scholarly journals Genomes from uncultivated prokaryotes: a comparison of metagenome-assembled and single-amplified genomes

2017 ◽  
Author(s):  
Johannes Alneberg ◽  
Christofer M. G. Karlsson ◽  
Anna-Maria Divne ◽  
Claudia Bergin ◽  
Felix Homa ◽  
...  

AbstractBackgroundProkaryotes dominate the biosphere and regulate biogeochemical processes essential to all life. Yet, our knowledge about their biology is for the most part limited to the minority that has been successfully cultured. Molecular techniques now allow for obtaining genome sequences of uncultivated prokaryotic taxa, facilitating in-depth analyses that may ultimately improve our understanding of these key organisms.ResultsWe compared results from two culture-independent strategies for recovering bacterial genomes: single-amplified genomes and metagenome-assembled genomes. Single-amplified genomes were obtained from samples collected at an offshore station in the Baltic Sea Proper and compared to previously obtained metagenome-assembled genomes from a time series at the same station. Among 16 single-amplified genomes analyzed, seven were found to match metagenome-assembled genomes, affiliated with a diverse set of taxa. Notably, genome pairs between the two approaches were nearly identical (>98.7% identity) across overlapping regions (30-80% of each genome). Within matching pairs, the single-amplified genomes were consistently smaller and less complete, whereas the genetic functional profiles were maintained. For the metagenome-assembled genomes, only on average 3.6% of the bases were estimated to be missing from the genomes due to wrongly binned contigs; the metagenome assembly was found to cause incompleteness to a higher degree than the binning procedure.ConclusionsThe strong agreement between the single-amplified and metagenome-assembled genomes emphasizes that both methods generate accurate genome information from uncultivated bacteria. Importantly, this implies that the research questions and the available resources are allowed to determine the selection of genomics approach for microbiome studies.


2021 ◽  
Vol 9 (3) ◽  
pp. 503
Author(s):  
Michael Bording-Jorgensen ◽  
Hannah Tyrrell ◽  
Colin Lloyd ◽  
Linda Chui

Acute gastroenteritis caused by Shiga toxin-producing Escherichia coli (STEC) affects more than 4 million individuals in Canada. Diagnostic laboratories are shifting towards culture-independent diagnostic testing; however, recovery of STEC remains an important aspect of surveillance programs. The objective of this study was to compare common broth media used for the enrichment of STEC. Clinical isolates including O157:H7 as well as non-O157 serotypes were cultured in tryptic soy (TSB), MacConkey (Mac), and Gram-negative (GN) broths and growth was compared using culture on sheep’s blood agar and real-time PCR (qPCR). In addition, a selection of the same isolates was spiked into negative stool and enriched in the same three broths, which were then evaluated using culture on CHROMagarTM STEC agar and qPCR. TSB was found to provide the optimal enrichment for growth of isolates with and without stool. The results from this study suggest that diagnostic laboratories may benefit from enriching STEC samples in TSB as a first line enrichment instead of GN or Mac.



2015 ◽  
Vol 7 ◽  
pp. e2015035 ◽  
Author(s):  
Rosangela Invernizzi ◽  
Federica Quaglia ◽  
Matteo Giovanni Della Porta

Myelodysplastic syndromes (MDS) are hematopoietic stem cell disorders characterized by dysplastic, ineffective, clonal and neoplastic hematopoiesis. MDS represent a complex hematological problem: differences in disease presentation, progression and outcome  have necessitated the use of classification systems to improve diagnosis, prognostication and treatment selection. However, since a single biological or genetic reliable diagnostic marker has not yet been discovered for MDS, quantitative and qualitative dysplastic morphological alterations of bone marrow precursors and of peripheral blood cells are still fundamental for diagnostic classification. In this paper World Health Organization (WHO) classification refinements and current minimal diagnostic criteria proposed by expert panels are highlighted and related problematic issues are discussed. The recommendations should facilitate diagnostic and prognostic evaluations in MDS and selection of patients for new effective targeted therapies. Although in the future morphology should be supplemented with new molecular techniques, the morphological approach, at least for the moment, is still the cornerstone for the diagnosis and classification of these disorders.



