scholarly journals Reproducibility and repeatability of six high-throughput 16S rDNA sequencing protocols for microbiota profiling

2017 ◽  
Author(s):  
Sajan C. Raju ◽  
Sonja Lagström ◽  
Pekka Ellonen ◽  
Willem M. de Vos ◽  
Johan G. Eriksson ◽  
...  
Keyword(s):  
Large Scale ◽  
Alpha Diversity ◽  
Epidemiological Studies ◽  
Molecular Techniques ◽  
16S Rdna Sequencing ◽  
High Reproducibility ◽  
Rdna Sequencing ◽  
Culture Independent ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

AbstractCulture-independent molecular techniques and advances in next generation sequencing (NGS) technologies make large-scale epidemiological studies on microbiota feasible. A challenge using NGS is to obtain high reproducibility and repeatability, which is mostly attained through robust amplification. We aimed to assess the reproducibility of saliva microbiota by comparing triplicate samples. The microbiota was produced with simplified in-house 16S amplicon assays taking advantage of large number of barcodes. The assays included primers with Truseq (TS-tailed) or Nextera (NX-tailed) adapters and either with dual index or dual index plus a 6-nt internal index. All amplification protocols produced consistent microbial profiles for the same samples. Although, in our study, reproducibility was highest for the TS-tailed method. Five replicates of a single sample, prepared with the TS-tailed 1-step protocol without internal index sequenced on the HiSeq platform provided high alpha-diversity and low standard deviation (mean Shannon and Inverse Simpson diversity was 3.19 ± 0.097 and 13.56 ± 1.634 respectively). Large-scale profiling of microbiota can consistently be produced by all 16S amplicon assays. The TS-tailed-1S dual index protocol is preferred since it provides repeatable profiles on the HiSeq platform and are less labour intensive.

scholarly journals Exotic airborne bacteria identified in urban resuspended dust by next generation sequencing

2019 ◽  
Vol 99 ◽  
pp. 04009
Author(s):  
Nora Kováts ◽  
Eszter Horváth ◽  
Katalin Hubai ◽  
András Hoffer ◽  
Beatrix Jancsek-Turóczi ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Large Scale ◽  
Rrna Gene ◽  
Pathogenic Microorganisms ◽  
Next Generation ◽  
Culture Independent ◽  
Next Generation Sequencing Ngs ◽  
Siberian Permafrost ◽  
Generation Sequencing ◽  
Airborne Transport

The airborne transport of bacteria is a well-known phenomenon, making it possible to exchange species between ecosystems, but it also provides a tool for spreading of pathogenic microorganisms. As part of a large-scale study, microbial community of inhalable and respirable fractions (PM1-10) of resuspended dust collected in Budapest (Hungary) has been characterised by culture-independent next generation sequencing (NGS) of variable 16S rRNA gene regions. Apart from common, mostly ubiqituos soil and organic material-dwelling bacteria, exotic airborne species have been identified, such as Variovorax ginsengisoli, previously isolated from Korean ginseng fields or Exiguobacterium sibiricum, isolated from the Siberian permafrost.

PSVII-9 Influence of lactose and milk oligosaccharides in whey permeate on jejunal mucosa-associated microbiota in nursery pigs during 7 to 11 kg BW

2021 ◽  
Vol 99 (Supplement_3) ◽  
pp. 407-407
Author(s):  
Ki Beom Jang ◽  
Sung Woo Kim
Keyword(s):  
Alpha Diversity ◽  
Jejunal Mucosa ◽  
16S Rdna Sequencing ◽  
Sequencing Data ◽  
Milk Oligosaccharides ◽  
Whey Permeate ◽  
Nursery Pigs ◽  
Rdna Sequencing ◽  
Acid Producing Bacteria ◽  
Diversity Estimates

