scholarly journals Application of Next-Generation Sequencing Technology Based on Single Gene Locus in Species Identification of Mixed Meat Products

2021 ◽  
Vol 2021 ◽  
pp. 1-5
Author(s):  
Xinmei Liu ◽  
Zhiyang Liu ◽  
Yiyu Cheng ◽  
Haijing Wu ◽  
Wei Shen ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
16S Rrna ◽  
Species Identification ◽  
Growth Hormone Receptor ◽  
Single Gene ◽  
Meat Products ◽  
Quantitative Detection ◽  
Next Generation ◽  
Next Generation Sequencing Technology ◽  
Generation Sequencing

Polymerase chain reaction (PCR) detection is a commonly used method for species identification of meat products. However, this method is not suitable for the analysis of meat products containing multiple mixtures. This study aimed to test whether next-generation sequencing (NGS) technology could be used as a method for the certification of mixed meat products. In this study, five kinds of common meat (pigs, cattle, sheep, chickens, and ducks) were mixed as samples with different proportions. The primers designed from mitochondrial 16S rRNA and nuclear genome gene (growth hormone receptor, GHR), respectively, were used to detect these meats. The sequencing results of NGS were analyzed using a self-designed bioinformatics program. The fragments with similar sequences were classified and compared with the database to determine their species. The results showed that all five kinds of meat components could be correctly identified using these two primers. The meat composition could be detected as low as 0.5% in the mixed samples using the NGS technology targeting GHR gene fragments, which was superior to those targeting mitochondrial 16S rRNA. However, the quantitative detection of species in the mixture was not likely to be quite accurate due to the amplification bias of PCR amplification. These results showed that the NGS technology could be applied to identify meat species in mixtures.

scholarly journals Amplification of the V5 – V8 region of the 16S rRNA gene effectively speciates medically important genital tract Lactobacillus species in the upper female genital tract

2019 ◽  
Author(s):  
Jessica L. O’Callaghan ◽  
Dana Willner ◽  
Melissa Buttini ◽  
Flavia Huygens ◽  
Elise S. Pelzer
Keyword(s):  
Next Generation Sequencing ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Genital Tract ◽  
Rrna Gene ◽  
Next Generation ◽  
Sequencing Technology ◽  
Next Generation Sequencing Technology ◽  
The 16S Rrna Gene ◽  
Generation Sequencing

ABSTRACTBackgroundThe endometrial cavity is an upper genital tract site largely heralded as sterile, however, advances in culture-independent, next generation sequencing technology have revealed that this site harbours a rich microbial community which includes multiple Lactobacillus species. These bacteria are considered to be the most common non-pathogenic genital tract commensals. Next-generation sequencing of the female lower genital tract has revealed significant variation amongst microbial community composition with respect to Lactobacillus sp. in samples collected from healthy and diseased women. The aim of this study was to evaluate the ability of the 16S rRNA gene to characterize genital tract lactobacilli to species-level taxonomy.MethodsSamples were interrogated for the presence of microbial DNA using two-step next generation sequencing technology to exploit the V5–V8 regions of the 16S rRNA gene and compared to standard speciation using qPCR.ResultsThe V5-V8 region of the 16S rRNA gene has sufficient sequence variation within frequently encountered genital tract lactobacilli to allow accurate determination of relative abundance within the community, and speciation for several key community members without completing additional experimentation.ConclusionsNext-generation sequencing of clinical genital tract isolates is an effective method for high throughput identification to species-level of key Lactobacillus sp.IMPORTANCEHuman microbiome experiments, including the low biomass organs such as the upper genital tract, require the development of consensus protocols to ensure accurate comparison between such studies and our data forms an important foundation for future protocols.This paper provides evidence to support the selection of the V5-V8 regions of the 16S rRNA gene improved Lactobacillus speciation using next generation sequencing technology. The choice of variable region for broad-range amplification in microbiome studies is important due to preferential primer binding associated with some genera based on nucleotide sequence patterns. By utilising the V5-V8 region, multiple species of Lactobacillus can be characterised with relative confidence.

scholarly journals Development of a reference data set for assigning Streptococcus and Enterococcus species based on next generation sequencing of the 16S–23S rRNA region

2019 ◽  
Vol 8 (1) ◽  
Author(s):  
Maja Kosecka-Strojek ◽  
Artur J. Sabat ◽  
Viktoria Akkerboom ◽  
Anna M. D. Kooistra-Smid ◽  
Jacek Miedzobrodzki ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Sequence Analysis ◽  
16S Rrna ◽  
Species Identification ◽  
23S Rrna ◽  
Clinical Samples ◽  
Next Generation ◽  
Enterococcal Species ◽  
Reference Sequences ◽  
Generation Sequencing

