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Auto Tag: Bacterial genome

September 22, 2019

A response to Lindsey et al. “Wolbachia pipientis should not be split into multiple species: A response to Ramírez-Puebla et al.”.

In Ramírez-Puebla et al. [18] we compared 34 Wolbachia genomes and constructed phylogenetic trees using genomic data. In general, our results were congruent with previously reported phy- logenetic trees [5,9]. Our datasets were carefully selected, checked and analyzed avoiding horizontally transferred genes. In the case of the wAna genome we did not use the raw data, but the assem- bled genome [22] and 31 genes were used to compare in a dataset of conserved proteins. To confirm our conclusions a new phyloge- nomic analysis was performed excluding the wAna strain in the dataset (Fig. 1). The same topology was obtained, therefore indi- cating that the results were not affected by the presence of this particular strain.

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September 22, 2019

Candidatus Dactylopiibacterium carminicum, a nitrogen-fixing symbiont of Dactylopius cochineal insects (Hemiptera: Coccoidea: Dactylopiidae)

The domesticated carmine cochineal Dactylopius coccus (scale insect) has commercial value and has been used for more than 500?years for natural red pigment production. Besides the domesticated cochineal, other wild Dactylopius species such as Dactylopius opuntiae are found in the Americas, all feeding on nutrient poor sap from native cacti. To compensate nutritional deficiencies, many insects harbor symbiotic bacteria which provide essential amino acids or vitamins to their hosts. Here, we characterized a symbiont from the carmine cochineal insects, Candidatus Dactylopiibacterium carminicum (betaproteobacterium, Rhodocyclaceae family) and found it in D. coccus and in D. opuntiae ovaries by fluorescent in situ hybridization, suggesting maternal inheritance. Bacterial genomes recovered from metagenomic data derived from whole insects or tissues both from D. coccus and from D. opuntiae were around 3.6?Mb in size. Phylogenomics showed that dactylopiibacteria constituted a closely related clade neighbor to nitrogen fixing bacteria from soil or from various plants including rice and other grass endophytes. Metabolic capabilities were inferred from genomic analyses, showing a complete operon for nitrogen fixation, biosynthesis of amino acids and vitamins and putative traits of anaerobic or microoxic metabolism as well as genes for plant interaction. Dactylopiibacterium nif gene expression and acetylene reduction activity detecting nitrogen fixation were evidenced in D. coccus hemolymph and ovaries, in congruence with the endosymbiont fluorescent in situ hybridization location. Dactylopiibacterium symbionts may compensate for the nitrogen deficiency in the cochineal diet. In addition, this symbiont may provide essential amino acids, recycle uric acid, and increase the cochineal life span.

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September 22, 2019

Genetic determinants of in vivo fitness and diet responsiveness in multiple human gut Bacteroides.

Libraries of tens of thousands of transposon mutants generated from each of four human gut Bacteroides strains, two representing the same species, were introduced simultaneously into gnotobiotic mice together with 11 other wild-type strains to generate a 15-member artificial human gut microbiota. Mice received one of two distinct diets monotonously, or both in different ordered sequences. Quantifying the abundance of mutants in different diet contexts allowed gene-level characterization of fitness determinants, niche, stability, and resilience and yielded a prebiotic (arabinoxylan) that allowed targeted manipulation of the community. The approach described is generalizable and should be useful for defining mechanisms critical for sustaining and/or approaches for deliberately reconfiguring the highly adaptive and durable relationship between the human gut microbiota and host in ways that promote wellness. Copyright © 2015, American Association for the Advancement of Science.

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September 22, 2019

Single Molecule Sequencing: new outlooks for solving genome assembly and transcripts identification challenges

In this review, we introduce a novel sequencing technology, named Single Molecule Real Time sequencing. Also called Single Molecule Sequencing, as it do not requires any amplification, this new technology is able to pro- duce much longer reads than previous NGS technologies such as Illumina. This read size improvements, which can reach 150 fold, will solve many challenges caused by the actual NGS technologies. Short NGS reads, reach- ing a maximum size of 300 bp, make it hard to reconstitute a whole genome and are always leading to fragmented genome assembly. It is also difficult to correctly infer transcript quantification and identification when there is a high isoforms diversity. Despite their higher error rate, long reads have shown very promising result concerning these actual issues. We show that longer reads can produce less fragmented assembly, with a better quality, but also sequence from start to end mRNA, making it much more easier to infer correct transcript quantification, and even allow new intron structure and so new isoforms discovery.

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September 22, 2019

ALE: a generic assembly likelihood evaluation framework for assessing the accuracy of genome and metagenome assemblies.

