scholarly journals The effect of reverse transcription enzymes and conditions on high throughput amplicon sequencing of the 16S rRNA

2019 ◽  
Author(s):  
Adam Šťovíček ◽  
Smadar Cohen-Chalamish ◽  
Osnat Gillor
Keyword(s):  
Microbial Communities ◽  
High Throughput ◽  
Ribosomal Rna ◽  
Reverse Transcription ◽  
High Throughput Sequencing ◽  
Dna Analysis ◽  
Amplicon Sequencing ◽  
Soil Microbiology ◽  
Active Population ◽  
Dna And Rna

It is assumed that the sequencing of ribosomes better reflects the active microbial community than the sequencing of the ribosomal RNA encoding genes. Yet, many studies exploring microbial communities in various environments, ranging from the human gut to deep oceans, questioned the validity of this paradigm due to the discrepancies between the DNA and RNA based communities. Here we focus on an often neglected key step in the analysis, the reverse transcription (RT) reaction. Previous studies showed that RT may introduce biases when expressed genes and ribosmal rRNA are quantified, yet its effect on microbial diversity and community composition was never tested. High throughput sequencing of ribosomal RNA is a valuable tool to understand microbial communities as it better describes the active population than DNA analysis. However, the necessary step of RT may introduce biases that have so far been poorly described. In this manuscript, we compare three RT enzymes, commonly used in soil microbiology, in two temperature modes to determine a potential source of bias due to non-standardized RT conditions. In our comparisons, we have observed up to 6 fold differences in bacterial class abundance. A temperature induced bias can be partially explained by G-C content of the affected bacterial groups, thus pointing towards a need for higher reaction temperatures. However, another source of bias was due to enzyme processivity differences. This bias is potentially hard to overcome and thus mitigating it might require the use of one enzyme for the sake of cross-study comparison.

scholarly journals The effect of reverse transcription enzymes and conditions on high throughput amplicon sequencing of the 16S rRNA

PeerJ ◽  
2019 ◽  
Vol 7 ◽  
pp. e7608
Author(s):  
Adam Šťovíček ◽  
Smadar Cohen-Chalamish ◽  
Osnat Gillor
Keyword(s):  
Microbial Communities ◽  
High Throughput ◽  
Ribosomal Rna ◽  
Reverse Transcription ◽  
High Throughput Sequencing ◽  
Dna Analysis ◽  
Amplicon Sequencing ◽  
Soil Microbiology ◽  
Active Population ◽  
Dna And Rna

It is assumed that the sequencing of ribosomes better reflects the active microbial community than the sequencing of the ribosomal RNA encoding genes. Yet, many studies exploring microbial communities in various environments, ranging from the human gut to deep oceans, questioned the validity of this paradigm due to the discrepancies between the DNA and RNA based communities. Here, we focus on an often neglected key step in the analysis, the reverse transcription (RT) reaction. Previous studies showed that RT may introduce biases when expressed genes and ribosmal rRNA are quantified, yet its effect on microbial diversity and community composition was never tested. High throughput sequencing of ribosomal RNA is a valuable tool to understand microbial communities as it better describes the active population than DNA analysis. However, the necessary step of RT may introduce biases that have so far been poorly described. In this manuscript, we compare three RT enzymes, commonly used in soil microbiology, in two temperature modes to determine a potential source of bias due to non-standardized RT conditions. In our comparisons, we have observed up to six fold differences in bacterial class abundance. A temperature induced bias can be partially explained by G-C content of the affected bacterial groups, thus pointing toward a need for higher reaction temperatures. However, another source of bias was due to enzyme processivity differences. This bias is potentially hard to overcome and thus mitigating it might require the use of one enzyme for the sake of cross-study comparison.

scholarly journals The effect of reverse transcription enzymes and conditions on high throughput amplicon sequencing of the 16S rRNA

2019 ◽  
Author(s):  
Adam Šťovíček ◽  
Smadar Cohen-Chalamish ◽  
Osnat Gillor
Keyword(s):  
Microbial Communities ◽  
High Throughput ◽  
Ribosomal Rna ◽  
Reverse Transcription ◽  
High Throughput Sequencing ◽  
Dna Analysis ◽  
Amplicon Sequencing ◽  
Soil Microbiology ◽  
Active Population ◽  
Dna And Rna

