scholarly journals Evaluation of the Effects of Library Preparation Procedure and Sample Characteristics on the Accuracy of Metagenomic Profiles

mSystems ◽  
2021 ◽  
Author(s):  
Christopher A. Gaulke ◽  
Emily R. Schmeltzer ◽  
Mark Dasenko ◽  
Brett M. Tyler ◽  
Rebecca Vega Thurber ◽  
...  
Keyword(s):  
Microbial Communities ◽  
Environmental Samples ◽  
Metagenomic Library ◽  
Preparation Procedure ◽  
Library Preparation ◽  
Preparation Methods ◽  
Sequencing Technologies ◽  
Sample Characteristics

Metagenomic library preparation methods and sequencing technologies continue to advance rapidly, allowing researchers to characterize microbial communities in previously underexplored environmental samples and systems. However, widely accepted standardized library preparation methods can be cost-prohibitive.

Application of metagenomics to assess microbial communities in water and other environmental matrices

2015 ◽  
Vol 96 (1) ◽  
pp. 121-129 ◽  
Author(s):  
Christopher Staley ◽  
Michael J. Sadowsky
Keyword(s):  
Public Health ◽  
Microbial Communities ◽  
Surface Waters ◽  
Environmental Samples ◽  
Subsequent Development ◽  
Complete Characterization ◽  
Human Pathogens ◽  
Environmental Matrices ◽  
Sequencing Technologies ◽  
Anthropogenic Inputs

The emergence of metagenomics-based approaches in biology has overcome historical culture-based biases in microbiological studies. This has also enabled a more comprehensive assessment of the microbial ecology of environmental samples. The subsequent development of next-generation sequencing technologies, able to produce hundreds of millions of sequences at improved cost and speed, necessitated a computational shift from user-supervised alignment and analysis pipelines, that were used previously for vector-based metagenomic studies that relied on Sanger sequencing. Current computational advances have expanded the scope of microbial biogeography studies and offered novel insights into microbial responses to environmental variation and anthropogenic inputs into ecosystems. However, new biostatistical and computational approaches are required to handle the large volume and complexity of these new multivariate datasets. While this has allowed more complete characterization of taxonomic, phylogenetic and functional microbial diversity, these tools are still limited by methodological biases, incomplete databases, and the high cost of fully characterizing environmental biodiversity. This review addresses the evolution of methods to monitor surface waters and characterize environmental samples through the recent computational advances in metagenomics, with an emphasis on the study of surface waters. These new methods have provided an abundance of opportunities to expand our understanding of the interaction between microbial communities and public health. Specifically, they have allowed for comprehensive monitoring of bacterial communities in surface waters for changes in community structure associated with faecal contamination and the presence of human pathogens, rather than relying on only a few indicator bacteria to direct public health concerns.

scholarly journals Library preparation and sequencing platform introduce bias in metagenomics characterisation of microbial communities

2019 ◽  
Author(s):  
Casper S. Poulsen ◽  
Sünje J. Pamp ◽  
Claus T. Ekstrøm ◽  
Frank M. Aarestrup
Keyword(s):  
Microbial Communities ◽  
Dna Isolation ◽  
Meta Analysis ◽  
Library Preparation ◽  
Sample Processing ◽  
Fecal Samples ◽  
Sequencing Platform ◽  
Sequencing Technologies ◽  
Systematic Effects

AbstractNext generation sequencing technologies have become increasingly used to describe microbial communities. Metagenomics characterization of microbiomes is associated with minimal manipulation during sample processing, which includes sampling, storage, DNA isolation, library preparation and sequencing, before the raw data are obtained. Here we assess the effect of library preparation using a kit with a polymerase chain reaction (PCR) step (Nextera) and two PCR-free kits (NEXTflex and KAPA), and the effect of sequencing platform (HiSeq and NextSeq) on the description of microbial communities in pig feces and sewage. Two pig fecal samples were obtained from different farms and two sewage samples were collected as inlet water at a local wastewater treatment facility. Samples were processed to both perform DNA-isolation immediately upon arrival in the lab and after storage for 64 hours at −80°C, DNA isolation was performed in duplicate.We find that both library preparation and sequencing platform had systematic effects on the microbial community description. The effects were at a level that made differentiating between the two pig fecal samples difficult. The sewage samples represented two very different communities and were at all times distinguishable from each other. We find that library preparation and sequencing platform introduced more variation than freezing the samples. The community changes did not seem associated with contamination during processing and distinct patterns connected specific types of organisms with a processing method, but it was difficult to generalize between samples. This highlights the need for continuous validation of the effect of sample processing in different types of samples and that all processing steps need to be considered when comparing between studies. We believe standardization of sample processing is key to generate comparable data within a study and that comparisons of differently generated data, e.g. in a meta-analysis, should be performed cautiously.

