scholarly journals DNA enrichment and tagmentation method for species-level identification and strain-level differentiation using ON-rep-seq

2019 ◽  
Vol 2 (1) ◽  
Author(s):  
Łukasz Krych ◽  
Josué L. Castro-Mejía ◽  
Laura M. Forero-Junco ◽  
Daniel N. Moesby ◽  
Morten B. Mikkelsen ◽  
...  
Keyword(s):  
Cost Effective ◽  
Species Level ◽  
Strain Level ◽  
Read Length ◽  
High Quality ◽  
Bacterial Dna ◽  
Sequencing Platform ◽  
Dna Library ◽  
Culture Independent ◽  
Level Identification

Abstract Despite the massive developments within culture-independent methods for detection of microorganisms during the last decade, culture-based methods remain a cornerstone in microbiology. Yet, the problem of rapid, accurate and inexpensive identification of bacterial isolates down to species/strain level remains unresolved. We have developed a new method for bacterial DNA enrichment and tagmentation allowing fast (<24 h) and cost-effective species level identification and strain level differentiation using the MinION portable sequencing platform (ON-rep-seq). DNA library preparation for 96 isolates takes less than 5 h and ensures highly reproducible distribution of reads that can be used to generate strain level specific read length counts profiles (LCp). We have developed a pipeline that by correcting reads error within peaks of LCp generates a set of high quality (>99%) consensus reads. Whereas, the information from high quality reads is used to retrieve species level taxonomy, comparison of LCp allows for strain level differentiation.

scholarly journals Finally, Bulk Typing of Bacterial Species down to Strain Level using ON-rep-seq

2018 ◽  
Author(s):  
Łukasz Krych ◽  
Josué L. Castro-Mejía ◽  
Daniel N. Moesby ◽  
Morten B. Mikkelsen ◽  
Morten A. Rasmussen ◽  
...  
Keyword(s):  
High Speed ◽  
Bacterial Species ◽  
Cost Effective ◽  
Species Level ◽  
Strain Level ◽  
Read Length ◽  
High Quality ◽  
Sequencing Platform ◽  
Culture Independent ◽  
Single Flow

AbstractDespite the massive developments within culture-independent methods for detection and quantification of microorganisms during the last decade, culture-based methods remain a cornerstone in microbiology. We have developed a new method for bacterial DNA enrichment and tagmentation allowing fast (< 24h) and cost-effective species level identification and strain level differentiation using the MinION portable sequencing platform (ON-rep-seq). DNA library preparation takes less than 5h and ensures highly reproducible distribution of reads that can be used to generate strain level specific read length counts profiles (LCp). We have developed a pipeline that by correcting the random error of reads within peaks of LCp generates a set (∼10 contigs per sample; 300bp - 3Kb) of high quality (>99%) consensus reads. Whereas, the information from high quality reads is used to retrieve species level taxonomy, comparison of LCp allows for strain level differentiation. With benchmarked 288 isolates identified on a single flow cell and a theoretical throughput to evaluate over 1000 isolates, our method allows for detailed bacterial identification for less than 2$ per sample at very high speed.

scholarly journals PseudomonasDiversity in Crude-Oil-Contaminated Intertidal Sand Samples Obtained after thePrestigeOil Spill

2010 ◽  
Vol 77 (3) ◽  
pp. 1076-1085 ◽  
Author(s):  
Magdalena Mulet ◽  
Zoyla David ◽  
Balbina Nogales ◽  
Rafael Bosch ◽  
Jorge Lalucat ◽  
...  
Keyword(s):  
Carbon Sources ◽  
Species Level ◽  
Gene Library ◽  
Direct Plating ◽  
Selective Media ◽  
Dna Library ◽  
Culture Independent ◽  
Northwestern Spain ◽  
Pcr Method ◽  
Culture Dependent