It is assumed that the sequencing of ribosomes better reflects the active microbial community than the sequencing of the ribosomal RNA encoding genes. Yet, many studies exploring microbial communities in various environments, ranging from the human gut to deep oceans, questioned the validity of this paradigm due to the discrepancies between the DNA and RNA based communities. Here we focus on an often neglected key step in the analysis, the reverse transcription (RT) reaction. Previous studies showed that RT may introduce biases when expressed genes and ribosmal rRNA are quantified, yet its effect on microbial diversity and community composition was never tested. High throughput sequencing of ribosomal RNA is a valuable tool to understand microbial communities as it better describes the active population than DNA analysis. However, the necessary step of RT may introduce biases that have so far been poorly described. In this manuscript, we compare three RT enzymes, commonly used in soil microbiology, in two temperature modes to determine a potential source of bias due to non-standardized RT conditions. In our comparisons, we have observed up to 6 fold differences in bacterial class abundance. A temperature induced bias can be partially explained by G-C content of the affected bacterial groups, thus pointing towards a need for higher reaction temperatures. However, another source of bias was due to enzyme processivity differences. This bias is potentially hard to overcome and thus mitigating it might require the use of one enzyme for the sake of cross-study comparison.

scholarly journals The effect of reverse transcription enzymes and conditions on high throughput amplicon sequencing of the 16S rRNA

2019 ◽  
Author(s):  
Adam Šťovíček ◽  
Smadar Cohen-Chalamish ◽  
Osnat Gillor
Keyword(s):  
Microbial Communities ◽  
High Throughput ◽  
Ribosomal Rna ◽  
Reverse Transcription ◽  
High Throughput Sequencing ◽  
Dna Analysis ◽  
Amplicon Sequencing ◽  
Soil Microbiology ◽  
Active Population ◽  
Dna And Rna

It is assumed that the sequencing of ribosomes better reflects the active microbial community than the sequencing of the ribosomal RNA encoding genes. Yet, many studies exploring microbial communities in various environments, ranging from the human gut to deep oceans, questioned the validity of this paradigm due to the discrepancies between the DNA and RNA based communities. Here we focus on an often neglected key step in the analysis, the reverse transcription (RT) reaction. Previous studies showed that RT may introduce biases when expressed genes and ribosomes are quantified, yet its effect on microbial diversity and community composition was never tested. High throughput sequencing of ribosomal RNA is a valuable tool to understand microbial communities as it better describes the active population than DNA analysis. However, the necessary step of RT may introduce biases that have so far been poorly described. In this manuscript, we compare three reverse transcription enzymes, commonly used in soil microbiology, in two temperature modes to determine a potential source of bias due to non-standardized reverse transcription conditions. In our comparisons, we have observed up to 6 fold differences in bacterial class abundance. A temperature induced bias can be partially explained by G-C content of the affected bacterial groups, thus pointing towards a need for higher reaction temperatures. However, another source of bias was due to enzyme processivity differences. This bias is potentially hard to overcome and thus mitigating it might require the use of one enzyme for the sake of cross-study comparison.

scholarly journals Extremely Halophilic Biohydrogen Producing Microbial Communities from High-Salinity Soil and Salt Evaporation Pond

Fuels ◽  
2021 ◽  
Vol 2 (2) ◽  
pp. 241-252
Author(s):  
Dyah Asri Handayani Taroepratjeka ◽  
Tsuyoshi Imai ◽  
Prapaipid Chairattanamanokorn ◽  
Alissara Reungsang
Keyword(s):  
Microbial Communities ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
High Salinity ◽  
Amplicon Sequencing ◽  
Spatial Proximity ◽  
Lignocellulosic Waste ◽  
Evaporation Pond ◽  
Operational Taxonomic Units ◽  
Determining Factor