Researchers need general purpose methods for objectively evaluating the accuracy of single and metagenome assemblies and for automatically detecting any errors they may contain. Current methods do not fully meet this need because they require a reference, only consider one of the many aspects of assembly quality or lack statistical justification, and none are designed to evaluate metagenome assemblies.In this article, we present an Assembly Likelihood Evaluation (ALE) framework that overcomes these limitations, systematically evaluating the accuracy of an assembly in a reference-independent manner using rigorous statistical methods. This framework is comprehensive, and integrates read quality, mate pair orientation and insert length (for paired-end reads), sequencing coverage, read alignment and k-mer frequency. ALE pinpoints synthetic errors in both single and metagenomic assemblies, including single-base errors, insertions/deletions, genome rearrangements and chimeric assemblies presented in metagenomes. At the genome level with real-world data, ALE identifies three large misassemblies from the Spirochaeta smaragdinae finished genome, which were all independently validated by Pacific Biosciences sequencing. At the single-base level with Illumina data, ALE recovers 215 of 222 (97%) single nucleotide variants in a training set from a GC-rich Rhodobacter sphaeroides genome. Using real Pacific Biosciences data, ALE identifies 12 of 12 synthetic errors in a Lambda Phage genome, surpassing even Pacific Biosciences’ own variant caller, EviCons. In summary, the ALE framework provides a comprehensive, reference-independent and statistically rigorous measure of single genome and metagenome assembly accuracy, which can be used to identify misassemblies or to optimize the assembly process.ALE is released as open source software under the UoI/NCSA license at http://www.alescore.org. It is implemented in C and Python.

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September 22, 2019

Composition and pathogenic potential of a microbial bioremediation product used for crude oil degradation.

A microbial bioremediation product (MBP) used for large-scale oil degradation was investigated for microbial constituents and possible pathogenicity. Aerobic growth on various media yielded >108 colonies mL-1. Full-length 16S rDNA sequencing and fatty acid profiling from morphologically distinct colonies revealed =13 distinct genera. Full-length 16S rDNA library sequencing, by either Sanger or long-read PacBio technology, suggested that up to 21% of the MBP was composed of Arcobacter. Other high abundance microbial constituents (>6%) included the genera Proteus, Enterococcus, Dysgonomonas and several genera in the order Bacteroidales. The MBP was most susceptible to ciprofloxacin, doxycycline, gentamicin, and meropenam. MBP exposure of human HT29 and A549 cells caused significant cytotoxicity, and bacterial growth and adherence. An acellular MBP filtrate was also cytotoxic to HT29, but not A549. Both MBP and filtrate exposures elevated the neutrophil chemoattractant IL-8. In endotracheal murine exposures, bacterial pulmonary clearance was complete after one-week. Elevation of pro-inflammatory cytokines IL-1ß, IL-6, and TNF-a, and chemokines KC and MCP-1 occurred between 2h and 48h post-exposure, followed by restoration to baseline levels at 96h. Cytokine/chemokine signalling was accompanied by elevated blood neutrophils and monocytes at 4h and 48h, respectively. Peripheral acute phase response markers were maximal at 24h. All indicators examined returned to baseline values by 168h. In contrast to HT29, but similar to A549 observations, MBP filtrate did not induce significant murine effects with the indicators examined. The results demonstrated the potentially complex nature of MBPs and transient immunological effects during exposure. Products containing microbes should be scrutinized for pathogenic components and subjected to characterisation and quality validation prior to commercial release.

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September 22, 2019

Introduction to isoform sequencing using Pacific Biosciences technology (Iso-Seq)

Alternative RNA splicing is a known phenomenon, but we still do not have a complete catalog of isoforms that explain variability in the human transcriptome. We have made significant progress in developing methods to study variability of the transcriptome, but we are far away of having a complete picture of the transcriptome. The initial methods to study gene expression were based on cloning of cDNAs and Sanger sequencing. The strategy was labor-intensive and expensive. With the development of microarrays, different methods based on exon arrays and tiling arrays provided valuable information about RNA expression. However, the microarray presented significant limitations. Most of the limitations became apparent by 2005, but it was not until 2008 that an alternative method to study the transcriptome was developed. RNA Sequencing using next-generation sequencing (RNA-Seq) quickly became the technology of choice for gene expression profiling. Recently, the precision and sensitivity of RNA-Seq have come into question, especially for transcriptome reconstruction. This chapter will describe a relatively new method, “Isoform Sequencing (Iso-Seq). Iso-Seq was developed by Pacific Biosciences (PacBio), and it is capable of identifying new isoforms with extraordinary precision due to its long-read technology. The technique to create libraries is straightforward, and the PacBio RS II instrument generates the information in hours. The bioinformatics analysis is performed using the freely available SMRT® Portal software. The SMRT Portal is easy to use and capable of performing all the steps necessary to analyze the raw data and to generate high-quality full-length isoforms. For the universal acceptance of the Iso-Seq method, the capacity of the SMRT Cells needs to improve at least 10- to 100-fold to make the system affordable and attractive to users.

