scholarly journals Accurate Estimation of Fungal Diversity and Abundance through Improved Lineage-Specific Primers Optimized for Illumina Amplicon Sequencing

2016 ◽  
Vol 82 (24) ◽  
pp. 7217-7226 ◽  
Author(s):  
D. Lee Taylor ◽  
William A. Walters ◽  
Niall J. Lennon ◽  
James Bochicchio ◽  
Andrew Krohn ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Large Subunit ◽  
Marker Gene ◽  
Soil Samples ◽  
Gene Copy ◽  
Accurate Estimation ◽  
Rrna Gene ◽  
Data Set ◽  
Mock Communities

ABSTRACTWhile high-throughput sequencing methods are revolutionizing fungal ecology, recovering accurate estimates of species richness and abundance has proven elusive. We sought to design internal transcribed spacer (ITS) primers and an Illumina protocol that would maximize coverage of the kingdom Fungi while minimizing nontarget eukaryotes. We inspected alignments of the 5.8S and large subunit (LSU) ribosomal genes and evaluated potential primers using PrimerProspector. We tested the resulting primers using tiered-abundance mock communities and five previously characterized soil samples. We recovered operational taxonomic units (OTUs) belonging to all 8 members in both mock communities, despite DNA abundances spanning 3 orders of magnitude. The expected and observed read counts were strongly correlated (r= 0.94 to 0.97). However, several taxa were consistently over- or underrepresented, likely due to variation in rRNA gene copy numbers. The Illumina data resulted in clustering of soil samples identical to that obtained with Sanger sequence clone library data using different primers. Furthermore, the two methods produced distance matrices with a Mantel correlation of 0.92. Nonfungal sequences comprised less than 0.5% of the soil data set, with most attributable to vascular plants. Our results suggest that high-throughput methods can produce fairly accurate estimates of fungal abundances in complex communities. Further improvements might be achieved through corrections for rRNA copy number and utilization of standardized mock communities.IMPORTANCEFungi play numerous important roles in the environment. Improvements in sequencing methods are providing revolutionary insights into fungal biodiversity, yet accurate estimates of the number of fungal species (i.e., richness) and their relative abundances in an environmental sample (e.g., soil, roots, water, etc.) remain difficult to obtain. We present improved methods for high-throughput Illumina sequencing of the species-diagnostic fungal ribosomal marker gene that improve the accuracy of richness and abundance estimates. The improvements include new PCR primers and library preparation, validation using a known mock community, and bioinformatic parameter tuning.

scholarly journals A novel metabarcoding strategy for studying nematode communities

2020 ◽  
Author(s):  
Md. Maniruzzaman Sikder ◽  
Mette Vestergård ◽  
Rumakanta Sapkota ◽  
Tina Kyndt ◽  
Mogens Nicolaisen
Keyword(s):  
18S Rrna ◽  
High Throughput Sequencing ◽  
Soil Samples ◽  
Coi Gene ◽  
Rrna Gene ◽  
Nematode Communities ◽  
Sequencing Platform ◽  
Spiked Soil ◽  
Primer Sets ◽  
Mock Communities

AbstractNematodes are widely abundant soil metazoa and often referred to as indicators of soil health. While recent advances in next-generation sequencing technologies have accelerated research in microbial ecology, the ecology of nematodes remains poorly elucidated, partly due to the lack of reliable and validated sequencing strategies. Objectives of the present study were (i) to compare commonly used primer sets and to identify the most suitable primer set for metabarcoding of nematodes; (ii) to establish and validate a high-throughput sequencing strategy for nematodes using Illumina paired-end sequencing. In this study, we tested four primer sets for amplicon sequencing: JB3/JB5 (mitochondrial, I3-M11 partition); SSU_04F/SSU_22R (18S rRNA, V1-V2 region); Nemf/18Sr2b (18S rRNA, V6-V8 region) from earlier studies; and MMSF/MMSR (18S rRNA, V4-V5 region), a newly developed primer set from this study. In order to test the primer sets, we used 22 samples of individual nematode species, 20 mock communities, 20 soil samples, 20 spiked soil samples (mock communities in soil), and 4 root/rhizosphere soil samples. We successfully amplified the target regions (I3-M11 partition of the COI gene; V1-V2, V4-V8 region of 18S rRNA gene) from these 86 DNA samples with the four different primer combinations and sequenced the amplicons on an Illumina MiSeq sequencing platform. We found that the MMSF/MMSR and Nemf/18Sr2b were efficient in detecting nematode compared to JB and SSU primer sets based on annotation of sequence reads at genus and in some cases at species level. Therefore, these primer sets are suggested for studies of nematode communities in agricultural environments.

