<h1>High-throughput sequencing comprehensively detects single-cell eukaryotes and parasites in biological or environmental samples</h1>
A high-throughput sequencing assay to comprehensively detect and characterize unicellular eukaryotes and helminths from biological and environmental samples
Microbiome, [9.133]
DOI: https://microbiomejournal.biomedcentral.com/articles/10.1186/s40168-018-0581-6
First author: Matthew V Cannon
Corresponding author: David Serre [email protected]
Principal units: University of Maryland, School of Medicine, Institute of Genomic Science
Other authors: Haikel Bogale, Lindsay Rutt, Michael Humphrys, Poonum Korpe, Priya Duggal, Jacques Ravel
<h2>Guide</h2>
Single-celled eukaryotes and parasites are important human pathogens, but lack convenient and specific detection methods;
This paper designs a number of pairs of 18S rRNA gene primers that can detect most single-celled eukaryotes and parasites, including apical compound gate, amoeba, diplodocus, mover order, parabasitoid phylum, nematodes, flat animals and microsporidia;
The length range and resolution of the amplification products were simulated and evaluated using the NCBI database, which were suitable for Illumina sequencing, and the natural sample evaluation results also covered most of the target taxons;
This method is an effective complement to the current 16S amplicon detection methods and helps to provide a more comprehensive understanding of the microbiome.
<h2>summary</h2>
Background: Several times serious human diseases are caused by eukaryotic parasites transmitted by mosquito vectors or water and food contamination. These pathogens are only a small proportion of eukaryotic single cells and parasites, and there are many very small creatures in the environment that have a general impact on health. We have good molecular biology analysis tools for bacteria, even fungi, but little is known about mostly fine single-cell eukaryotes and parasites: detecting these species is usually done with a microscope, unable to distinguish between related species, and only specific pathogenic bacteria can be detected at the molecular level.
Results: We propose a high-throughput sequencing method similar to 16S sequencing that can detect a wide range of single-cell eukaryotes and parasites, including species taxa of all human parasites. We designed and evaluated taxa-specific PCR primers to amplify 8 main taxa (apical subphylum Apicomplexa, Amoeba, Diplomonadida, Kinetoplastida, Phylum Parabasalia, Nematodes Nematodata, Platyhelminthes, and Microsporidia microsporidia). Using primers to screen the DNA of clinical, biological, and environmental samples, sequencing results analysis shows that most of the target classifications of self-knowledge and end-reporting can be identified.
Conclusion: This high-throughput analysis method can detect and identify eukaryotic parasites and related species in samples of complex organisms or environments. This approach is easy to develop and is an effective complement to today's approach, providing a more comprehensive perspective on the microbiome.
<h2>The main result</h2>
<h3>Table 1. Characteristics and specificity of primers</h3>
Table 1 shows the amplification of each pair of primers, as well as the electronic assessment of amplification range, information content, and specificity. The number of scalable species (species disclosed in the NCBI) is electronically simulated, and the DNA sequence matches into individual genera or species proportions, which belong to the target taxon. The last two columns are primer sequences.
<h3>Figure 1. Method flowchart</h3>
Pattern diagrams illustrate the process of augmentation, labeling, mixing, and sequencing.
<h3>Table 2. Examples of single-cell eukaryotic and parasite identification in biological or environmental samples</h3>
Each class of sample and each class of primers can be expanded up to 5 species to match the DNA sequence, which is more consistent with the NCBI identification ratio. When a DNA sequence matches more than one genus, the result is indicated. Detailed results are shown in Schedule 4.
<h3>Figure 2. Reconstruct the evolutionary tree of amplification sequences and sequences in NCBI</h3>
a. Adjacency trees show annotated amoeba sequences (black squares) and amplified sequences in human feces (green diamonds)
b. Adjacent trees show annotated dipterocarp sequences (black squares), fecal amplified sequences (green diamonds or circles), and amplified sequences (brown diamonds) in three Potomaco River samples.
<h2>method</h2>
<h3>PCR primer design</h3>
We designed primers to amplify human parasites: Apicomplexa, Amoeba, Diplomonidae of the yeast genus Blastocystis Diplomonadida, Kinetoplastida, Phylum Parabasalia, Nematodata Nematodata, Platyhelminthes, and microspores Microsporidia. For our purposes, primers need to meet the following requirements: 1. 2. All species in the target population need to be amplified, with very few non-specific amplifications; 2. The sequences of species to be identified have sufficient distinguishable genetic information; The amplification product should be short enough to be sequenced by Illumina.
<h3>Electronic assessment</h3>
Based on the relevant species in the NCBI public database, the reference process is https://github.com/MVesuviusC/2018_methods_paper to evaluate the heterogeneity of primers, amplification degree, amplification product effectiveness, etc
Main analysis process:
Analysis of natural samples
PCR amplification and high-throughput sequencing
Bioinformatics analysis
Evolutionary analysis
<h2>summary</h2>
This method, which provides species information on single-celled eukaryotes and parasites in samples, complements existing bacterial research and increases our understanding of the microbiome in human and animal health.
<h2>Warm-hearted daily</h2>
New primers extend the 18S rRNA sequencing detection range
Original title: High-throughput sequencing comprehensive detection of single-cell eukaryotes and parasites in biological or environmental samples
(1) Single-celled eukaryotes and parasites are important human pathogens, but lack convenient and specific detection methods;
(2) This paper designed a multi-pair of 18S rRNA gene primers to detect most single-cell eukaryotes and parasites, including apical compound animals, amoeba, yeast, dipterans, gotuss, nematodes, flat animals and microsporidium;
(3) The length range, resolution and other indicators of the amplification products were simulated and evaluated using the NCBI database, which were suitable for Illumina sequencing, and the natural sample evaluation results also covered most of the target taxons;
(4) This method is an effective supplement to the current sequencing of rRNA amplicons, which contributes to a more comprehensive understanding of the microbiome.
<h2>bibliography</h2>
New Primers Extend the 18S rRNA Sequencing Detection Range https://www.mr-gut.cn/papers/read/1058127404?kf=mobile.search
A high-throughput sequencing assay to comprehensively detect and characterize unicellular eukaryotes and helminths from biological and environmental samples. Microbiome, [9.133] DOI: https://microbiomejournal.biomedcentral.com/articles/10.1186/s40168-018-0581-6
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