2021 ◽  
Author(s):  
Pavithra Anantharaman Sudhakari ◽  
Bhaskar Chandra Mohan Ramisetty

Plasmids are acellular propagating entities that depend, as molecular parasites, on bacteria for propagation. The conflict between the bacterial genome and the parasitic plasmids allows the emergence of genetic arms such as Colicin (Col) operons. Endonuclease Col operons encode three proteins; an endonuclease colicin (cleaves nucleic acids), an immunity protein (inactivates its cognate colicin), and lysis protein (aids in colicin release via host cell lysis). Col operons are efficient plasmid-maintenance systems; (i) the plasmid cured cells are killed by the colicins; (ii) damaged cells lyse and releases the colicins that eliminate the competitors; and (iii) the released plasmids invade new bacteria. Surprisingly, some bacterial genomes have Col operons. The eco-evolutionary drive and physiological relevance of genomic Col operons are unknown. We investigated plasmidic and genomic Col operons using sequence analyses from an eco-evolutionary perspective. We found 1,248 genomic and plasmidic colicins across 30 bacterial genera. Although 51% of the genomes harbor colicins, the majority of the genomic colicins lacked a functional lysis gene, suggesting the negative selection of lethal genes. The immunity gene of the Col operon protects the cured host thereby eliminating the metabolic burden due to plasmid. We show mutual exclusivity of col operons on genomes and plasmids. We propose anti-addiction hypothesis for genomic colicins. Using a stochastic agent-based model, we show that the genomic colicins confer an advantage to the host genome in terms of immunity to the toxin and elimination of plasmid burden. Col operons are genetic arms that regulate the ecological interplay of bacterial genomes and plasmids.



2013 ◽  
Vol 88 (4) ◽  
pp. 506-510 ◽  
Author(s):  
I. Rząd ◽  
P. Hofsoe ◽  
R. Panicz ◽  
J.K. Nowakowski

AbstractUnlike the sporocyst stages, adult leucochloridiid digeneans are difficult to differentiate. Sporocyst broodsacs can be identified on the basis of their colour and banding pattern, but in the absence of broodsacs and when experimental infection cannot be performed, tentative morphological identification needs to be verified, and molecular techniques offer a tool to do this. In this study, adult leucochloridiid digeneans were collected from the great tit (Parus major) found dead at three localities at or near the Baltic Sea coast (Hel, Bukowo-Kopań and Szczecin) in northern Poland. On the basis of differences in their morphological characters, Hel specimens were tentatively assigned to Leucochloridium perturbatum, Bukowo-Kopań and Szczecin specimens being identified tentatively as L. paradoxum. Subsequent ribosomal DNA sequence analysis confirmed the identification of these leucochloridiid flukes. Nucleotide sequences discriminating between the two species were identical to those used by earlier authors as characteristic of two distinctly different sporocyst broodsacs representing L. perturbatum and L. paradoxum.



2001 ◽  
Vol 41 (3) ◽  
pp. 299 ◽  
Author(s):  
J. E. Thies ◽  
E. M. Holmes ◽  
A. Vachot

The symbiosis between legumes and their specific root-nodule bacteria, rhizobia, has been employed to improve agricultural productivity for most of the 20th century. During this time, great advances have been made in our knowledge of both plant and bacterial genomes, the biochemistry of the symbiosis, plant and bacterial signaling and the measurement of nitrogen fixation. However, knowledge of the ecology of the bacterial symbiont has lagged behind, largely due to a lack of practical techniques that can be used to monitor and assess the performance of these bacteria in the field. Most techniques developed in the last few decades have relied on somehow ‘marking’ individual strains to allow us to follow their fate in the field environment. Such techniques, while providing knowledge of the success or failure of specific strains in a range of environments, have not allowed insight into the nature of the pre-existing rhizobial populations in these sites, nor the interaction between marked strains and the background population. The advent of molecular techniques has revolutionised the study of Rhizobium ecology by allowing us to follow the flux of a variety of ecotypes within a particular site and to examine how introduced rhizobia interact with a genetically diverse background. In addition, molecular techniques have increased our understanding of how individual strains and populations of root-nodule bacteria respond to changes in the environment and how genetic diversity evolves in field sites over time. This review focuses on recently developed molecular techniques that hold promise for continuing to develop our understanding of Rhizobium ecology and how these can be used to address a range of applied problems to yield new insights into rhizobial life in soil and as legume symbionts.



2021 ◽  
Vol 16 (4) ◽  
pp. 271-288
Author(s):  
Ian Gassiep ◽  
Delaney Burnard ◽  
Michelle J Bauer ◽  
Robert E Norton ◽  
Patrick N Harris

Melioidosis is an emerging infectious disease with an estimated global burden of 4.64 million disability-adjusted life years per year. A major determinant related to poor disease outcomes is delay to diagnosis due to the fact that identification of the causative agent Burkholderia pseudomallei may be challenging. Over the last 25 years, advances in molecular diagnostic techniques have resulted in the potential for rapid and accurate organism detection and identification direct from clinical samples. While these methods are not yet routine in clinical practice, laboratory diagnosis of infectious diseases is transitioning to culture-independent techniques. This review article aims to evaluate molecular methods for melioidosis diagnosis direct from clinical samples and discuss current and future utility and limitations.