scholarly journals Multivariate Analysis of Complex DNA Sequence Electropherograms for High-Throughput Quantitative Analysis of Mixed Microbial Populations

2007 ◽  
Vol 73 (15) ◽  
pp. 4975-4983 ◽  
Author(s):  
Pål Trosvik ◽  
Beate Skånseng ◽  
Kjetill S. Jakobsen ◽  
Nils C. Stenseth ◽  
Tormod Næs ◽  
...  
Keyword(s):  
Dna Sequence ◽  
High Throughput ◽  
Anaerobic Fermentation ◽  
Marker Gene ◽  
Microbial Populations ◽  
Series Data ◽  
Accurate Estimation ◽  
Infection Model ◽  
Rrna Gene ◽  
Specific Marker

ABSTRACT High-throughput quantification of genetically coherent units (GCUs) is essential for deciphering population dynamics and species interactions within a community of microbes. Current techniques for microbial community analyses are, however, not suitable for this kind of high-throughput application. Here, we demonstrate the use of multivariate statistical analysis of complex DNA sequence electropherograms for the effective and accurate estimation of relative genotype abundance in cell samples from mixed microbial populations. The procedure is no more labor-intensive than standard automated DNA sequencing and provides a very effective means of quantitative data acquisition from experimental microbial communities. We present results with the Campylobacter jejuni strain-specific marker gene gltA, as well as the 16S rRNA gene, which is a universal marker across bacterial assemblages. The statistical models computed for these genes are applied to genetic data from two different experimental settings, namely, a chicken infection model and a multispecies anaerobic fermentation model, demonstrating collection of time series data from model bacterial communities. The method presented here is, however, applicable to any experimental scenario where the interest is quantification of GCUs in genetically heterogeneous DNA samples.

scholarly journals Run-to-Run Sequencing Variation Can Introduce Taxon-Specific Bias in the Evaluation of Fungal Microbiomes

2018 ◽  
Vol 2 (3) ◽  
pp. 165-170 ◽  
Author(s):  
Zewei Song ◽  
Dan Schlatter ◽  
Daryl M. Gohl ◽  
Linda L. Kinkel
Keyword(s):  
Quality Control ◽  
Microbial Communities ◽  
High Throughput Sequencing ◽  
Marker Gene ◽  
Soil Samples ◽  
The Other ◽  
Potential Bias ◽  
Mock Community ◽  
Control Information ◽  
Mock Communities

The routine use of high-throughput sequencing to profile microbial communities necessitates improved protocols for detecting and adjusting for variation among sequencing runs for marker gene analysis. Although mock communities are widely used as a control among runs, the composition and diversity of mock communities, in most cases, are orders of magnitude lower than the actual samples. We demonstrated that replicated biological samples (“technical replicates”) are superior to a mock community in detecting variation and potential bias among sequencing runs. We present a case in which technical replicates of three soil samples were sequenced in three MiSeq runs containing samples from multiple experiments. The technical replicate samples revealed a potentially biased, outlier sequencing run, from which several Ascomycota taxa were substantially underestimated. Similar bias was seen in the other samples sequenced but was not detected using the mock community. Our study demonstrates that using technical replicates along with traditional mock communities provide additional quality control information and aid in detecting outlier sequencing runs.

scholarly journals Comparison of invertebrate diversity in lake waters and their resting eggs in sediments, as revealed by high-throughput sequencing (HTS)

