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RNA-seq03:21

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RNA sequencing, or RNA-Seq, is a high-throughput sequencing technology used to study the transcriptome of a cell. Transcriptomics helps to interpret the functional elements of a genome and identify the molecular constituents of an organism. Additionally, it also helps in understanding the development of an organism and the occurrence of diseases. 
Before the discovery of RNA-seq, microarray-based methods and Sanger sequencing were used for transcriptome analysis. However, while...
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Next-generation Sequencing of 16S Ribosomal RNA Gene Amplicons
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Ecological Observations Based on Functional Gene Sequencing Are Sensitive to the Amplicon Processing Method.

Fabien Cholet1, Agata Lisik2, Hélène Agogué3

  • 1Infrastructure and Environment, James Watt School of Engineering, University of Glasgowgrid.8756.c, Glasgow, Scotland.

Msphere
|August 8, 2022
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Summary
This summary is machine-generated.

The amplicon sequence variant (ASV) method offers better standardization for functional gene analysis than operational taxonomic units (OTUs). This study compares ASV and OTU approaches for microbial nitrogen-cycling genes, recommending ASVs for improved data reliability and reproducibility.

Keywords:
amplicon sequence variantamplicon sequencingcoastal sedimentsfunctional genesnitrogen cycle

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Area of Science:

  • Microbial ecology
  • Bioinformatics
  • Molecular biology

Background:

  • Traditional operational taxonomic units (OTUs) based on sequence similarity thresholds are debated for functional gene analysis due to lack of consensus.
  • The amplicon sequence variant (ASV) method, using noise-error models for abundance profiles, presents a potential alternative for higher resolution.
  • Standardization in microbial amplicon sequencing data analysis is crucial for reliability and reproducibility, especially for functional genes.

Purpose of the Study:

  • To compare the performance of OTU and ASV-based amplicon reconstruction methods for functional genes.
  • To evaluate taxonomy assignment methods, Naive Bayesian Classifier (NBC) and Bayesian Lowest Common Ancestor (BLCA), in functional gene analysis.
  • To assess the impact of sedimentary structures on microbial nitrogen-cycling communities using different reconstruction approaches.

Main Methods:

  • Comparison of OTU (various thresholds) and ASV methods for reconstructing functional gene communities (amoA, nxrB, nirS, nirK, nrfA).
  • Application of NBC and BLCA algorithms for taxonomy assignment.
  • Analysis of microbial communities from the Brouage mudflat, France, focusing on nitrogen-cycling genes and sedimentary structures.

Main Results:

  • Significant effects of sedimentary structure on community composition were observed for several genes (AOA amoA, AOB amoA, nirS) using ASVs and specific OTU thresholds.
  • Discrepancies were found between ASV and OTU methods for AOB amoA and nrfA, while nirK and nxrB showed consistent results across methods.
  • BLCA provided more phylogenetically relevant classifications when reference databases matched the study environment; NBC performed better with dissimilar sequences.

Conclusions:

  • The ASV approach offers superior standardization potential for functional gene amplicon data compared to OTUs.
  • The choice of reconstruction method (ASV vs. OTU) and taxonomy assignment algorithm can influence ecological conclusions.
  • A framework for quality filtering based on protein sequence verification is proposed to enhance data quality and standardization.