2020 ◽  
Vol 3 (2) ◽  
pp. 39 ◽  
Author(s):  
Anna Ojo-Okunola ◽  
Shantelle Claassen-Weitz ◽  
Kilaza S. Mwaikono ◽  
Sugnet Gardner-Lubbe ◽  
Heather J. Zar ◽  
...  

Culture-independent molecular techniques have advanced the characterization of environmental and human samples including the human milk (HM) bacteriome. However, extraction of high-quality genomic DNA that is representative of the bacterial population in samples is crucial. Lipids removal from HM prior to DNA extraction is common practice, but this may influence the bacterial population detected. The objective of this study was to compare four commercial DNA extraction kits and lipid removal in relation to HM bacterial profiles. Four commercial DNA extraction kits, QIAamp® DNA Microbiome Kit, ZR Fungal/Bacterial DNA MiniPrep™, QIAsymphony DSP DNA Kit and ZymoBIOMICS™ DNA Miniprep Kit, were assessed using milk collected from ten healthy lactating women. The kits were evaluated based on their ability to extract high quantities of pure DNA from HM and how well they extracted DNA from bacterial communities present in a commercial mock microbial community standard spiked into HM. Finally, the kits were evaluated by assessing their extraction repeatability. Bacterial profiles were assessed using Illumina MiSeq sequencing targeting the V4 region of the 16S rRNA gene. The ZR Fungal/Bacterial DNA MiniPrep™ and ZymoBIOMICS™ DNA Miniprep (Zymo Research Corp., Irvine, CA, USA) kits extracted the highest DNA yields with the best purity. DNA extracted using ZR Fungal/Bacterial DNA MiniPrep™ best represented the bacteria in the mock community spiked into HM. In un-spiked HM samples, DNA extracted using the QIAsymphony DSP DNA kit showed statistically significant differences in taxa prevalence from DNA extracted using ZR Fungal/Bacterial DNA MiniPrep™ and ZymoBIOMICS™ DNA Miniprep kits. The only difference between skim and whole milk is observed in bacterial profiles with differing relative abundances of Enhydrobacter and Acinetobacter. DNA extraction, but not lipids removal, substantially influences bacterial profiles detected in HM samples, emphasizing the need for careful selection of a DNA extraction kit to improve DNA recovery from a range of bacterial taxa.



2009 ◽  
Vol 75 (13) ◽  
pp. 4435-4442 ◽  
Author(s):  
Lisa M. Steinberg ◽  
John M. Regan

ABSTRACT Methanogens are of great importance in carbon cycling and alternative energy production, but quantitation with culture-based methods is time-consuming and biased against methanogen groups that are difficult to cultivate in a laboratory. For these reasons, methanogens are typically studied through culture-independent molecular techniques. We developed a SYBR green I quantitative PCR (qPCR) assay to quantify total numbers of methyl coenzyme M reductase α-subunit (mcrA) genes. TaqMan probes were also designed to target nine different phylogenetic groups of methanogens in qPCR assays. Total mcrA and mcrA levels of different methanogen phylogenetic groups were determined from six samples: four samples from anaerobic digesters used to treat either primarily cow or pig manure and two aliquots from an acidic peat sample stored at 4°C or 20°C. Only members of the Methanosaetaceae, Methanosarcina, Methanobacteriaceae, and Methanocorpusculaceae and Fen cluster were detected in the environmental samples. The three samples obtained from cow manure digesters were dominated by members of the genus Methanosarcina, whereas the sample from the pig manure digester contained detectable levels of only members of the Methanobacteriaceae. The acidic peat samples were dominated by both Methanosarcina spp. and members of the Fen cluster. In two of the manure digester samples only one methanogen group was detected, but in both of the acidic peat samples and two of the manure digester samples, multiple methanogen groups were detected. The TaqMan qPCR assays were successfully able to determine the environmental abundance of different phylogenetic groups of methanogens, including several groups with few or no cultivated members.