2020 ◽  
pp. 19
Author(s):  
Xiaoyan Wang ◽  
Qing Wang ◽  
Yufeng Yang ◽  
Wenbo Yu
Keyword(s):  
High Throughput ◽  
Water Samples ◽  
High Throughput Sequencing ◽  
Essential Feature ◽  
Resting Eggs ◽  
Freshwater Ecosystems ◽  
Rrna Gene ◽  
Promising Alternative ◽  
Invertebrate Diversity ◽  
Significant Difference

Aquatic invertebrate diversity reflects water quality and the health of aquatic ecosystems and should be monitored as an essential feature of freshwater ecosystems. The resting eggs of aquatic invertebrates in sediments populate the overlying water. The diversity of invertebrates in waters and their resting eggs in sediments in Baiyangdian Lake, Xiongan, North China, were assessed using high-throughput sequencing (HTS) with a pair of 18S rRNA gene adaptor-linked primers. The total of 99 operational taxonomic units (OTUs) derived from 353,755 invertebrate sequences (mostly zooplankton) were revealed by this study. A total of 50 species in the water samples including 20 rotifers, 11 copepods, 1 cladoceran and 18 other species were sorted out. In the sediment 37 species, including 21 rotifers, 3 copepods, 1 cladoceran and 12 other species, were identified. There were 24 species in common between water and corresponding sediments. Invertebrate OTU richness in water samples was higher than that in sediments (p < 0.01), while there was no significant difference in the Shannon-Wiener index. These results suggest that HTS is a promising alternative for efficient biodiversity assessment and monitoring.

scholarly journals Microbial Composition of Human Appendices from Patients following Appendectomy

mBio ◽  
2013 ◽  
Vol 4 (1) ◽  
Author(s):  
Caitriona M. Guinane ◽  
Amany Tadrous ◽  
Fiona Fouhy ◽  
C. Anthony Ryan ◽  
Eugene M. Dempsey ◽  
...  
Keyword(s):  
Human Health ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Evolutionary Development ◽  
Rrna Gene ◽  
Limited Data ◽  
Microbial Composition ◽  
Content Type ◽  
Study Results ◽  
Human Appendix

ABSTRACT The human appendix has historically been considered a vestige of evolutionary development with an unknown function. While limited data are available on the microbial composition of the appendix, it has been postulated that this organ could serve as a microbial reservoir for repopulating the gastrointestinal tract in times of necessity. We aimed to explore the microbial composition of the human appendix, using high-throughput sequencing of the 16S rRNA gene V4 region. Seven patients, 5 to 25 years of age, presenting with symptoms of acute appendicitis were included in this study. Results showed considerable diversity and interindividual variability among the microbial composition of the appendix samples. In general, however, Firmicutes was the dominant phylum, with the majority of additional sequences being assigned at various levels to Proteobacteria, Bacteroidetes, Actinobacteria, and Fusobacteria. Despite the large diversity in the microbiota found within the appendix, however, a few major families and genera were found to comprise the majority of the sequences present. Interestingly, also, certain taxa not generally associated with the human intestine, including the oral pathogens Gemella, Parvimonas, and Fusobacterium, were identified among the appendix samples. The prevalence of genera such as Fusobacterium could also be linked to the severity of inflammation of the organ. We conclude that the human appendix contains a robust and varied microbiota distinct from the microbiotas in other niches within the human microbiome. The microbial composition of the human appendix is subject to extreme variability and comprises a diversity of biota that may play an important, as-yet-unknown role in human health. IMPORTANCE There are currently limited data available on the microbial composition of the human appendix. It has been suggested, however, that it may serve as a “safe house” for commensal bacteria that can reinoculate the gut at need. The present study is the first comprehensive view of the microbial composition of the appendix as determined by high-throughput sequencing. We have determined that the human appendix contains a wealth of microbes, including members of 15 phyla. Important information regarding the associated bacterial diversity of the appendix which will help determine the role, if any, the appendix microbiota has in human health is presented.

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