scholarly journals MinION Sequencing of colorectal cancer tumour microbiomes – a comparison with amplicon-based and RNA-Sequencing

2019 ◽  
Author(s):  
William S Taylor ◽  
John Pearson ◽  
Allison Miller ◽  
Sebastian Schmeier ◽  
Frank A Frizelle ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
16S Rrna ◽  
Rna Sequencing ◽  
Amplicon Sequencing ◽  
Read Length ◽  
Bacterial Strains ◽  
Average Read Length ◽  
Colorectal Tumour ◽  
16S Sequencing ◽  
16S Rrna Amplicon Sequencing

AbstractRecent evidence suggests a role for the gut microbiome in the development and progression of colorectal cancer. In this study we compare MinION sequencing with meta-transcriptomics and amplicon-based sequencing for microbiome analysis of colorectal tumour tissue samples. DNA and RNA were extracted from 11 colorectal tumour samples. RNA-Sequencing, 16S rRNA amplicon sequencing and MinION genomic sequencing was carried out and resulting data used as input for taxonomic classification using Kraken2. Taxonomic concordance between the three platforms at different taxonomic levels was tested on a per sample basis. The average number of reads per sample using RNA-Sequencing was more than 129 times that generated using MinION sequencing. However, the average read length of MinION sequences was more than 13 times that of RNA or 16S rRNA amplicon sequencing. 16S sequencing was less informative beyond the genus level, and both RNA-Sequencing and MinION sequencing could detect more phyla and genera in the same samples, compared to 16S sequencing. Long-read sequences generated using MinION sequencing can compensate for low numbers of reads for bacterial classification. MinION sequencing can discriminate between bacterial strains and plasmids and shows potential as a tool for microbiome sequencing of colorectal cancers in a clinical setting.

scholarly journals Choice of Commercial DNA Extraction Method Does Not Affect 16S Sequencing Outcomes in Cloacal Swabs

Animals ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 1372
Author(s):  
Emily Van Syoc ◽  
Natália Carrillo Gaeta ◽  
Erika Ganda
Keyword(s):  
16S Rrna ◽  
Dna Extraction ◽  
Amplicon Sequencing ◽  
Extraction Methods ◽  
Negative Control ◽  
Careful Consideration ◽  
16S Sequencing ◽  
16S Rrna Amplicon Sequencing ◽  
Microbiome Research ◽  
Negative Controls

As the applications of microbiome science in agriculture expand, laboratory methods should be constantly evaluated to ensure optimization and reliability of downstream results. Most animal microbiome research uses fecal samples or rectal swabs for profiling the gut bacterial community; however, in birds, this is difficult given the unique anatomy of the cloaca where the fecal, urinary, and reproductive tracts converge into one orifice. Therefore, avian gut microbiomes are usually sampled from cloacal swabs, creating a need to evaluate sample preparation methods to optimize 16S sequencing. We compared four different DNA extraction methods from two commercially available kits on cloacal swabs from 10 adult commercial laying hens and included mock communities and negative controls, which were then subjected to 16S rRNA amplicon sequencing. Extracted DNA yield and quality, diversity analyses, and contaminants were assessed. Differences in DNA quality and quantity were observed, and all methods needed further purification for optimal sequencing, suggesting contaminants due to cloacal contents, method reagents, and/or environmental factors. However, no differences were observed in alpha or beta diversity between methods. Importantly, multiple bacterial contaminants were detected in each mock community and negative control, indicating the prevalence of laboratory and handling contamination as well as method-specific reagent contamination. We found that although the extraction methods resulted in different extraction quality and yield, overall sequencing results were not affected, and we did not identify any method that would be an inappropriate choice in extracting DNA from cloacal swabs for 16S rRNA sequencing. Overall, our results highlight the need for careful consideration of positive and negative controls in addition to DNA isolation method and lend guidance to future microbiome research in poultry.

scholarly journals Analysis of changes in microbiome compositions related to the prognosis of colorectal cancer patients based on tissue-derived 16S rRNA sequences

