scholarly journals Evaluation of the effects of library preparation procedure and sample characteristics on the accuracy of metagenomic profiles

2021 ◽  
Author(s):  
Christopher Gaulke ◽  
Emily R Schmeltzer ◽  
Mark Dasenko ◽  
Brett M Tyler ◽  
Rebecca Vega Thurber ◽  
...  
Keyword(s):  
Microbial Community ◽  
Cost Effective ◽  
Low Complexity ◽  
Careful Consideration ◽  
Metagenomic Data ◽  
Metagenomic Sequencing ◽  
Library Preparation ◽  
Dna Amount ◽  
Preparation Methods ◽  
Shotgun Metagenomic Sequencing

Shotgun metagenomic sequencing has transformed our understanding of microbial community ecology. However, preparing metagenomic libraries for high-throughput DNA sequencing remains a costly, labor-intensive, and time-consuming procedure, which in turn limits the utility of metagenomes. Several library preparation procedures have recently been developed to offset these costs, but it is unclear how these newer procedures compare to current standards in the field. In particular, it is not clear if all such procedures perform equally well across different types of microbial communities, or if features of the biological samples being processed (e.g., DNA amount) impact the accuracy of the approach. To address these questions, we assessed how five different shotgun DNA sequence library preparation methods, including the commonly used Nextera® Flex kit, perform when applied to metagenomic DNA. We measured each method's ability to produce metagenomic data that accurately represents the underlying taxonomic and genetic diversity of the community. We performed these analyses across a range of microbial community types (e.g., soil, coral-associated, mouse-gut-associated) and input DNA amounts. We find that the type of community and amount of input DNA influence each method’s performance, indicating that careful consideration may be needed when selecting between methods, especially for low complexity communities. However, cost-effective preparation methods we assessed are generally comparable to the current gold standard Nextera® DNA Flex kit for high-complexity communities. Overall, the results from this analysis will help expand and even facilitate access to metagenomic approaches in future studies.

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 Evaluation of the CosmosID Bioinformatics Platform for Prosthetic Joint-Associated Sonicate Fluid Shotgun Metagenomic Data Analysis

2018 ◽  
Vol 57 (2) ◽  
Author(s):  
Qun Yan ◽  
Yu Mi Wi ◽  
Matthew J. Thoendel ◽  
Yash S. Raval ◽  
Kerryl E. Greenwood-Quaintance ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Metagenomic Data ◽  
Metagenomic Sequencing ◽  
Antibacterial Resistance ◽  
Sequencing Data ◽  
Bacterial Detection ◽  
Shotgun Metagenomic Sequencing ◽  
Prosthetic Joint ◽  
Validation Set ◽  
Fluid Culture

ABSTRACT We previously demonstrated that shotgun metagenomic sequencing can detect bacteria in sonicate fluid, providing a diagnosis of prosthetic joint infection (PJI). A limitation of the approach that we used is that data analysis was time-consuming and specialized bioinformatics expertise was required, both of which are barriers to routine clinical use. Fortunately, automated commercial analytic platforms that can interpret shotgun metagenomic data are emerging. In this study, we evaluated the CosmosID bioinformatics platform using shotgun metagenomic sequencing data derived from 408 sonicate fluid samples from our prior study with the goal of evaluating the platform vis-à-vis bacterial detection and antibiotic resistance gene detection for predicting staphylococcal antibacterial susceptibility. Samples were divided into a derivation set and a validation set, each consisting of 204 samples; results from the derivation set were used to establish cutoffs, which were then tested in the validation set for identifying pathogens and predicting staphylococcal antibacterial resistance. Metagenomic analysis detected bacteria in 94.8% (109/115) of sonicate fluid culture-positive PJIs and 37.8% (37/98) of sonicate fluid culture-negative PJIs. Metagenomic analysis showed sensitivities ranging from 65.7 to 85.0% for predicting staphylococcal antibacterial resistance. In conclusion, the CosmosID platform has the potential to provide fast, reliable bacterial detection and identification from metagenomic shotgun sequencing data derived from sonicate fluid for the diagnosis of PJI. Strategies for metagenomic detection of antibiotic resistance genes for predicting staphylococcal antibacterial resistance need further development.

scholarly journals Shotgun metagenomic sequencing data of sunflower rhizosphere microbial community in South Africa

Data in Brief ◽  
2020 ◽  
Vol 31 ◽  
pp. 105831
Author(s):  
Olubukola Oluranti Babalola ◽  
Temitayo Tosin Alawiye ◽  
Carlos Rodriguez Lopez ◽  
Ayansina Segun Ayangbenro
Keyword(s):  
South Africa ◽  
Microbial Community ◽  
Metagenomic Sequencing ◽  
Sequencing Data ◽  
Shotgun Metagenomic Sequencing ◽  
Rhizosphere Microbial Community

scholarly journals Premature neonatal gut microbial community patterns supporting an epithelial TLR-mediated pathway for necrotizing enterocolitis

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Alexander G. Shaw ◽  
Kathleen Sim ◽  
Graham Rose ◽  
David J. Wooldridge ◽  
Ming-Shi Li ◽  
...  
Keyword(s):  
Microbial Community ◽  
Bayesian Regression ◽  
Validation Dataset ◽  
Metagenomic Sequencing ◽  
Faecal Microbiota ◽  
Community Patterns ◽  
Cpg Dna ◽  
Shotgun Metagenomic Sequencing ◽  
Low Levels ◽  
Stimulation Of

