Why are universal 16s rdna primers used in your experiment

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16S ribosomal RNA

why are universal 16s rdna primers used in your experiment

What Is 16S rDNA

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Next-generation sequencing technologies have revolutionized the methods for studying microbial ecology by enabling high-resolution community profiling. However, the use of these technologies in unraveling the plant microbiome remains challenging. Therefore, we experimentally tested a series of commonly used primers for the analysis of plant-associated bacterial communities using pyrosequencing. Results indicate that experimental evaluation of primers provide valuable information that could contribute in the selection of suitable primer pairs for 16S rDNA metabarcoding studies in plant-microbiota research. Furthermore, we show that primer pair FR outperforms all other primer pairs in our study in the elimination of non-target DNA and retrieval of bacterial OTUs. The development and implementation of next-generation sequencing technologies NGS and their corresponding bioinformatics tools have revolutionized the methods for studying microbial ecology by enabling high-resolution community profiling Margulies et al. Currently, the most used next generation sequencing platforms in the profiling of microbiomes are the pyrosequencing technology Margulies et al.

Targeted amplicon sequencing of the 16S ribosomal RNA gene is one of the key tools for studying microbial diversity. The accuracy of this approach strongly depends on the choice of primer pairs and, in particular, on the balance between efficiency, specificity and sensitivity in the amplification of the different bacterial 16S sequences contained in a sample. There is thus the need for computational methods to design optimal bacterial 16S primers able to take into account the knowledge provided by the new sequencing technologies. We propose here a computational method for optimizing the choice of primer sets, based on multi-objective optimization, which simultaneously: 1 maximizes efficiency and specificity of target amplification; 2 maximizes the number of different bacterial 16S sequences matched by at least one primer; 3 minimizes the differences in the number of primers matching each bacterial 16S sequence. Our algorithm can be applied to any desired amplicon length without affecting computational performance. Results show that our strategy is able to find better primer pairs than the ones available in the literature according to all three optimization criteria. We also experimentally validated three of the primer pairs identified by our method on multiple bacterial species, belonging to different genera and phyla.

B. They will anneal to unique sequences of genes encoding 16S rRNA in specific to highly conserved areas of the gene that encodes bacterial 16S rRNA.
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Next-generation sequencing NGS technology has transformed metagenomics because the high-throughput data allow an in-depth exploration of a complex microbial community. Assembling 16S rDNA segments into longer sequences has been proposed for improving species identification. Current approaches, however, either suffer from amplification bias due to one single primer or insufficient 16S rDNA reads in whole genome sequencing data. Multiple primers were used to amplify different 16S rDNA segments for sequencing, followed by read classification and assembly. This permitted targeted sequencing while reducing primer bias. For test samples containing four known bacteria, accurate and near full-length 16S rDNAs of three known bacteria were obtained.

Selection of optimal primer pairs in 16S rRNA gene sequencing is a pivotal issue in microorganism diversity analysis. However, limited effort has been put into investigation of specific primer sets for analysis of the bacterial diversity of aging flue-cured tobaccos AFTs , as well as prediction of the function of the bacterial community. Results revealed that the primer set FR covering the amplification region V5V6V7 gave a more accurate picture of the bacterial community structure of AFTs, with lower co-amplification levels of chloroplast and mitochondrial genes, and more genera covered than when using the other primers. In addition, functional gene prediction suggested that the microbiome of AFTs was involved in kinds of interested pathways. A high abundance of functional genes involved in nitrogen metabolism was detected in AFTs, reflecting a high level of bacteria involved in degrading harmful nitrogen compounds and generating nitrogenous nutrients for others. Additionally, the functional genes involved in biosynthesis of valuable metabolites and degradation of toxic compounds provided information that the AFTs possess a huge library of microorganisms and genes that could be applied to further studies.

The genes coding for it are referred to as 16S rRNA gene and are used in reconstructing phylogenies , due to the slow rates of evolution of this region of the gene. Fox were two of the people who pioneered the use of 16S rRNA in phylogenetics in Multiple sequences of the 16S rRNA gene can exist within a single bacterium. The 16S rRNA gene is used for phylogenetic studies [6] as it is highly conserved between different species of bacteria and archaea. The most common primer pair was devised by Weisburg et al. The two primers are almost identical, but 27F has an M instead of a C.



Optimizing PCR primers targeting the bacterial 16S ribosomal RNA gene

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I want to know the reason why we particularly use 16S rRNA for identifying makes it possible to construct *universal primers* that can amplify 16S rRNA genes.
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