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Sukjung Choi ◽  
Jongsuk Chung ◽  
Mi-La Cho ◽  
Donghyun Park ◽  
Sun Shim Choi
Keyword(s):  
Colorectal Cancer ◽  
16S Rrna ◽  
Pathogenic Bacteria ◽  
Fusobacterium Nucleatum ◽  
Amplicon Sequencing ◽  
Normal Tissues ◽  
16S Rrna Amplicon Sequencing ◽  
Tumor Tissues ◽  
Colorectal Cancer Patients ◽  
Host Genes

Abstract Background Comparing the microbiome compositions obtained under different physiological conditions has frequently been attempted in recent years to understand the functional influence of microbiomes in the occurrence of various human diseases. Methods In the present work, we analyzed 102 microbiome datasets containing tumor- and normal tissue-derived microbiomes obtained from a total of 51 Korean colorectal cancer (CRC) patients using 16S rRNA amplicon sequencing. Two types of comparisons were used: ‘normal versus (vs.) tumor’ comparison and ‘recurrent vs. nonrecurrent’ comparison, for which the prognosis of patients was retrospectively determined. Results As a result, we observed that in the ‘normal vs. tumor’ comparison, three phyla, Firmicutes, Actinobacteria, and Bacteroidetes, were more abundant in normal tissues, whereas some pathogenic bacteria, including Fusobacterium nucleatum and Bacteroides fragilis, were more abundant in tumor tissues. We also found that bacteria with metabolic pathways related to the production of bacterial motility proteins or bile acid secretion were more enriched in tumor tissues. In addition, the amount of these two pathogenic bacteria was positively correlated with the expression levels of host genes involved in the cell cycle and cell proliferation, confirming the association of microbiomes with tumorigenic pathway genes in the host. Surprisingly, in the ‘recurrent vs. nonrecurrent’ comparison, we observed that these two pathogenic bacteria were more abundant in the patients without recurrence than in the patients with recurrence. The same conclusion was drawn in the analysis of both normal and tumor-derived microbiomes. Conclusions Taken together, it seems that understanding the composition of tissue microbiomes is useful for predicting the prognosis of CRC patients.

scholarly journals Technical challenges regarding the use of formalin-fixed paraffin embedded (FFPE) tissue specimens for the detection of bacterial alterations in colorectal cancer

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Suk Yee Lam ◽  
Athanasia Ioannou ◽  
Prokopis Konstanti ◽  
Thijmen Visseren ◽  
Michail Doukas ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
16S Rrna ◽  
Nested Pcr ◽  
Amplicon Sequencing ◽  
16S Rrna Amplicon Sequencing ◽  
Bacterial Contaminants ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Ffpe Tissues ◽  
Formalin Fixed

Abstract Background Formalin-fixed paraffin embedded (FFPE) tissues may provide an exciting resource to study microbial associations in human disease, but the use of these low biomass specimens remains challenging. We aimed to reduce unintentional bacterial interference in molecular analysis of FFPE tissues and investigated the feasibility of conducting quantitative polymerase chain reaction (qPCR) and 16S rRNA amplicon sequencing using 14 colorectal cancer, 14 normal adjacent and 13 healthy control tissues. Results Bacterial contaminants from the laboratory environment and the co-extraction of human DNA can affect bacterial analysis. The application of undiluted template improves bacterial DNA amplification, allowing the detection of specific bacterial markers (Escherichia coli and Faecalibacterium prausnitzii) by qPCR. Nested and non-nested PCR-based 16S rRNA amplicon sequencing approaches were employed, showing that bacterial communities of tissues and paired paraffin controls cluster separately at genus level on weighted Unifrac in both non-nested (R2 = 0.045; Pr(> F) = 0.053) and nested (R2 = 0.299; Pr(> F) = 0.001) PCR datasets. Nevertheless, considerable overlap of bacterial genera within tissues was seen with paraffin, DNA extraction negatives (non-nested PCR) or PCR negatives (nested PCR). Following mathematical decontamination, no differences in α- and β diversity were found between tumor, normal adjacent and control tissues. Conclusions Bacterial marker analysis by qPCR seems feasible using non-normalized template, but 16S rRNA amplicon sequencing remains challenging. Critical evaluation of laboratory procedures and incorporation of positive and negative controls for bacterial analysis of FFPE tissues are essential for quality control and to account for bacterial contaminants.

scholarly journals Bacterial Diversity of Breast Milk in Healthy Spanish Women: Evolution from Birth to Five Years Postpartum

