scholarly journals Amplicon-sequencing of raw milk microbiota: impact of DNA extraction and library-PCR

2021 ◽  
Author(s):  
Annemarie Siebert ◽  
Katharina Hofmann ◽  
Lena Staib ◽  
Etienne V. Doll ◽  
Siegfried Scherer ◽  
...  
Keyword(s):  
Sample Preparation ◽  
Dna Extraction ◽  
Raw Milk ◽  
Amplicon Sequencing ◽  
Rrna Gene ◽  
Bacterial Dna ◽  
Microbiome Analysis ◽  
Eukaryotic Dna ◽  
Selective Lysis ◽  
The Impact

Abstract The highly complex raw milk matrix challenges the sample preparation for amplicon-sequencing due to low bacterial counts and high amounts of eukaryotic DNA originating from the cow. In this study, we optimized the extraction of bacterial DNA from raw milk for microbiome analysis and evaluated the impact of cycle numbers in the library-PCR. The selective lysis of eukaryotic cells by proteinase K and digestion of released DNA before bacterial lysis resulted in a high reduction of mostly eukaryotic DNA and increased the proportion of bacterial DNA. Comparative microbiome analysis showed that a combined enzymatic and mechanical lysis procedure using the DNeasy® PowerFood® Microbial Kit with a modified protocol was best suitable to achieve high DNA quantities after library-PCR and broad coverage of detected bacterial biodiversity. Increasing cycle numbers during library-PCR systematically altered results for species and beta-diversity with a tendency to overrepresentation or underrepresentation of particular taxa. To limit PCR bias, high cycle numbers should thus be avoided. An optimized DNA extraction yielding sufficient bacterial DNA and enabling higher PCR efficiency is fundamental for successful library preparation. We suggest that a protocol using ethylenediaminetetraacetic acid (EDTA) to resolve casein micelles, selective lysis of somatic cells, extraction of bacterial DNA with a combination of mechanical and enzymatic lysis, and restriction of PCR cycles for analysis of raw milk microbiomes is optimal even for samples with low bacterial numbers. Key points • Sample preparation for high-throughput 16S rRNA gene sequencing of raw milk microbiota. • Reduction of eukaryotic DNA by enzymatic digestion. • Shift of detected microbiome caused by high cycle numbers in library-PCR.

scholarly journals Development of a robust protocol for the characterization of the pulmonary microbiota

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Nathan Dumont-Leblond ◽  
Marc Veillette ◽  
Christine Racine ◽  
Philippe Joubert ◽  
Caroline Duchaine
Keyword(s):  
Amplicon Sequencing ◽  
Rrna Gene ◽  
Validity Of Results ◽  
Healthy Human ◽  
Bacterial Dna ◽  
Starting Point ◽  
Sampling Procedures ◽  
Negative Controls ◽  
The Impact

AbstractThe lack of methodological standardization diminishes the validity of results obtained and the conclusions drawn when studying the lung microbiota. We report the validation of a complete 16S rRNA gene amplicon sequencing workflow, from patient recruitment to bioinformatics, tailored to the constrains of the pulmonary environment. We minimize the impact of contaminants and establish negative controls to track and account for them at every step. Enzymatic and mechanical homogenization combined to commercially available extraction kits allow for a fast and reliable extraction of bacterial DNA. The DNA extraction kits have a significant impact on the bacterial composition of the controls. The bacterial signatures of extracted cancerous and healthy human tissues from 5 patients are highly distinguishable from methodological controls. Our work expands our understanding of low microbial burdened environments analysis. This article is to be a starting point towards methodological standardization and the implementation of proper sampling procedures in the study of lung microbiota.

Microbial Associations of Four Species of Algal Symbiont-bearing Foraminifers from the Florida Reef Tract, Usa

2019 ◽  
Vol 49 (2) ◽  
pp. 178-190
Author(s):  
Makenna M. Martin ◽  
Christina A. Kellogg ◽  
Pamela Hallock
Keyword(s):  
Dna Extraction ◽  
Variable Region ◽  
Florida Keys ◽  
Amplicon Sequencing ◽  
Rrna Gene ◽  
Bacterial Dna ◽  
Microbiome Composition ◽  
Algal Symbiont ◽  
Reef Tract ◽  
Bacterial Dna Extraction