ABSTRACTThe Galicia seashore, in northwestern Spain, was one of the shorelines affected by thePrestigeoil spill in November 2002. The diversity of autochthonousPseudomonaspopulations present at two beaches (Carnota municipality) was analyzed using culture-independent and culture-dependent methods. The first analysis involved the screening of anrpoDgene library. The second involved the isolation of 94Pseudomonasstrains that were able to grow on selective media by direct plating or after serial enrichments on several carbon sources: biphenyl, gentisate, hexadecane, methylnaphthalene, naphthalene, phenanthrene, salicylate, xylene, and succinate. Eight denitrifyingPseudomonasstrains were also isolated by their ability to grow anaerobically with nitrate. The calculated coverage index forPseudomonasspecies was 89% when clones and isolates were considered together, and there were 29 phylospecies detected. The most abundant were members of the speciesP. stutzeri,P.putida,P. anguilliseptica, andP. oleovorans. Thirty-one isolates could not be identified at the species level and were considered representatives of 16 putative novelPseudomonasspecies. One isolate was considered representative of a novelP. stutzerigenomovar. Concordant results were obtained when the diversities of the cloned DNA library and the cultured strains were compared. The clone library obtained by therpoDPCR method was a useful tool for evaluatingPseudomonascommunities and also for microdiversity studies ofPseudomonaspopulations.

scholarly journals Species-Level Resolution of Female Bladder Microbiota from 16S rRNA Amplicon Sequencing

mSystems ◽  
2021 ◽  
Author(s):  
Carter Hoffman ◽  
Nazema Y. Siddiqui ◽  
Ian Fields ◽  
W. Thomas Gregory ◽  
Holly M. Simon ◽  
...  
Keyword(s):  
16S Rrna ◽  
Amplicon Sequencing ◽  
Species Level ◽  
Classification Schemes ◽  
Common Culture ◽  
16S Rrna Amplicon Sequencing ◽  
Female Bladder ◽  
Culture Independent ◽  
Level Identification

Accurate species-level identification from culture-independent techniques is of importance for microbial niches that are less well characterized, such as that of the bladder. 16S rRNA amplicon sequencing, a common culture-independent way to identify bacteria, is often critiqued for lacking species-level resolution. Here, we extensively evaluate classification schemes for species-level bacterial annotation of 16S amplicon data from bladder bacteria.

scholarly journals Development of a Two-Color Fluorescence In Situ Hybridization Technique for Species-Level Identification of Human-Infectious Cryptosporidium spp.

2009 ◽  
Vol 75 (18) ◽  
pp. 5996-5998 ◽  
Author(s):  
A. Alagappan ◽  
P. L. Bergquist ◽  
B. C. Ferrari
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Cryptosporidium Parvum ◽  
Cost Effective ◽  
Species Level ◽  
Hybridization Technique ◽  
Cryptosporidium Spp ◽  
Simultaneous Identification ◽  
Level Identification

ABSTRACT A two-color fluorescence in situ hybridization assay that allows for the simultaneous identification of Cryptosporidium parvum and C. hominis was developed. The assay is a simple, rapid, and cost-effective tool for the detection of the major Cryptosporidium species of concern to public health.

scholarly journals Comparison of three amplicon sequencing approaches to determine staphylococcal populations on human skin

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Charlotte Marie Ahle ◽  
Kristian Stødkilde-Jørgensen ◽  
Anja Poehlein ◽  
Wolfgang R. Streit ◽  
Jennifer Hüpeden ◽  
...  
Keyword(s):  
Human Skin ◽  
Skin Diseases ◽  
Cost Effective ◽  
Amplicon Sequencing ◽  
Species Level ◽  
Skin Microbiome ◽  
Staphylococcal Species ◽  
Culture Independent ◽  
Skin Samples ◽  
Mock Communities