Nutrients ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 2414
Author(s):  
Laura Sanjulián ◽  
Alexandre Lamas ◽  
Rocío Barreiro ◽  
Alberto Cepeda ◽  
Cristina A. Fente ◽  
...  
Keyword(s):  
Breast Milk ◽  
16S Rrna ◽  
Human Milk ◽  
Alpha Diversity ◽  
Amplicon Sequencing ◽  
Maternal Body Mass Index ◽  
16S Rrna Amplicon Sequencing ◽  
Spanish Women ◽  
Calcium Magnesium ◽  
Abundant Genus

The objective of this work was to characterize the microbiota of breast milk in healthy Spanish mothers and to investigate the effects of lactation time on its diversity. A total of ninety-nine human milk samples were collected from healthy Spanish women and were assessed by means of next-generation sequencing of 16S rRNA amplicons and by qPCR. Firmicutes was the most abundant phylum, followed by Bacteroidetes, Actinobacteria, and Proteobacteria. Accordingly, Streptococcus was the most abundant genus. Lactation time showed a strong influence in milk microbiota, positively correlating with Actinobacteria and Bacteroidetes, while Firmicutes was relatively constant over lactation. 16S rRNA amplicon sequencing showed that the highest alpha-diversity was found in samples of prolonged lactation, along with wider differences between individuals. As for milk nutrients, calcium, magnesium, and selenium levels were potentially associated with Streptococcus and Staphylococcus abundance. Additionally, Proteobacteria was positively correlated with docosahexaenoic acid (DHA) levels in breast milk, and Staphylococcus with conjugated linoleic acid. Conversely, Streptococcus and trans-palmitoleic acid showed a negative association. Other factors such as maternal body mass index or diet also showed an influence on the structure of these microbial communities. Overall, human milk in Spanish mothers appeared to be a complex niche shaped by host factors and by its own nutrients, increasing in diversity over time.

scholarly journals Connecting structure to function with the recovery of over 1000 high-quality metagenome-assembled genomes from activated sludge using long-read sequencing

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Caitlin M. Singleton ◽  
Francesca Petriglieri ◽  
Jannie M. Kristensen ◽  
Rasmus H. Kirkegaard ◽  
Thomas Y. Michaelsen ◽  
...  
Keyword(s):  
16S Rrna ◽  
Wastewater Treatment Plants ◽  
In Situ Hybridisation ◽  
Amplicon Sequencing ◽  
Rrna Genes ◽  
Fluorescence In Situ Hybridisation ◽  
Sequencing Data ◽  
High Quality ◽  
16S Rrna Amplicon Sequencing ◽  
Long Read

AbstractMicroorganisms play crucial roles in water recycling, pollution removal and resource recovery in the wastewater industry. The structure of these microbial communities is increasingly understood based on 16S rRNA amplicon sequencing data. However, such data cannot be linked to functional potential in the absence of high-quality metagenome-assembled genomes (MAGs) for nearly all species. Here, we use long-read and short-read sequencing to recover 1083 high-quality MAGs, including 57 closed circular genomes, from 23 Danish full-scale wastewater treatment plants. The MAGs account for ~30% of the community based on relative abundance, and meet the stringent MIMAG high-quality draft requirements including full-length rRNA genes. We use the information provided by these MAGs in combination with >13 years of 16S rRNA amplicon sequencing data, as well as Raman microspectroscopy and fluorescence in situ hybridisation, to uncover abundant undescribed lineages belonging to important functional groups.

Advantage of 16S rRNA amplicon sequencing in Helicobacter pylori diagnosis

Helicobacter ◽  
2021 ◽  
Author(s):  
Boldbaatar Gantuya ◽  
Hashem B. El Serag ◽  
Batsaikhan Saruuljavkhlan ◽  
Dashdorj Azzaya ◽  
Takashi Matsumoto ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
16S Rrna ◽  
Amplicon Sequencing ◽  
16S Rrna Amplicon Sequencing

scholarly journals Prokaryotic Communities in the Thalassohaline Tuz Lake, Deep Zone, and Kayacik, Kaldirim and Yavsan Salterns (Turkey) Assessed by 16S rRNA Amplicon Sequencing