Abstract This study aimed to evaluate supplemental effects of milk carbohydrates in whey permeate on jejunal mucosa-associated microbiota in nursery pigs during 7 to 11 kg BW. A total of 720 pigs at 7.5 kg BW were allotted to 6 treatments (6 pens/treatment and 20 pigs/pen). Treatments were 6 levels of whey permeate supplementation (0, 3.75, 7.50, 11.25, 15.00, and 18.75%) and fed to pigs for 11 d. On d 11, 36 pigs representing median BW of each pen were euthanized to collect the jejunal mucosa to evaluate microbiota in the jejunum by 16S rDNA sequencing. Data were analyzed using contrasts in MIXED procedure of SAS. Whey permeate contained 76.3% lactose and 0.4% milk oligosaccharides. Increasing whey permeate supplementation from 0 to 18.75% did not affect the alpha-diversity estimates of microbiota. Whey permeate supplementation tended to decrease (P = 0.073, 1.59 to 1.22) Firmicutes:Bacteroidetes compared with no addition of whey permeate. Increasing whey permeate supplementation tended to linearly increase Bifidobacteriaceae (P = 0.089, 0.73 to 1.11), decrease Enterobacteriaceae (P = 0.091, 1.04 to 0.52), decrease Stretococcaceae (P = 0.094, 1.50 to 0.71), and caused quadratic changes (P < 0.05) on Lactobacillaceae (maximum: 9.14% at 12.91% whey permeate). Increasing whey permeate supplementation caused a quadratic change (P < 0.05) on Lactobacillus_Salivarius (maximum: 0.92% at 7.35% whey permeate) and tended to cause quadratic changes on Lactobacillus_Rogosae (P = 0.083; maximum: 0.53% at 8.45% whey permeate) and Lactobacillus_Mucosae (P = 0.092; maximum: 0.70% at 6.98% whey permeate). In conclusion, supplementation of whey permeate as sources of lactose and milk oligosaccharides at a range from 7 to 13% seems to be beneficial to nursery pigs by increasing the abundance of lactic acid-producing bacteria in the jejunal mucosa.

EARRINGS: an efficient and accurate adapter trimmer entails no a priori adapter sequences

2021 ◽  
Author(s):  
Ting-Hsuan Wang ◽  
Cheng-Ching Huang ◽  
Jui-Hung Hung
Keyword(s):  
Open Source Software ◽  
Large Scale ◽  
A Priori ◽  
Supplementary Information ◽  
Supplementary Data ◽  
Comparable Accuracy ◽  
Meta Analyses ◽  
Next Generation Sequencing Ngs ◽  
Adapter Trimming ◽  
Generation Sequencing

Abstract Motivation Cross-sample comparisons or large-scale meta-analyses based on the next generation sequencing (NGS) involve replicable and universal data preprocessing, including removing adapter fragments in contaminated reads (i.e. adapter trimming). While modern adapter trimmers require users to provide candidate adapter sequences for each sample, which are sometimes unavailable or falsely documented in the repositories (such as GEO or SRA), large-scale meta-analyses are therefore jeopardized by suboptimal adapter trimming. Results Here we introduce a set of fast and accurate adapter detection and trimming algorithms that entail no a priori adapter sequences. These algorithms were implemented in modern C++ with SIMD and multithreading to accelerate its speed. Our experiments and benchmarks show that the implementation (i.e. EARRINGS), without being given any hint of adapter sequences, can reach comparable accuracy and higher throughput than that of existing adapter trimmers. EARRINGS is particularly useful in meta-analyses of a large batch of datasets and can be incorporated in any sequence analysis pipelines in all scales. Availability and implementation EARRINGS is open-source software and is available at https://github.com/jhhung/EARRINGS. Supplementary information Supplementary data are available at Bioinformatics online.

scholarly journals Comparison of oral microbiome profiles in stimulated and unstimulated saliva, tongue, and mouth-rinsed water

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Ryutaro Jo ◽  
Yuichiro Nishimoto ◽  
Kouta Umezawa ◽  
Kazuma Yama ◽  
Yuto Aita ◽  
...  
Keyword(s):  
Microbial Diversity ◽  
Large Scale ◽  
Epidemiological Studies ◽  
Oral Microbiome ◽  
Tongue Coating ◽  
Microbial Composition ◽  
Microbiome Analysis ◽  
Unstimulated Saliva ◽  
Encoding Genes ◽  
Generation Sequencing