Reconstructing single genomes from complex microbial communities

2016 ◽  
Vol 58 (3) ◽  
Author(s):  
Dongwan D. Kang ◽  
Edward M. Rubin ◽  
Zhong Wang
Keyword(s):  
Microbial Communities ◽  
Environmental Samples ◽  
Research Community ◽  
Functional Metagenomics ◽  
High Quality ◽  
Microbial Genomes ◽  
Sequencing Technologies ◽  
Approaches To Study ◽  
Generation Sequencing ◽  
Pathway Level

AbstractHigh throughput next generation sequencing technologies have enabled cultivation-independent approaches to study microbial communities in environmental samples. To date much of functional metagenomics has been limited to the gene or pathway level. Recent breakthroughs in metagenome binning have made it feasible to reconstruct high quality, individual microbial genomes from complex communities with thousands of species. In this review we aim to compare several automated metagenome binning software tools for their performance, and provide a practical guide for the metagenomics research community to carry out successful binning analyses.

scholarly journals Comparison of library preparation methods reveals their impact on interpretation of metatranscriptomic data

BMC Genomics ◽  
2014 ◽  
Vol 15 (1) ◽  
pp. 912 ◽  
Author(s):  
Adriana Alberti ◽  
Caroline Belser ◽  
Stéfan Engelen ◽  
Laurie Bertrand ◽  
Céline Orvain ◽  
...  
Keyword(s):  
Library Preparation ◽  
Preparation Methods

Phylum-Specific Primer Design and Implication in Quantification of the Microbial Community Structure in GuJingGong Pit Mud

2014 ◽  
Vol 1051 ◽  
pp. 311-316 ◽  
Author(s):  
Xi Mei Luo ◽  
Zhi Lei Gao ◽  
Hui Min Zhang ◽  
An Jun Li ◽  
Hong Kui He ◽  
...  
Keyword(s):  
Community Structure ◽  
Microbial Community ◽  
Microbial Communities ◽  
Microbial Community Structure ◽  
Real Time Quantitative Pcr ◽  
Sequencing Technologies ◽  
Specific Pcr ◽  
Pit Mud ◽  
Almost All ◽  
Polymerase Chain Reaction Primers

In recent years, despite the significant improvement of sequencing technologies such as the pyrosequencing, rapid evaluation of microbial community structures remains very difficult because of the abundance and complexity of organisms in almost all natural microbial communities. In this paper, a group of phylum-specific primers were elaborately designed based on a single nucleotide discrimination technology to quantify the main microbial community structure from GuJingGong pit mud samples using the real-time quantitative PCR (qPCR). Specific PCR (polymerase chain reaction) primers targeting a particular group would provide promising sensitivity and more in-depth assessment of microbial communities.

scholarly journals Facile, High Quality Sequencing of Bacterial Genomes from Small Amounts of DNA

2014 ◽  
Vol 2014 ◽  
pp. 1-8
Author(s):  
Momchilo Vuyisich ◽  
Ayesha Arefin ◽  
Karen Davenport ◽  
Shihai Feng ◽  
Cheryl Gleasner ◽  
...  
Keyword(s):  
Genomic Dna ◽  
De Novo ◽  
Gc Content ◽  
Library Preparation ◽  
Sequencing Data ◽  
Bacterial Genomes ◽  
Dna Amount ◽  
High Quality ◽  
Preparation Methods