Extreme halophiles offer the advantage to save on the costs of sterilization and water for biohydrogen production from lignocellulosic waste after the pretreatment process with their ability to withstand extreme salt concentrations. This study identifies the dominant hydrogen-producing genera and species among the acclimatized, extremely halotolerant microbial communities taken from two salt-damaged soil locations in Khon Kaen and one location from the salt evaporation pond in Samut Sakhon, Thailand. The microbial communities’ V3–V4 regions of 16srRNA were analyzed using high-throughput amplicon sequencing. A total of 345 operational taxonomic units were obtained and the high-throughput sequencing confirmed that Firmicutes was the dominant phyla of the three communities. Halanaerobium fermentans and Halanaerobacter lacunarum were the dominant hydrogen-producing species of the communities. Spatial proximity was not found to be a determining factor for similarities between these extremely halophilic microbial communities. Through the study of the microbial communities, strategies can be developed to increase biohydrogen molar yield.

Bioinformatics Analysis of The Rhizosphere Microbiota of Dangshan Su Pear in Different Soil Types

2020 ◽  
Vol 15 (5) ◽  
pp. 503-514
Author(s):  
Xiaojing Ma ◽  
Sambhaji Balaso Thakar ◽  
Huimin Zhang ◽  
Zequan Yu ◽  
Li Meng ◽  
...  
Keyword(s):  
Microbial Communities ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Terrestrial Ecosystems ◽  
Amplicon Sequencing ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Soil Types ◽  
Plant Materials ◽  
Microbial Structures

Background: The rhizosphere microbiota are of vital importance for plant growth and health in terrestrial ecosystems. There have been extensive studies aiming to identify the microbial communities as well as their relationship with host plants in different soil types. Objective: In the present study, we have employed the high-throughput sequencing technology to investigate the composition and structure of rhizosphere microbiota prosperous at the root of Dangshan Su pear growing in sandy soil and clay soil. Methods: A high-throughput amplicon sequencing survey of the bacterial 16S rRNA genes and fungal ITS regions from rhizosphere microbiota was firstly performed. Subsequently, several common bacterial and fungal communities were found to be essential to Dangshan Su pear by using a series of bioinformatics and statistics tools. Finally, the soil-preferred microbiota were identified through variance analysis and further characterized in the genus level. Result: Dangshan Su pears host rich and diverse microbial communities in thin layer of soil adhering to their roots. The composition of dominant microbial phyla is similar across different soil types, but the quantity of each microbial community varies significantly. Specially, the relative abundance of Firmicutes increases from 9.69% to 61.66% as the soil ecosystem changes from clay to sandy, which can be not only conducive to the degradation of complex plant materials, but also responsible for the disinfestation of pathogens. Conclusion: Our results have a symbolic significance for the potential efforts of rhizosphere microbiota on the soil bioavailability and plant health. Through selecting soil types and altering microbial structures, the improvement of fruit quality of Dangshan Su pear is expected to be achieved.

scholarly journals Different Amplicon Targets for Sequencing-Based Studies of Fungal Diversity

2017 ◽  
Vol 83 (17) ◽  
Author(s):  
Francesca De Filippis ◽  
Manolo Laiola ◽  
Giuseppe Blaiotta ◽  
Danilo Ercolini
Keyword(s):  
Microbial Ecology ◽  
High Throughput ◽  
Biological Samples ◽  
Fungal Diversity ◽  
High Throughput Sequencing ◽  
Amplicon Sequencing ◽  
Fungal Species ◽  
Fungal Communities ◽  
Its2 Region ◽  
Mock Community