Abstract Epidemiological studies using saliva have revealed relationships between the oral microbiome and many oral and systemic diseases. However, when collecting from a large number of participants such as a large-scale cohort study, the time it takes to collect saliva can be a problem. Mouth-rinsed water, which is water that has been used to rinse the oral cavity, can be used as an alternative method for collecting saliva for oral microbiome analysis because it can be collected in a shorter time than saliva. The purpose of this study was to verify whether mouth-rinsed water is a suitable saliva substitute for analyzing the oral microbiome. We collected samples of mouth-rinsed water, stimulated saliva, unstimulated saliva, and tongue coating from 10 systemic healthy participants, and compared the microbial diversity and composition of the samples using next-generation sequencing of 16S rRNA-encoding genes. The results showed that the microbial diversity of mouth-rinsed water was similar to that of unstimulated and stimulated saliva, and significantly higher than that of tongue-coating samples. The microbial composition at the species level of mouth-rinsed water also showed a very high correlation with the composition of unstimulated and stimulated saliva. These results suggest that the mouth-rinsed water is a suitable collection method instead of saliva for oral microbiome analysis.

scholarly journals An Update on Zoonotic Cryptosporidium Species and Genotypes in Humans

Animals ◽  
2021 ◽  
Vol 11 (11) ◽  
pp. 3307
Author(s):  
Una Ryan ◽  
Alireza Zahedi ◽  
Yaoyu Feng ◽  
Lihua Xiao
Keyword(s):  
Next Generation Sequencing ◽  
Genome Sequencing ◽  
Molecular Detection ◽  
Epidemiological Studies ◽  
Cryptosporidium Species ◽  
Transmission Dynamics ◽  
Whole Genome ◽  
Zoonotic Pathogen ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

The enteric parasite, Cryptosporidium is a major cause of diarrhoeal illness in humans and animals worldwide. No effective therapeutics or vaccines are available and therefore control is dependent on understanding transmission dynamics. The development of molecular detection and typing tools has resulted in the identification of a large number of cryptic species and genotypes and facilitated our understanding of their potential for zoonotic transmission. Of the 44 recognised Cryptosporidium species and >120 genotypes, 19 species, and four genotypes have been reported in humans with C. hominis, C. parvum, C. meleagridis, C. canis and C. felis being the most prevalent. The development of typing tools that are still lacking some zoonotic species and genotypes and more extensive molecular epidemiological studies in countries where the potential for transmission is highest are required to further our understanding of this important zoonotic pathogen. Similarly, whole-genome sequencing (WGS) and amplicon next-generation sequencing (NGS) are important for more accurately tracking transmission and understanding the mechanisms behind host specificity.

scholarly journals Next-generation sequencing in neuropathological diagnosis of infections of the nervous system

2016 ◽  
Author(s):  
Steven L. Salzberg ◽  
Florian Breitwieser ◽  
Anupama Kumar ◽  
Haiping Hao ◽  
Peter Burger ◽  
...  
Keyword(s):  
Spinal Cord ◽  
Nervous System ◽  
Next Generation Sequencing ◽  
Large Scale ◽  
Infectious Agent ◽  
Next Generation ◽  
Dna And Rna ◽  
Wide Range ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

Objective: To determine the feasibility of next-generation sequencing (NGS) microbiome approaches in the diagnosis of infectious disorders in brain or spinal cord biopsies in patients with suspected central nervous system (CNS) infections. Methods: In a prospective-pilot study, we applied NGS in combination with a new computational analysis pipeline to detect the presence of pathogenic microbes in brain or spinal cord biopsies from ten patients with neurological problems indicating possible infection but for whom conventional clinical and microbiology studies yielded negative or inconclusive results. Results: Direct DNA and RNA sequencing of brain tissue biopsies generated 8.3 million to 29.1 million sequence reads per sample, which successfully identified with high confidence the infectious agent in three patients, identified possible pathogens in two more, and helped to understand neuropathological processes in three others, demonstrating the power of large-scale unbiased sequencing as a novel diagnostic tool. Validation techniques confirmed the pathogens identified by NGS in each of the three positive cases. Clinical outcomes were consistent with the findings yielded by NGS on the presence or absence of an infectious pathogenic process in eight of ten cases, and were non-contributory in the remaining two. Conclusions: NGS-guided metagenomic studies of brain, spinal cord or meningeal biopsies offer the possibility for dramatic improvements in our ability to detect (or rule out) a wide range of CNS pathogens, with potential benefits in speed, sensitivity, and cost. NGS-based microbiome approaches present a major new opportunity to investigate the potential role of infectious pathogens in the pathogenesis of neuroinflammatory disorders.

