contributed to the evolutionary success of invertebrates. Taken together, antimicrobial peptide (AMP) precursors of defensin, mytilin, myticin and mytimycin can represent about 40% of the hemocyte transcriptome in mussels injected with viral-like and bacterial preparations, and unique profiles of myticin C variants are expressed in single mussels. Based on amplicon pyrosequencing, we have ascertained and compared the natural and Vibrio-induced diversity of AMP transcripts in mussel hemocytes from three European regions.Methodology/principal findingsHemolymph was collected from mussels farmed in the coastal regions of Palavas (France), Vigo (Spain) and Venice (Italy). To represent the AMP families known in M. galloprovincialis, nine transcript sequences have been selected, amplified from hemocyte RNA and subjected to pyrosequencing. Hemolymph from farmed (offshore) and wild (lagoon) Venice mussels, both injected with 10(7) Vibrio cells, were similarly processed. Amplicon pyrosequencing emphasized the AMP transcript diversity, with Single Nucleotide Changes (SNC) minimal for mytilin B/C and maximal for arthropod-like defensin and myticin C. Ratio of non-synonymous vs. synonymous changes also greatly differed between AMP isotypes. Overall, each amplicon revealed similar levels of nucleotidic variation across geographical regions, with two main sequence patterns confirmed for mytimycin and no substantial changes after immunostimulation.Conclusions/significanceBarcoding and bidirectional pyrosequencing allowed us to map and compare the transcript diversity of known mussel AMPs. Though most of the genuine cds variation was common to the analyzed samples we could estimate from 9 to 106 peptide variants in hemolymph pools representing 100 mussels, depending on the AMP isoform and sampling site. In this study, no prevailing SNC patterns related to geographical origin or Vibrio injection emerged. Whether or not the contact with potential pathogens can increase the amount of AMP transcript variants in mussels requires additional study.

Project description:BackgroundTo date, evidence for the relative prevalence or rarity of molecular convergent and parallel evolution is conflicting, and understanding of how these processes contribute to adaptation is limited. We compared four high-elevation anuran species (Bufo tibetanus, Nanorana parkeri, Rana kukunoris and Scutiger boulengeri) from the Tibetan region, and examined convergent and parallel amino acid substitutions between them and how they may have contributed to high-elevation adaptation.ResultsGenomic data of the four high-elevation species and eight of their low-elevation close relatives were gathered. A total of 1098 orthologs shared by all species were identified. We first conducted pairwise comparisons using Zhang and Kumar's test. Then, the Rconv index was calculated and convergence/divergence correlation plotting was conducted. Furthermore, genes under positive selection and with elevated evolutionary rate were examined. We detected a large number of amino acid sites with convergent or parallel substitutions. Several pairs of high-elevation species, in particular, R. kukunoris vs N. parkeri and B. tibetanus vs S. boulengeri, had excessive amounts of convergent substitutions compared to neutral expectation. Nevertheless, these sites were mostly concentrated in a small number of genes (3-32), and no genome-wide convergence was detected. Furthermore, the majority of these convergent genes were neither under detectable positive selection nor had elevated evolutionary rates, although functional prediction analysis suggested some of the convergent genes could potentially contribute to high-elevation adaptation.ConclusionsThere is a substantial amount of convergent evolution at the amino-acid level among high-elevation amphibians, although these sites are concentrated in a few genes, not widespread across the genomes. This may attribute to the fact that all the target species are from the same environment. The relative prevalence of convergent substitutions among high-elevation amphibians provides an excellent opportunity for further study of molecular convergent evolution.

Project description:Among molecular-based techniques for fungal identification, Sanger sequencing of the primary universal fungal DNA barcode, the internal transcribed spacer (ITS) region (ITS1, 5.8S, ITS2), is commonly used in clinical routine laboratories due to its simplicity, universality, efficacy, and affordability for fungal species identification. However, Sanger sequencing fails to identify mixed ITS sequences in the case of mixed infections. To overcome this limitation, different high-throughput sequencing technologies have been explored. The nanopore-based technology is now one of the most promising long-read sequencing technologies on the market as it has the potential to sequence the full-length ITS region in a single read. In this study, we established a workflow for species identification using the sequences of the entire ITS region generated by nanopore sequencing of both pure yeast isolates and mocked mixed species reads generated with different scenarios. The species used in this study included Candida albicans (n = 2), Candida tropicalis (n = 1), Nakaseomyces glabratus (formerly Candida glabrata) (n = 1), Trichosporon asahii (n = 2), Pichia kudriavzevii (formerly Candida krusei) (n = 1), and Cryptococcus neoformans (n = 1). Comparing various methods to generate the consensus sequence for fungal species identification, the results from this study indicate that read clustering using a modified version of the NanoCLUST pipeline is more sensitive than Canu or VSEARCH, as it classified species accurately with a lower abundance cluster of reads (3% abundance compared to 10% with VSEARCH). The modified NanoCLUST also reduced the number of classified clusters compared to VSEARCH, making the subsequent BLAST+ analysis faster. Subsampling of the datasets, which reduces the size of the datasets by approximately tenfold, did not significantly affect the identification results in terms of the identified species name, percent identity, query coverage, percentage of reads in the classified cluster, and the number of clusters. The ability of the method to distinguish mixed species within sub-populations of large datasets has the potential to aid computer analysis by reducing the required processing power. The herein presented new sequence analysis pipeline will facilitate better interpretation of fungal sequence data for species identification.

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Parallel identification of novel antimicrobial peptide sequences from multiple anuran species by targeted DNA sequencing.

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Parallel identification of novel antimicrobial peptide sequences from multiple anuran species by targeted DNA sequencing.

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