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September 22, 2019

Metagenomic SMRT sequencing-based exploration of novel lignocellulose-degrading capability in wood detritus from Torreya nucifera in Bija forest on Jeju Island.

Lignocellulose, mostly composed of cellulose, hemicellulose and lignin generated through secondary growth of woody plant, is considered as promising resources for bio-fuel. In order to use lignocellulose as a biofuel, the biodegradation besides high-cost chemical treatments were applied, but its knowledge on decomposition of lignocellulose occurring in a natural environment were insufficient. We analyzed 16S rRNA gene and metagenome to understand how the lignocellulose are decomposed naturally in decayed Torreya nucifera (L) of Bija forest (Bijarim) in Gotjawal, an ecologically distinct environment. A total of 464,360 reads were obtained from 16S rRNA gene sequencing, representing diverse phyla; Proteobacteria (51%), Bacteroidetes (11%) and Actinobacteria (10%). The metagenome analysis using Single Molecules Real-Time Sequencing revealed that the assembled contigs determined by originated from Proteobacteria (58%) and Actinobacteria (10.3%). Carbohydrate Active enZYmes (CAZy) and Protein families (Pfam) based analysis showed that Proteobacteria was involved in degrading whole lignocellulose and Actinobacteria played a role only in a part of hemicellulose degradation. Combining these results, it suggested that Proteobacteria and Actinobacteria had selective biodegradation potential for different lignocellulose substrate. Thus, it is considered that understanding of the systemic microbial degradation pathways may be a useful strategy for recycle of lignocellulosic biomass and the microbial enzymes in Bija forest can be useful natural resources in industrial processes.

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September 22, 2019

Next-generation sequencing for pathogen detection and identification

Over the past decade, the field of genomics has seen such drastic improvements in sequencing chemistries that high-throughput sequencing, or next-generation sequencing (NGS), is being applied to generate data across many disciplines. NGS instruments are becoming less expensive, faster, and smaller, and therefore are being adopted in an increasing number of laboratories, including clinical laboratories. Thus far, clinical use of NGS has been mostly focused on the human genome, for purposes such as characterizing the molecular basis of cancer or for diagnosing and understanding the basis of rare genetic disorders. There are, however, an increasing number of examples whereby NGS is employed to discover novel pathogens, and these cases provide precedent for the use of NGS in microbial diagnostics. NGS has many advantages over traditional microbial diagnostic methods, such as unbiased rather than pathogen-specific protocols, ability to detect fastidious or non-culturable organisms, and ability to detect co-infections. One of the most impressive advantages of NGS is that it requires little or no prior knowledge of the pathogen, unlike many other diagnostic assays; therefore for pathogen discovery, NGS is very valuable. However, despite these advantages, there are challenges involved in implementing NGS for routine clinical microbiological diagnosis. We discuss these advantages and challenges in the context of recently described research studies.

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September 22, 2019

A novel enrichment strategy reveals unprecedented number of novel transcription start sites at single base resolution in a model prokaryote and the gut microbiome.

The initiating nucleotide found at the 5′ end of primary transcripts has a distinctive triphosphorylated end that distinguishes these transcripts from all other RNA species. Recognizing this distinction is key to deconvoluting the primary transcriptome from the plethora of processed transcripts that confound analysis of the transcriptome. The currently available methods do not use targeted enrichment for the 5’end of primary transcripts, but rather attempt to deplete non-targeted RNA.We developed a method, Cappable-seq, for directly enriching for the 5′ end of primary transcripts and enabling determination of transcription start sites at single base resolution. This is achieved by enzymatically modifying the 5′ triphosphorylated end of RNA with a selectable tag. We first applied Cappable-seq to E. coli, achieving up to 50 fold enrichment of primary transcripts and identifying an unprecedented 16539 transcription start sites (TSS) genome-wide at single base resolution. We also applied Cappable-seq to a mouse cecum sample and identified TSS in a microbiome.Cappable-seq allows for the first time the capture of the 5′ end of primary transcripts. This enables a unique robust TSS determination in bacteria and microbiomes.  In addition to and beyond TSS determination, Cappable-seq depletes ribosomal RNA and reduces the complexity of the transcriptome to a single quantifiable tag per transcript enabling digital profiling of gene expression in any microbiome.