scholarly journals INNV-04. CONGENITAL SPINAL GLIOBLASTOMA WITH SUSTAINED RESPONSE TO TROPOMYOSIN RECEPTOR KINASE (TRK) INHIBITION: A CASE REPORT

2020 ◽  
Vol 22 (Supplement_2) ◽  
pp. ii117-ii117
Author(s):  
Christina Coleman Abadi ◽  
Peter Sun ◽  
Caroline Hastings
Keyword(s):  
Targeted Therapy ◽  
Gene Fusion ◽  
Large Scale ◽  
Response To Treatment ◽  
Molecular Characteristics ◽  
Molecular Testing ◽  
High Grade ◽  
Molecular Features ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

Abstract Large scale genomic efforts have shown that a significant percentage of infant high-grade gliomas (HGG) carry targetable gene fusions. Given the rarity of congenital spinal HGGs, little is known about the molecular features of these tumors and their response to treatment. We describe a case of a patient with congenital spinal glioblastoma. A full-term female infant with symmetric IUGR was noted to have weakness with little movement of her upper extremities. MRI of the spine showed an infiltrative and hemorrhagic intraspinal cervicothoracic neoplasm with a large hemorrhagic intramedullary component. A biopsy was done and revealed a high-grade glial neoplasm with pan-TRK expression. Next generation sequencing (NGS) confirmed a MEF2D-NTRK1 gene fusion. Prior to the result of her molecular testing, the patient clinically deteriorated with decreased movement and increased apneic episodes despite steroid treatment. She was then started on treatment with carboplatin and etoposide per Macy et al 1. After one cycle of chemotherapy and given the results of NGS, targeted therapy with larotrectinib was added to her treatment. The patient showed a response (clinical and imaging) following cycle 1 with chemotherapy, and a marked response following cycle 2 with both chemotherapy and targeted TRK inhibition. The patient completed six cycles of chemotherapy and has continued on daily oral larotrectinib. She continues to tolerate the targeted therapy well and currently is 8 months old, growing and developing well. She has regained much of her movement and is eating and growing with apparent normal development. This case provides an example of a patient with congenital glioblastoma with TRK gene fusion that showed an impressive response to targeted therapy combined with more conventional chemotherapy. 1Macy M, et al. Clinical and molecular characteristics of congenital glioblastoma. Neuro Oncol. Jul;14 (7) 931–941, 2012).

scholarly journals Tendentious effects of automated and manual metagenomic DNA purification protocols on broiler gut microbiome taxonomic profiling

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Gabor Fidler ◽  
Emese Tolnai ◽  
Aniko Stagel ◽  
Judit Remenyik ◽  
Laszlo Stundl ◽  
...  
Keyword(s):  
Large Scale ◽  
Magnetic Bead ◽  
Positive Influence ◽  
Optimal Choice ◽  
Rrna Gene ◽  
Taxonomic Profiling ◽  
High Consistency ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing ◽  
Significant Positive Influence

AbstractHere, we developed protocols to improve sensitivity, rigor and comparability of 16S rRNA gene amplification-based next-generation sequencing (NGS) results. A thorough study was performed by evaluating extraction efficiency with respect to the yield, purity, fragmentation of the purified DNA, and sequencing metrics considering the number of quality reads, amplicon sequence variants (ASVs), community structure and biodiversity. We identified batch-effects that significantly bias broiler gastrointestinal tract (GIT) community compositions and made recommendations to improve sensitivity, consistency, and cross-study comparability. We found that the purity of the extracted nucleic acid had a strong effect on the success rate of downstream library preparations. The preparation of stool bacterial suspensions from feces showed a significant positive influence on community biodiversity by enriching Gram-negative bacteria and cataloguing low abundant taxa with greater success than direct processing of fecal material. Applications relying on the automated Roche MagNa Pure 24 magnetic-bead based method provided results with high consistency therefore it seems to be the optimal choice in large-scale studies for investigating broiler GIT microbiota.