Abstract Background Many members of Streptococcus and Enterococcus genera are clinically relevant opportunistic pathogens warranting accurate and rapid identification for targeted therapy. Currently, the developed method based on next generation sequencing (NGS) of the 16S–23S rRNA region proved to be a rapid, reliable and precise approach for species identification directly from polymicrobial and challenging clinical samples. The introduction of this new method to routine diagnostics is hindered by a lack of the reference sequences for the 16S–23S rRNA region for many bacterial species. The aim of this study was to develop a careful assignment for streptococcal and enterococcal species based on NGS of the 16S–23S rRNA region. Methods Thirty two strains recovered from clinical samples and 19 reference strains representing 42 streptococcal species and nine enterococcal species were subjected to bacterial identification by four Sanger-based sequencing methods targeting the genes encoding (i) 16S rRNA, (ii) sodA, (iii) tuf and (iv) rpoB; and NGS of the 16S–23S rRNA region. Results This study allowed obtainment and deposition of reference sequences of the 16S–23S rRNA region for 15 streptococcal and 3 enterococcal species followed by enrichment for 27 and 6 species, respectively, for which reference sequences were available in the databases. For Streptococcus, NGS of the 16S–23S rRNA region was as discriminative as Sanger sequencing of the tuf and rpoB genes allowing for an unambiguous identification of 93% of analyzed species. For Enterococcus, sodA, tuf and rpoB genes sequencing allowed for identification of all species, while the NGS-based method did not allow for identification of only one enterococcal species. For both genera, the sequence analysis of the 16S rRNA gene was endowed with a low identification potential and was inferior to that of other tested identification methods. Moreover, in case of phylogenetically related species the sequence analysis of only the intergenic spacer region was not sufficient enough to precisely identify Streptococcus strains at the species level. Conclusions Based on the developed reference dataset, clinically relevant streptococcal and enterococcal species can now be reliably identified by 16S–23S rRNA sequences in samples. This study will be useful for introduction of a novel diagnostic tool, NGS of the 16S–23S rRNA region, which undoubtedly is an improvement for reliable culture-independent species identification directly from polymicrobially constituted clinical samples.

A Novel Method for Microsatellite Instability Detection by Liquid Biopsy Based on Next- Generation Sequencing

2020 ◽  
Vol 15 ◽  
Author(s):  
Zheng Jiang ◽  
Hui Liu ◽  
Siwen Zhang ◽  
Jia Liu ◽  
Weitao Wang ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Microsatellite Instability ◽  
Liquid Biopsy ◽  
Tumor Tissue ◽  
High Sensitivity ◽  
Next Generation ◽  
Tissue Samples ◽  
Next Generation Sequencing Technology ◽  
Medication Selection ◽  
Generation Sequencing

Background: Microsatellite instability (MSI) is a prognostic biomarker used to guide medication selection in multiple cancers, such as colorectal cancer. Traditional PCR with capillary electrophoresis and next-generation sequencing using paired tumor tissue and leukocyte samples are the main approaches for MSI detection due to their high sensitivity and specificity. Currently, patient tissue samples are obtained through puncture or surgery, which causes injury and risk of concurrent disease, further illustrating the need for MSI detection by liquid biopsy. Methods: We propose an analytic method using paired plasma/leukocyte samples and MSI detection using next-generation sequencing technology. Based on the theoretical progress of oncogenesis, we hypothesized that the microsatellite site length in plasma equals the combination of the distribution of tumor tissue and leukocytes. Thus, we defined a window-judgement method to identify whether biomarkers were stable. Results: Compared to traditional PCR as the standard, we evaluated three methods in 20 samples (MSI-H:3/MSS:17): peak shifting method using tissue vs. leukocytes, peak shifting method using plasma vs. leukocytes, and our method using plasma vs. leukocytes. Compared to traditional PCR, we observed a sensitivity of 100%, 0%, and 100%, and a specificity of 100.00%, 94.12%, and 88.24%, respectively. Conclusion: Our method has the advantage of possibly detecting MSI in a liquid biopsy and provides a novel direction for future studies to increase the specificity of the method.

Molecular marker information from de novo assembled transcriptomes of chilli pepper (Capsicum annuum L.) varieties based on next-generation sequencing technology

2014 ◽  
Vol 12 (S1) ◽  
pp. S83-S86 ◽  
Author(s):  
Yul-Kyun Ahn ◽  
Swati Tripathi ◽  
Young-Il Cho ◽  
Jeong-Ho Kim ◽  
Hye-Eun Lee ◽  
...  
Keyword(s):  
Molecular Markers ◽  
Next Generation Sequencing ◽  
De Novo ◽  
Transcriptome Assembly ◽  
Sequence Variant ◽  
Nucleotide Polymorphisms ◽  
Next Generation ◽  
Chilli Pepper ◽  
Next Generation Sequencing Technology ◽  
Generation Sequencing

Next-generation sequencing technique has been known as a useful tool for de novo transcriptome assembly, functional annotation of genes and identification of molecular markers. This study was carried out to mine molecular markers from de novo assembled transcriptomes of four chilli pepper varieties, the highly pungent ‘Saengryeg 211’ and non-pungent ‘Saengryeg 213’ and variably pigmented ‘Mandarin’ and ‘Blackcluster’. Pyrosequencing of the complementary DNA library resulted in 361,671, 274,269, 279,221, and 316,357 raw reads, which were assembled in 23,607, 19,894, 18,340 and 20,357 contigs, for the four varieties, respectively. Detailed sequence variant analysis identified numerous potential single-nucleotide polymorphisms (SNPs) and simple sequence repeats (SSRs) for all the varieties for which the primers were designed. The transcriptome information and SNP/SSR markers generated in this study provide valuable resources for high-density molecular genetic mapping in chilli pepper and Quantitative trait loci analysis related to fruit qualities. These markers for pepper will be highly valuable for marker-assisted breeding and other genetic studies.

Next Generation Sequencing (NGS) approach applied to species identification in mixed processed seafood products

Food Control ◽  
2021 ◽  
pp. 108590
Author(s):  
Roberta Piredda ◽  
Anna Mottola ◽  
Giulia Cipriano ◽  
Roberto Carlucci ◽  
Giuseppina Ciccarese ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Species Identification ◽  
Next Generation ◽  
Seafood Products ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing
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