Sequencing bacterial genomes has traditionally required large amounts of genomic DNA (~1 μg). There have been few studies to determine the effects of the input DNA amount or library preparation method on the quality of sequencing data. Several new commercially available library preparation methods enable shotgun sequencing from as little as 1 ng of input DNA. In this study, we evaluated the NEBNext Ultra library preparation reagents for sequencing bacterial genomes. We have evaluated the utility of NEBNext Ultra for resequencing andde novoassembly of four bacterial genomes and compared its performance with the TruSeq library preparation kit. The NEBNext Ultra reagents enable high quality resequencing andde novoassembly of a variety of bacterial genomes when using 100 ng of input genomic DNA. For the two most challenging genomes (Burkholderiaspp.), which have the highest GC content and are the longest, we also show that the quality of both resequencing andde novoassembly is not decreased when only 10 ng of input genomic DNA is used.

scholarly journals A phylogenetic transform enhances analysis of compositional microbiota data

2016 ◽  
Author(s):  
Justin D Silverman ◽  
Alex Washburne ◽  
Sayan Mukherjee ◽  
Lawrence A David
Keyword(s):  
Microbial Communities ◽  
Relative Abundance ◽  
Dominant Role ◽  
Phylogenetic Information ◽  
Abundance Data ◽  
Sequencing Technologies ◽  
Environmental Selection ◽  
Health And Disease ◽  
High Throughput Dna Sequencing ◽  
Spurious Results

ABSTRACTHigh-throughput DNA sequencing technologies have revolutionized the study of microbial communities (microbiota) and have revealed their importance in both human health and disease. However, due to technical limitations, data from microbiota surveys reflect the relative abundance of bacterial taxa and not their absolute levels. It is well known that applying common statistical methods, such as correlation or hypothesis testing, to relative abundance data can lead to spurious results. Here, we introduce the PhILR transform, a data transform that utilizes microbial phylogenetic information. This transform enables off-the-shelf statistical tools to be applied to microbiota surveys free from artifacts usually associated with analysis of relative abundance data. Using environmental and human-associated microbial community datasets as benchmarks, we find that the PhILR transform significantly improves the performance of distance-based and machine learning-based statistics, boosting the accuracy of widely used algorithms on reference benchmarks by 90%. Because the PhILR transform relies on bacterial phylogenies, statistics applied in the PhILR coordinate system are also framed within an evolutionary perspective. Regression on PhILR transformed human microbiota data identified evolutionarily neighboring bacterial clades that may have differentiated to adapt to distinct body sites. Variance statistics showed that the degree of covariation of bacterial clades across human body sites tended to increase with phylogenetic relatedness between clades. These findings support the hypothesis that environmental selection, not competition between bacteria, plays a dominant role in structuring human-associated microbial communities.

scholarly journals CAMISIM: Simulating metagenomes and microbial communities

2018 ◽  
Author(s):  
Adrian Fritz ◽  
Peter Hofmann ◽  
Stephan Majda ◽  
Eik Dahms ◽  
Johannes Dröge ◽  
...  
Keyword(s):  
Microbial Communities ◽  
De Novo ◽  
Real Data ◽  
Small Data ◽  
Data Sets ◽  
Sequencing Data ◽  
Taxonomic Profiling ◽  
Benchmark Data ◽  
Sequencing Technologies ◽  
Wide Range

Shotgun metagenome data sets of microbial communities are highly diverse, not only due to the natural variation of the underlying biological systems, but also due to differences in laboratory protocols, replicate numbers, and sequencing technologies. Accordingly, to effectively assess the performance of metagenomic analysis software, a wide range of benchmark data sets are required. Here, we describe the CAMISIM microbial community and metagenome simulator. The software can model different microbial abundance profiles, multi-sample time series and differential abundance studies, includes real and simulated strain-level diversity, and generates second and third generation sequencing data from taxonomic profiles or de novo. Gold standards are created for sequence assembly, genome binning, taxonomic binning, and taxonomic profiling. CAMSIM generated the benchmark data sets of the first CAMI challenge. For two simulated multi-sample data sets of the human and mouse gut microbiomes we observed high functional congruence to the real data. As further applications, we investigated the effect of varying evolutionary genome divergence, sequencing depth, and read error profiles on two popular metagenome assemblers, MEGAHIT and metaSPAdes, on several thousand small data sets generated with CAMISIM. CAMISIM can simulate a wide variety of microbial communities and metagenome data sets together with truth standards for method evaluation. All data sets and the software are freely available at: https://github.com/CAMI-challenge/CAMISIM

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