scholarly journals Genomic evidence for divergent co-infections of SARS-CoV-2 lineages

2021 ◽  
Author(s):  
Hang-Yu Zhou ◽  
Ye-Xiao Cheng ◽  
Lin Xu ◽  
Jia-Ying Li ◽  
Chen-Yue Tao ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Large Scale ◽  
Rna Viruses ◽  
Average Rate ◽  
Genomic Analysis ◽  
Distribution Method ◽  
Reference Framework ◽  
Next Generation Sequencing Ngs ◽  
Ngs Data ◽  
Generation Sequencing

Recently, patients co-infected by two SARS-CoV-2 lineages have been sporadically reported. Concerns are raised because previous studies have demonstrated co-infection may contribute to the recombination of RNA viruses and cause severe clinic symptoms. In this study, we have estimated the compositional lineage(s), tendentiousness, and frequency of co-infection events in population from a large-scale genomic analysis for SARS-CoV-2 patients. SARS-CoV-2 lineage(s) infected in each sample have been recognized from the assignment of within-host site variations into lineage-defined feature variations by introducing a hypergeometric distribution method. Of all the 29,993 samples, 53 (~0.18%) co-infection events have been identified. Apart from 52 co-infections with two SARS-CoV-2 lineages, one sample with co-infections of three SARS-CoV-2 lineages was firstly identified. As expected, the co-infection events mainly happened in the regions where have co-existed more than two dominant SARS-CoV-2 lineages. However, co-infection of two sub-lineages in Delta lineage were detected as well. Our results provide a useful reference framework for the high throughput detecting of SARS-CoV-2 co-infection events in the Next Generation Sequencing (NGS) data. Although low in average rate, the co-infection events showed an increasing tendency with the increased diversity of SARS-CoV-2. And considering the large base of SARS-CoV-2 infections globally, co-infected patients would be a nonnegligible population. Thus, more clinical research is urgently needed on these patients.

scholarly journals A Longitudinal Study of the Human Oropharynx Microbiota Over Time Reveals a Common Core and Significant Variations With Self-Reported Disease

2021 ◽  
Vol 11 ◽  
Author(s):  
Lydia Luise Bach ◽  
Asha Ram ◽  
Umer Z. Ijaz ◽  
Thomas J. Evans ◽  
Jan Lindström
Keyword(s):  
Microbial Communities ◽  
Community Composition ◽  
Common Core ◽  
Temporal Stability ◽  
Alpha Diversity ◽  
Molecular Techniques ◽  
Human Pathogens ◽  
Healthy Human ◽  
Culture Independent ◽  
Over Time

Our understanding of human microbial communities, in particular in regard to diseases is advancing, yet the basic understanding of the microbiome in healthy subjects over time remains limited. The oropharynx is a key target for colonization by several important human pathogens. To understand how the oropharyngeal microbiome might limit infections, and how intercurrent infections might be associated with its composition, we characterized the oropharyngeal microbiome of 18 healthy adults, sampled weekly over a 40-weeks using culture-independent molecular techniques. We detected nine phyla, 202 genera and 1438 assignments on OTU level, dominated by Firmicutes, Bacteroidetes, and Proteobacteria on phylum level. Individual microbiomes of participants were characterized by levels of high alpha diversity (mean = 204.55 OTUs, sd = 35.64), evenness (19.83, sd = 9.74) and high temporal stability (mean Pearson’s correlation between samples of 0.52, sd = 0.060), with greater differences in microbiome community composition between than within individuals. Significant changes in community composition were associated with disease states, suggesting that it is possible to detect specific changes in OTU abundance and community composition during illness. We defined the common core microbiota by varying occurrence and abundance thresholds showing that individual core microbiomes share a substantial number of OTUs across participants, chiefly Streptococci and Veillonella. Our results provide insights into the microbial communities that characterize the healthy human oropharynx, community structure and variability, and provide new approaches to define individual and shared cores. The wider implications of this result include the potential for modeling the general dynamics of oropharynx microbiota both in health and in response to antimicrobial treatments or probiotics.

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