Abstract Background Necrotising enterocolitis (NEC) is a devastating bowel disease, primarily affecting premature infants, with a poorly understood aetiology. Prior studies have found associations in different cases with an overabundance of particular elements of the faecal microbiota (in particular Enterobacteriaceae or Clostridium perfringens), but there has been no explanation for the different results found in different cohorts. Immunological studies have indicated that stimulation of the TLR4 receptor is involved in development of NEC, with TLR4 signalling being antagonised by the activated TLR9 receptor. We speculated that differential stimulation of these two components of the signalling pathway by different microbiota might explain the dichotomous findings of microbiota-centered NEC studies. Here we used shotgun metagenomic sequencing and qPCR to characterise the faecal microbiota community of infants prior to NEC onset and in a set of matched controls. Bayesian regression was used to segregate cases from control samples using both microbial and clinical data. Results We found that the infants suffering from NEC fell into two groups based on their microbiota; one with low levels of CpG DNA in bacterial genomes and the other with high abundances of organisms expressing LPS. The identification of these characteristic communities was reproduced using an external metagenomic validation dataset. We propose that these two patterns represent the stimulation of a common pathway at extremes; the LPS-enriched microbiome suggesting overstimulation of TLR4, whilst a microbial community with low levels of CpG DNA suggests reduction of the counterbalance to TLR4 overstimulation. Conclusions The identified microbial community patterns support the concept of NEC resulting from TLR-mediated pathways. Identification of these signals suggests characteristics of the gastrointestinal microbial community to be avoided to prevent NEC. Potential pre- or pro-biotic treatments may be designed to optimise TLR signalling.

scholarly journals Metagenome-Assembled Genomes Isolated from a Human Fecal Diarrhea Sample

2021 ◽  
Vol 10 (19) ◽  
Author(s):  
Tshepiso Pleasure Ateba ◽  
Kazeem Adekunle Alayande ◽  
Ngoma Lubanza ◽  
Mulunda Mwanza
Keyword(s):  
Infectious Disease ◽  
Microbial Community ◽  
Fecal Sample ◽  
Metagenomic Sequencing ◽  
Affected Child ◽  
Shotgun Metagenomic Sequencing

ABSTRACT Diarrheal infection is the second leading infectious disease that is killing children under the age of 5 years. This study investigates the microbial community within a fecal sample from a diarrhea-affected child through shotgun metagenomic sequencing.

scholarly journals The maintenance of microbial community in human fecal samples by a cost effective preservation buffer

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Chongming Wu ◽  
Tianda Chen ◽  
Wenyi Xu ◽  
Tingting Zhang ◽  
Yuwei Pei ◽  
...  
Keyword(s):  
Storage Temperature ◽  
Cost Effective ◽  
Human Saliva ◽  
Fecal Microbiota ◽  
Microbial Consortia ◽  
Metagenomic Sequencing ◽  
Fecal Samples ◽  
Preservation Methods ◽  
Shotgun Metagenomic Sequencing ◽  
Cost Efficient

AbstractIn the burgeoning microbiome field, powerful sequencing approaches and accompanied bioanalytical methods have made tremendous contributions to the discoveries of breakthroughs, which favor to unravel the intimate interplay between gut microbiota and human health. The proper preservation of samples before being processed is essential to guarantee the authenticity and reliability of microbiome studies. Hence, the development of preservation methods is extremely important to hold samples eligible for the consequent analysis, especially population cohort-based investigations or those spanning species or geography, which frequently facing difficulties in suppling freezing conditions. Although there are several commercial products available, the exploration of cost-efficient and ready-to-use preservation methods are still in a large demand. Here, we performed shotgun metagenomic sequencing and demonstrated that microbial consortia in human fecal samples were substantially preserved within a temporary storage of 4 h, independent of the storage temperature. We also verified a previous reported self-made preservation buffer (PB buffer) could not only preserve fecal microbiota at room temperature up to 4 weeks but also enable samples to endure a high temperature condition which mimics temperature variations in summer logistics. Moreover, PB buffer exhibited suitability for human saliva as well. Collectively, PB buffer may be a valuable choice to stabilize samples if neither freezing facilities nor liquid nitrogen is available.

scholarly journals Metagenome Analysis of Surface Waters of the Shardara Reservoir, the Largest Artificial Reservoir in Southern Kazakhstan

2020 ◽  
Vol 9 (11) ◽  
Author(s):  
Madina S. Alexyuk ◽  
Andrey P. Bogoyavlenskiy ◽  
Pavel G. Alexyuk ◽  
Yergali S. Moldakhanov ◽  
Vladimir E. Berezin
Keyword(s):  
Microbial Community ◽  
Surface Waters ◽  
Metagenomic Analysis ◽  
Metagenomic Sequencing ◽  
Metagenome Analysis ◽  
Artificial Reservoir ◽  
Shotgun Metagenomic Sequencing ◽  
Hydropower Engineering

Here, we present a metagenomic analysis of the microflora of the surface waters of the Shardara reservoir, the largest artificial reservoir in Southern Kazakhstan, created to meet irrigation and hydropower engineering needs. In this case, shotgun metagenomic sequencing of the microbial community DNA was used.

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