Reuse Recipe Document for: Directional high-throughput sequencing of RNAs without gene-specific primers

2015 ◽  
Vol 82 (2) ◽  
pp. 491-501 ◽  
Author(s):  
Ida Karlsson ◽  
Véronique Edel-Hermann ◽  
Nadine Gautheron ◽  
Mikael Brandström Durling ◽  
Anna-Karin Kolseth ◽  
...  

ABSTRACTFusariumis a large and diverse genus of fungi of great agricultural and economic importance, containing many plant pathogens and mycotoxin producers. To date, high-throughput sequencing ofFusariumcommunities has been limited by the lack of genus-specific primers targeting regions with high discriminatory power at the species level. In the present study, we evaluated twoFusarium-specific primer pairs targeting translation elongation factor 1 (TEF1). We also present the new primer pair Fa+7/Ra+6. MockFusariumcommunities reflecting phylogenetic diversity were used to evaluate the accuracy of the primers in reflecting the relative abundance of the species. TEF1 amplicons were subjected to 454 high-throughput sequencing to characterizeFusariumcommunities. Field samples from soil and wheat kernels were included to test the method on more-complex material. For kernel samples, a single PCR was sufficient, while for soil samples, nested PCR was necessary. The newly developed primer pairs Fa+7/Ra+6 and Fa/Ra accurately reflectedFusariumspecies composition in mock DNA communities. In field samples, 47Fusariumoperational taxonomic units were identified, with the highestFusariumdiversity in soil. TheFusariumcommunity in soil was dominated by members of theFusarium incarnatum-Fusarium equisetispecies complex, contradicting findings in previous studies. The method was successfully applied to analyzeFusariumcommunities in soil and plant material and can facilitate further studies ofFusariumecology.


2020 ◽  
Author(s):  
Lin zheng ◽  
Ling-Wei Zhu ◽  
Jia-yao Guan ◽  
Ying Wang ◽  
Jie Jing ◽  
...  

Abstract Background: In August 2018, a large number of migratory birds died in Chifeng, Neimeng. We were entrusted by local animal disease prevention and control center to collect the migratory bird epidemic materials and their living environmental water, and in 2019, we collect the local migratory bird stool and their living environmental water again. The bacterial communities in migratory bird epidemic materials, water and aquatic plants are profifiled by high-throughput sequencing of the V3–V4 hypervariable region of 16S rDNA gene.Results: We found that the dominant phylum between migratory bird epidemic materials, water and aquatic plant were Proteobacteia, Bacteroidetes, Firmicutes, Fusobacteria and Verrucomicrobia in 2018. One year later, we found that the dominant phylum between migratory bird stool, water and aquatic plant were Proteobacteia, Firmicutes, Cyanobacteria, Bacteroidetes. The relative abundance among bacterial phylum notably differed between two years. The relative abundance of Fusobacteria and Verrucomicrobia were higher in samples in 2018, while that of Cyanobacteria was higher in water, 2019. The relative abundance of Fusobacteria in migratory bird epidemic materials began to decline in the later period over time. At genus level, the relative abundance of Vibrio, Clostridium and other patnogenic bacteria decreased markedly and disappeared in 2019. The salt content and pH show a downward trend.Conclusions: Differences in diet and geographical location can lead to diversification in migratory bird intestinal flora. It is necessary to pay attention to diversification in intestinal flora of migratory birds, especially the abundance of vibrio in intestine. The overall structure of intestinal flora and relative abundance changes of various species are showed intuitively by 16S rDNA amplicon. But comparing to PCR which using specific primers, the accuracy and sensitivity are relatively poor. In the case of purposeful detection, it can be used in combination with both 16S rDNA amplicon and PCR which using specific primers. They are more accurately, because of supplement each other.


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