Abstract Background Staphylococci are important members of the human skin microbiome. Many staphylococcal species and strains are commensals of the healthy skin microbiota, while few play essential roles in skin diseases such as atopic dermatitis. To study the involvement of staphylococci in health and disease, it is essential to determine staphylococcal populations in skin samples beyond the genus and species level. Culture-independent approaches such as amplicon next-generation sequencing (NGS) are time- and cost-effective options. However, their suitability depends on the power of resolution. Results Here we compare three amplicon NGS schemes that rely on different targets within the genes tuf and rpsK, designated tuf1, tuf2 and rpsK schemes. The schemes were tested on mock communities and on human skin samples. To obtain skin samples and build mock communities, skin swab samples of healthy volunteers were taken. In total, 254 staphylococcal strains were isolated and identified to the species level by MALDI-TOF mass spectrometry. A subset of ten strains belonging to different staphylococcal species were genome-sequenced. Two mock communities with nine and eighteen strains, respectively, as well as eight randomly selected skin samples were analysed with the three amplicon NGS methods. Our results imply that all three methods are suitable for species-level determination of staphylococcal populations. However, the novel tuf2-NGS scheme was superior in resolution power. It unambiguously allowed identification of Staphylococcus saccharolyticus and distinguish phylogenetically distinct clusters of Staphylococcus epidermidis. Conclusions Powerful amplicon NGS approaches for the detection and relative quantification of staphylococci in human samples exist that can resolve populations to the species and, to some extent, to the subspecies level. Our study highlights strengths, weaknesses and pitfalls of three currently available amplicon NGS approaches to determine staphylococcal populations. Applied to the analysis of healthy and diseased skin, these approaches can be useful to attribute host-beneficial and -detrimental roles to skin-resident staphylococcal species and subspecies.

The Formability and Elongation of Aluminum Alloys AA5083 and AA3003 for Micro-Truss Sandwiches Manufacturing

2020 ◽  
Vol 38 (9A) ◽  
pp. 1396-1405
Author(s):  
Arwa F. Tawfeeq ◽  
Matthew R. Barnett
Keyword(s):  
Aluminum Alloy ◽  
Strain Rate ◽  
Cost Effective ◽  
Tensile Tests ◽  
Truss Structures ◽  
High Quality ◽  
Trade Off ◽  
Clad Layer ◽  
Higher Temperature ◽  
Different Shapes

The development in the manufacturing of micro-truss structures has demonstrated the effectiveness of brazing for assembling these sandwiches, which opens new opportunities for cost-effective and high-quality truss manufacturing. An evolving idea in micro-truss manufacturing is the possibility of forming these structures in different shapes with the aid of elevated temperature. This work investigates the formability and elongation of aluminum alloy sheets typically used for micro-truss manufacturing, namely AA5083 and AA3003. Tensile tests were performed at a temperature in the range of 25-500 ○C and strain rate in the range of 2x10-4 -10-2 s-1. The results showed that the clad layer in AA3003 exhibited an insignificant effect on the formability and elongation of AA3003. The formability of the two alloys was improved significantly with values of m as high as 0.4 and 0.13 for AA5083 and AA3003 at 500 °C. While the elongation of both AA5083 and AA3003 was improved at a higher temperature, the elongation of AA5083 was inversely related to strain rate. It was concluded that the higher the temperature is the better the formability and elongation of the two alloys but at the expense of work hardening. This suggests a trade-off situation between formability and strength. 

Quality Improvement of the DNA extracted by boiling method in Gram negative bacteria

2017 ◽  
Vol 6 (04) ◽  
pp. 5347 ◽  
Author(s):  
Omar B. Ahmed* ◽  
Anas S. Dablool
Keyword(s):  
Dna Extraction ◽  
Control Method ◽  
Extraction Procedure ◽  
Cost Effective ◽  
High Yield ◽  
Gram Negative Bacteria ◽  
Bacterial Dna ◽  
Gram Negative ◽  
E Coli

Several methods of Deoxyribonucleic acid (DNA) extraction have been applied to extract bacterial DNA. The amount and the quality of the DNA obtained for each one of those methods are variable. The study aimed to evaluate bacterial DNA extraction using conventional boiling method followed by alcohol precipitation. DNA extraction from Gram negative bacilli was extracted and precipitated using boiling method with further precipitation by ethanol. The extraction procedure performed using the boiling method resulted in high DNA yields for both E. coli and K. pneumoniae bacteria in (199.7 and 285.7μg/ml, respectively) which was close to control method (229.3 and 440.3μg/ml). It was concluded that after alcohol precipitation boiling procedure was easy, cost-effective, and applicable for high-yield quality of DNA in Gram-negative bacteria.