Via Latgalica ◽  
2013 ◽  
pp. 8
Author(s):  
Lidija Leikuma

The article presents the conclusions the author has come to during the implementation of the project “Development of Research Infrastructure for Education in the Humanities in Eastern Latvia, Lithuania”) the code name of which is “LLIII-207 HipiLatLit”. On the basis of 1,000,000 (one million) word usage instances of “Frequency Dictionary of the Written Sources of the Lithuanian Language” (“Dažninis rašytinės lietuvių kalbos žodynas” (Compiler A. Utka; http://donelaitis.vdu.lt/publikacijos/Dazninis_zodynas.pdf) an original translating dictionary containing 10,000 units has been compiled. For the most frequently used words of the base (Lithuanian) language the counterparts have been given in the two standardised variants of the Latvian language – the Latvian literary language and the Latgalian written or literary language. The ways of searching and possibilities of fi nding the equivalents are shown by the insight into the laboratory of creation of the electronic “Lithuanian-Latvian- Latgalian Dictionary”, focusing on the Latgalian part of the dictionary in more detail. Since a wide-scale and linguistically correct collection of the Latgalian vocabulary is still not available, in the process of the development of the dictionary it was necessary to deal with a number of theoretical and practical problems. For solving of these problems the conclusions of the Latvian (Alīse Laua, Ruta Veidemane, Liene Roze, Inta Freimane) and non-Latvian (Valery Berkov, Vladimir Dubichinski, Evalda Jakaitienė) lexicologists and lexicographers have been applied. In the project much has been done for clearing up and specifying the semantic counterparts of the base language in accordance with the qualities of the corpus. The linguistic processing of the selected lexemes and homogeneous interpretation of the material needed multiple checking of the material as it is given in dictionaries and scrupulous comparison. The semantic structure coincides for unambiguous words, i.e. vocabulary of general use, for other cases translating and explaining equivalents have been searched for. Nowadays not everything corresponds any longer to the recordings in the earlier lexicographic sources (“The Lithuanian-Latvian dictionary” (1995), “The Latvian Literary Language Dictionary” (1972–1996)) – the languages have been changing. Also the Latgalian part of the dictionary presents the changes of the lexical composition and word meanings. It has been tried to display it with appropriate markings as to the restrictions of the word usage. Although in the Latgalian language at times there are no necessary terms or names for the abstract concepts, however, it is possible to find the necessary counterparts for everything. The lexicographic finish of the material enclosed is explained in more detail. The most important dictionaries for the project under implementation have been actualised. Up to now, two translating trilingual dictionaries have been published (Jānis Kurmins “Słownik polsko łacinsko łotewski” (1858) and Eduards Kozlovskis “Krìwu- Latgališu-Wòcu wòrdinica” (1918)) where one of the languages is Latgalian; topical entries of these dictionaries have been dealt with in more detail. Also other developments have been analysed, namely those containing the Latgalian vocabulary, critical comments being given on potential neologisms, namely nonce-words. The majority of the collections of the Latgalian vocabulary are not extensive, and their producers have generally not been the Baltic linguists. The two former lexicographic sources have a normative nature: Pīters Strods “Pareizraksteibas vōrdneica" (1933) and Mikelis Bukšs and Juris Placinskis “Latgaļu volūdas gramatika un pareizraksteibas vōrdneica” (1973). In selecting the Latgalian counterparts for the new dictionary both the former normative sources have been taken into account and traditions have been observed, the area of the usage of lexemes, the significance of the word or the form, the frequency of the usage, etc. have been respected. The possibilities of the Latgalian language to present the semantic counterparts for the relevant lexemes of the Lithuanian and Latvian languages have been characterised in more detail. In compliance with the base language both the equivalent Latgalian vocabulary and that one having no direct equivalent have been shown, the principles for selection of synonyms and word variants have been explained, the necessity of inclusion of the word variants in the translating dictionary has been substantiated. The less the language has been cultivated, the higher number of variants it has, although the option of choice of variants is unsatisfactory mostly for practitioners. Some uncertainty in the Latgalian language at the moment is inevitable: due to uncertain status of the language and the variable use as regards the intensity of utilisation, the language has been standardised and codified deficiently up to now. In this article a special attention has been paid to the analysis of the very material of the Latgalian language, to the synonyms of the Latgalian equivalents, the explanations as regards indications of the restrictions on the word usage. Occasionally more detailed comments have been given for the relevant examples. Due to the rich use of marking “The Lithuanian-Latvian-Latgalian Dictionary” differs from the traditional translating dictionaries where appropriate signs are used less frequently, however, cultivation of the language is promoted just by evaluation of the vocabulary layers, analysis of potentials, predictions of the further changes. Such problem situations as, for example, usefulness of inclusion of a greater number of Slavisms, a lack of consequence as to indications of the stylistic shade of colloquial speech, the uncertain coherence between the colloquialisms and barbarisms have been discussed in the article. The place of the literarisms has been dealt with in the system of the Latgalian language, their fitting and “inevitability" nowadays have been analysed. Some of the uncertainties remained in the development of the dictionary have been outlined. Evaluation has also been provided for the selection of the Latgalian vocabulary, thus giving new, clearer and more certain impulses as to the content for cultivators of the Latgalian language. The compiled electronic “Lithuanian-Latvian-Latgalian Dictionary” will be useful both for practical use and research in lexicology and lexicography. Its Latgalian part can be used as a basis for the further lexicographic developments.



2010 ◽  
Vol 42 (6) ◽  
pp. 878-887 ◽  
Author(s):  
Penny R. Hirsch ◽  
Tim H. Mauchline ◽  
Ian M. Clark


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