ABSTRACT Target-gene amplicon sequencing is the most exploited high-throughput sequencing application in microbial ecology. The targets are taxonomically relevant genes, with 16S rRNA being the gold standard for bacteria. As for fungi, the most commonly used target is the internal transcribed spacer (ITS). However, the uneven ITS length among species may promote preferential amplification and sequencing and incorrect estimation of their abundance. Therefore, the use of different targets is desirable. We evaluated the use of three different target amplicons for the characterization of fungal diversity. After an in silico primer evaluation, we compared three amplicons (the ITS1-ITS2 region [ITS1-2], 18S ribosomal small subunit RNA, and the D1/D2 domain of the 26S ribosomal large subunit RNA), using biological samples and a mock community of common fungal species. All three targets allowed for accurate identification of the species present. Nevertheless, high heterogeneity in ITS1-2 length was found, and this caused an overestimation of the abundance of species with a shorter ITS, while both 18S and 26S amplicons allowed for more reliable quantification. We demonstrated that ITS1-2 amplicon sequencing, although widely used, may lead to an incorrect evaluation of fungal communities, and efforts should be made to promote the use of different targets in sequencing-based microbial ecology studies. IMPORTANCE Amplicon-sequencing approaches for fungi may rely on different targets affecting the diversity and abundance of the fungal species. An increasing number of studies will address fungal diversity by high-throughput amplicon sequencing. The description of the communities must be accurate and reliable in order to draw useful insights and to address both ecological and biological questions. By analyzing a mock community and several biological samples, we demonstrate that using different amplicon targets may change the results of fungal microbiota analysis, and we highlight how a careful choice of the target is fundamental for a thorough description of the fungal communities.

scholarly journals Novel Approach to Study the Diversity of Soil Microbial Communities in Qatar

2020 ◽  
Author(s):  
Jane Oja ◽  
Sakeenah Adenan ◽  
Abdel-Fattah Talaat ◽  
Juha Alatalo
Keyword(s):  
Microbial Communities ◽  
High Throughput ◽  
Mycorrhizal Fungi ◽  
High Throughput Sequencing ◽  
Soil Microbial Communities ◽  
Arbuscular Mycorrhizal ◽  
Soil Microbial ◽  
Fungal Abundance ◽  
Novel Approach ◽  
Soil Climate

A broad diversity of microorganisms can be found in soil, where they are essential for nutrient cycling and energy transfer. Recent high-throughput sequencing methods have greatly advanced our knowledge about how soil, climate and vegetation variables structure the composition of microbial communities in many world regions. However, we are lacking information from several regions in the world, e.g. Middle-East. We have collected soil from 19 different habitat types for studying the diversity and composition of soil microbial communities (both fungi and bacteria) in Qatar and determining which edaphic parameters exert the strongest influences on these communities. Preliminary results indicate that in overall bacteria are more abundant in soil than fungi and few sites have notably higher abundance of these microbes. In addition, we have detected some soil patameters, which tend to have reduced the overall fungal abundance and enhanced the presence of arbuscular mycorrhizal fungi and N-fixing bacteria. More detailed information on the diversity and composition of soil microbial communities is expected from the high-throughput sequenced data.

scholarly journals Application of a High-Throughput Amplicon Sequencing Method to Chart the Bacterial Communities that Are Associated with European Fermented Meats from Different Origins

Foods ◽  
2020 ◽  
Vol 9 (9) ◽  
pp. 1247
Author(s):  
Emiel Van Reckem ◽  
Christina Charmpi ◽  
David Van der Veken ◽  
Wim Borremans ◽  
Luc De Vuyst ◽  
...  
Keyword(s):  
Microbial Communities ◽  
High Throughput ◽  
Bacterial Communities ◽  
Geographical Origin ◽  
Salt Content ◽  
Amplicon Sequencing ◽  
Taxonomic Resolution ◽  
Future Application ◽  
Processing Conditions ◽  
The Impact

Insight into the microbial species diversity of fermented meats is not only paramount to gain control over quality development, but also to better understand the link with processing technology and geographical origin. To study the composition of the microbial communities, the use of culture-independent methods is increasingly popular but often still suffers from drawbacks, such as a limited taxonomic resolution. This study aimed to apply a previously developed high-throughput amplicon sequencing (HTS) method targeting the 16S rRNA and tuf genes to characterize the bacterial communities in European fermented meats in greater detail. The data obtained broadened the view on the microbial communities that were associated with the various products examined, revealing the presence of previously underreported subdominant species. Moreover, the composition of these communities could be linked to the specificities of individual products, in particular pH, salt content, and geographical origin. In contrast, no clear links were found between the volatile organic compound profiles of the different products and the country of origin, distinct processing conditions, or microbial communities. Future application of the HTS method offers the potential to further unravel complex microbial communities in fermented meats, as well as to assess the impact of different processing conditions on microbial consortia.

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