scholarly journals Microbiota identified from preserved Anopheles

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Bianca E Silva ◽  
Zvifadzo Matsena Zingoni ◽  
Lizette L. Koekemoer ◽  
Yael L. Dahan-Moss
Keyword(s):  
Microbial Diversity ◽  
Vector Competence ◽  
Mosquito Species ◽  
Anopheles Funestus ◽  
Cost Effective ◽  
Diversity Indices ◽  
Malaria Vectors ◽  
Microbial Composition ◽  
Culture Independent ◽  
Culture Dependent

Abstract Background Mosquito species from the Anopheles gambiae complex and the Anopheles funestus group are dominant African malaria vectors. Mosquito microbiota play vital roles in physiology and vector competence. Recent research has focused on investigating the mosquito microbiota, especially in wild populations. Wild mosquitoes are preserved and transported to a laboratory for analyses. Thus far, microbial characterization post-preservation has been investigated in only Aedes vexans and Culex pipiens. Investigating the efficacy of cost-effective preservatives has also been limited to AllProtect reagent, ethanol and nucleic acid preservation buffer. This study characterized the microbiota of African Anopheles vectors: Anopheles arabiensis (member of the An. gambiae complex) and An. funestus (member of the An. funestus group), preserved on silica desiccant and RNAlater® solution. Methods Microbial composition and diversity were characterized using culture-dependent (midgut dissections, culturomics, MALDI-TOF MS) and culture-independent techniques (abdominal dissections, DNA extraction, next-generation sequencing) from laboratory (colonized) and field-collected mosquitoes. Colonized mosquitoes were either fresh (non-preserved) or preserved for 4 and 12 weeks on silica or in RNAlater®. Microbiota were also characterized from field-collected An. arabiensis preserved on silica for 8, 12 and 16 weeks. Results Elizabethkingia anophelis and Serratia oryzae were common between both vector species, while Enterobacter cloacae and Staphylococcus epidermidis were specific to females and males, respectively. Microbial diversity was not influenced by sex, condition (fresh or preserved), preservative, or preservation time-period; however, the type of bacterial identification technique affected all microbial diversity indices. Conclusions This study broadly characterized the microbiota of An. arabiensis and An. funestus. Silica- and RNAlater®-preservation were appropriate when paired with culture-dependent and culture-independent techniques, respectively. These results broaden the selection of cost-effective methods available for handling vector samples for downstream microbial analyses.

scholarly journals 2D-3D integration of hexagonal boron nitride and a high-κ dielectric for ultrafast graphene-based electro-absorption modulators

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Hitesh Agarwal ◽  
Bernat Terrés ◽  
Lorenzo Orsini ◽  
Alberto Montanaro ◽  
Vito Sorianello ◽  
...  
Keyword(s):  
Boron Nitride ◽  
Hafnium Oxide ◽  
High Speed ◽  
Hexagonal Boron Nitride ◽  
Temperature Stability ◽  
Cost Effective ◽  
Fold Increase ◽  
Building Blocks ◽  
High Quality ◽  
New Device

AbstractElectro-absorption (EA) waveguide-coupled modulators are essential building blocks for on-chip optical communications. Compared to state-of-the-art silicon (Si) devices, graphene-based EA modulators promise smaller footprints, larger temperature stability, cost-effective integration and high speeds. However, combining high speed and large modulation efficiencies in a single graphene-based device has remained elusive so far. In this work, we overcome this fundamental trade-off by demonstrating the 2D-3D dielectric integration in a high-quality encapsulated graphene device. We integrated hafnium oxide (HfO2) and two-dimensional hexagonal boron nitride (hBN) within the insulating section of a double-layer (DL) graphene EA modulator. This combination of materials allows for a high-quality modulator device with high performances: a ~39 GHz bandwidth (BW) with a three-fold increase in modulation efficiency compared to previously reported high-speed modulators. This 2D-3D dielectric integration paves the way to a plethora of electronic and opto-electronic devices with enhanced performance and stability, while expanding the freedom for new device designs.

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