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September 22, 2019

Single molecule real-time (SMRT) sequencing comes of age: applications and utilities for medical diagnostics.

Short read massive parallel sequencing has emerged as a standard diagnostic tool in the medical setting. However, short read technologies have inherent limitations such as GC bias, difficulties mapping to repetitive elements, trouble discriminating paralogous sequences, and difficulties in phasing alleles. Long read single molecule sequencers resolve these obstacles. Moreover, they offer higher consensus accuracies and can detect epigenetic modifications from native DNA. The first commercially available long read single molecule platform was the RS system based on PacBio’s single molecule real-time (SMRT) sequencing technology, which has since evolved into their RSII and Sequel systems. Here we capsulize how SMRT sequencing is revolutionizing constitutional, reproductive, cancer, microbial and viral genetic testing.© The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research.

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September 22, 2019

Metataxonomic and metagenomic approaches vs. culture-based techniques for clinical pathology.

Diagnoses that are both timely and accurate are critically important for patients with life-threatening or drug resistant infections. Technological improvements in High-Throughput Sequencing (HTS) have led to its use in pathogen detection and its application in clinical diagnoses of infectious diseases. The present study compares two HTS methods, 16S rRNA marker gene sequencing (metataxonomics) and whole metagenomic shotgun sequencing (metagenomics), in their respective abilities to match the same diagnosis as traditional culture methods (culture inference) for patients with ventilator associated pneumonia (VAP). The metagenomic analysis was able to produce the same diagnosis as culture methods at the species-level for five of the six samples, while the metataxonomic analysis was only able to produce results with the same species-level identification as culture for two of the six samples. These results indicate that metagenomic analyses have the accuracy needed for a clinical diagnostic tool, but full integration in diagnostic protocols is contingent on technological improvements to decrease turnaround time and lower costs.

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September 22, 2019

Effects of antibiotic on microflora in ileum and cecum for broilers by 16S rRNA sequence analysis.

An experiment was conducted to analyze and compare the microbial composition, abundance, dynamic distribution, and functions without and with antibiotic fed to broilers. A 16S rRNA-sequencing approach was used to evaluate the bacterial composition of the gut of male broilers under different groups. A total of 240 1-day old AA male broilers were randomly assigned to two groups, with 120 broilers per group. The treatment group was administered an antibiotic with their feed, while the control group was not administered antibiotic (control group). A total of 10 replicates were assessed per treatment. The control group was fed a basal diet containing corn, soybean meal, and cottonseed meal and met the nutritional requirement. The antibiotic group was fed 100 mg/kg aureomycin (based on the basal diet). The trial lasted 42 days. Operational taxonomic unit partition and classification, alpha diversity, taxonomic composition, beta diversity, and microflora comparative analyses along with key species screening were performed for all of the treatment groups. Our data indicate that aureomycin treatment in broilers is directly correlated with variations of the gut content of specific bacterial taxa, and herein provide insights into the impact of antibiotic on microbial communities in cecum and ileum of broiler chickens.© 2018 Japanese Society of Animal Science.

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September 22, 2019

The effects of probiotics administration on the milk production, milk components and fecal bacteria microbiota of dairy cows

Probiotics administration can improve host health. This study aims to determine the effects of probiotics (Lactobacillus casei Zhang and Lactobacillus plantarum P-8) administration on milk production, milk functional components, milk composition, and fecal microbiota of dairy cows. Variations in the fecal bacteria microbiota between treatments were assessed based on 16S rRNA profiles determined by PacBio single molecule real-time sequencing technology. The probiotics supplementation significantly increased the milk production and the contents of milk immunoglobulin G (IgG), lactoferrin (LTF), lysozyme (LYS) and lactoperoxidase (LP), while the somatic cell counts (SCC) significantly decreased (P < 0.01). However, no significant difference was found in the milk fat, protein and lactose contents (P > 0.05). Although the probiotics supplementation did not change the fecal bacteria richness and diversity, significantly more rumen fermentative bacteria (Bacteroides, Roseburia, Ruminococcus, Clostridium, Coprococcus and Dorea) and beneficial bacteria (Faecalibacterium prausnitzii) were found in the probiotics treatment group. Meanwhile, some opportunistic pathogens e.g. Bacillus cereus, Cronobacter sakazakii and Alkaliphilus oremlandii, were suppressed. Additionally, we found some correlations between the milk production, milk components and fecal bacteria. To sum up, our study demonstrated the beneficial effects of probiotics application in improving the quality and quantity of cow milk production.

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