2021 ◽  
Vol 9 (7) ◽  
pp. 1525
Author(s):  
Can Akpolat ◽  
Ana Beatriz Fernández ◽  
Pinar Caglayan ◽  
Baris Calli ◽  
Meral Birbir ◽  
...  
Keyword(s):  
16S Rrna ◽  
Salt Lake ◽  
Amplicon Sequencing ◽  
Deep Zone ◽  
Biotechnological Potential ◽  
16S Rrna Amplicon Sequencing ◽  
Physico Chemical ◽  
Potential Applications ◽  
A Minor ◽  
Tuz Lake

Prokaryotic communities and physico-chemical characteristics of 30 brine samples from the thalassohaline Tuz Lake (Salt Lake), Deep Zone, Kayacik, Kaldirim, and Yavsan salterns (Turkey) were analyzed using 16S rRNA amplicon sequencing and standard methods, respectively. Archaea (98.41% of reads) was found to dominate in these habitats in contrast to the domain Bacteria (1.38% of reads). Representatives of the phylum Euryarchaeota were detected as the most predominant, while 59.48% and 1.32% of reads, respectively, were assigned to 18 archaeal genera, 19 bacterial genera, 10 archaeal genera, and one bacterial genus that were determined to be present, with more than 1% sequences in the samples. They were the archaeal genera Haloquadratum, Haloarcula, Halorhabdus, Natronomonas, Halosimplex, Halomicrobium, Halorubrum, Halonotius, Halolamina, Halobacterium, and Salinibacter within the domain Bacteria. The genera Haloquadratum and Halorhabdus were found in all sampling sites. While Haloquadratum, Haloarcula, and Halorhabdus were the most abundant genera, two uncultured Tuz Lake Halobacteria (TLHs) 1 and 2 were detected in high abundance, and an additional uncultured haloarchaeal TLH-3 was found as a minor abundant uncultured taxon. Their future isolation in pure culture would permit us to expand our knowledge on hypersaline thalassohaline habitats, as well as their ecological role and biomedical and biotechnological potential applications.

16S rRNA Amplicon Sequencing for Metagenomics

2018 ◽  
pp. 135-161
Author(s):  
Henrik Christensen ◽  
Anna Jasmine Andersson ◽  
Steffen Lynge Jørgensen ◽  
Josef Korbinian Vogt
Keyword(s):  
16S Rrna ◽  
Amplicon Sequencing ◽  
16S Rrna Amplicon Sequencing

scholarly journals Dramatic differences in gut bacterial densities help to explain the relationship between diet and habitat in rainforest ants

2017 ◽  
Author(s):  
Jon G Sanders ◽  
Piotr Lukasik ◽  
Megan E Frederickson ◽  
Jacob A Russell ◽  
Ryuichi Koga ◽  
...  
Keyword(s):  
16S Rrna ◽  
Microbial Diversity ◽  
Tropical Rainforest ◽  
Amplicon Sequencing ◽  
Sequencing Data ◽  
Lowland Tropical Forest ◽  
16S Rrna Amplicon Sequencing ◽  
Microbial Symbionts ◽  
Microbial Symbiosis ◽  
Diversity Profiles

AbstractAbundance is a key parameter in microbial ecology, and important to estimates of potential metabolite flux, impacts of dispersal, and sensitivity of samples to technical biases such as laboratory contamination. However, modern amplicon-based sequencing techniques by themselves typically provide no information about the absolute abundance of microbes. Here, we use fluorescence microscopy and quantitative PCR as independent estimates of microbial abundance to test the hypothesis that microbial symbionts have enabled ants to dominate tropical rainforest canopies by facilitating herbivorous diets, and compare these methods to microbial diversity profiles from 16S rRNA amplicon sequencing. Through a systematic survey of ants from a lowland tropical forest, we show that the density of gut microbiota varies across several orders of magnitude among ant lineages, with median individuals from many genera only marginally above detection limits. Supporting the hypothesis that microbial symbiosis is important to dominance in the canopy, we find that the abundance of gut bacteria is positively correlated with stable isotope proxies of herbivory among canopy-dwelling ants, but not among ground-dwelling ants. Notably, these broad findings are much more evident in the quantitative data than in the 16S rRNA sequencing data. Our results help to resolve a longstanding question in tropical rainforest ecology, and have broad implications for the interpretation of sequence-based surveys of microbial diversity.

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