Abstract While microbiome research is a rapidly expanding field of study, relatively little is known of the microbiomes associated with Foraminifera. This preliminary study investigated microbes associated with four species of Foraminifera, representing two taxonomic orders, which host three kinds of algal endosymbionts. A major objective was to explore potential influences on the microbiome composition, including phylogenetic relatedness among the host species, similarities in algal symbionts hosted, and environmental conditions from which the specimens were collected. Samples examined from two locations along the middle Florida Keys reef tract included 45 foraminiferal specimens and four environmental samples. Bacterial DNA extraction from individual specimens was followed by amplification and amplicon sequencing of the V4 variable region of the 16S rRNA gene; results were obtained from 21 specimens. The Order Miliolida, Family Soritidae, was represented by 5–8 specimens of each of three species: Archaias angulatus and Cyclorbiculina compressa, which both host chlorophyte symbionts, and Sorites orbiculus, which hosts dinoflagellate symbionts. Three Ar. angulatus specimens from which the microbiome was successfully sequenced shared 177 OTUs. Six C. compressa specimens successfully sequenced shared 58 OTUs, of which 31 were also shared by the three specimens of Ar. angulatus. Four successfully sequenced S. orbiculus specimens shared 717 unique OTUs. The 13 soritid specimens shared 26 OTUs, 23 of which represented Proteobacteria, predominantly of the bacterial family Rhodobacteraceae. The fourth foraminiferal species, Amphistegina gibbosa (Order Rotaliida) hosts diatom endosymbionts. Bacterial DNA extraction was attempted on 16 Am. gibbosa, including both normal-appearing and partly-bleached specimens. Only six OTUs, four of which represented Proteobacteria, were found in all eight specimens successfully sequenced. The partly bleached specimens shared nearly twice as many unique microbial OTUs (32) as the normal-appearing specimens (19). All Am. gibbosa specimens shared only four microbial OTUs with the soritid species, three of which may have been contaminants, indicating minimal commonality between the microbiomes of Am. gibbosa and the soritid taxa.

scholarly journals An inter-laboratory study to investigate the impact of the bioinformatics component on microbiome analysis using mock communities

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Denise M. O’Sullivan ◽  
Ronan M. Doyle ◽  
Sasithon Temisak ◽  
Nicholas Redshaw ◽  
Alexandra S. Whale ◽  
...  
Keyword(s):  
Microbial Community ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Relative Abundance ◽  
Amplicon Sequencing ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Microbiome Analysis ◽  
The Impact

AbstractDespite the advent of whole genome metagenomics, targeted approaches (such as 16S rRNA gene amplicon sequencing) continue to be valuable for determining the microbial composition of samples. Amplicon microbiome sequencing can be performed on clinical samples from a normally sterile site to determine the aetiology of an infection (usually single pathogen identification) or samples from more complex niches such as human mucosa or environmental samples where multiple microorganisms need to be identified. The methodologies are frequently applied to determine both presence of micro-organisms and their quantity or relative abundance. There are a number of technical steps required to perform microbial community profiling, many of which may have appreciable precision and bias that impacts final results. In order for these methods to be applied with the greatest accuracy, comparative studies across different laboratories are warranted. In this study we explored the impact of the bioinformatic approaches taken in different laboratories on microbiome assessment using 16S rRNA gene amplicon sequencing results. Data were generated from two mock microbial community samples which were amplified using primer sets spanning five different variable regions of 16S rRNA genes. The PCR-sequencing analysis included three technical repeats of the process to determine the repeatability of their methods. Thirteen laboratories participated in the study, and each analysed the same FASTQ files using their choice of pipeline. This study captured the methods used and the resulting sequence annotation and relative abundance output from bioinformatic analyses. Results were compared to digital PCR assessment of the absolute abundance of each target representing each organism in the mock microbial community samples and also to analyses of shotgun metagenome sequence data. This ring trial demonstrates that the choice of bioinformatic analysis pipeline alone can result in different estimations of the composition of the microbiome when using 16S rRNA gene amplicon sequencing data. The study observed differences in terms of both presence and abundance of organisms and provides a resource for ensuring reproducible pipeline development and application. The observed differences were especially prevalent when using custom databases and applying high stringency operational taxonomic unit (OTU) cut-off limits. In order to apply sequencing approaches with greater accuracy, the impact of different analytical steps needs to be clearly delineated and solutions devised to harmonise microbiome analysis results.

scholarly journals The microbial community of the gut differs between piglets fed sow milk, milk replacer or bovine colostrum

2017 ◽  
Vol 117 (7) ◽  
pp. 964-978 ◽  
Author(s):  
Ann-Sofie R. Poulsen ◽  
Nadieh de Jonge ◽  
Sugiharto Sugiharto ◽  
Jeppe L. Nielsen ◽  
Charlotte Lauridsen ◽  
...  
Keyword(s):  
Gut Microbiota ◽  
Amplicon Sequencing ◽  
Bovine Colostrum ◽  
Milk Replacer ◽  
Rrna Gene ◽  
Bacterial Dna ◽  
Faecal Samples ◽  
Milk Replacers ◽  
The 16S Rrna Gene ◽  
Gut Microbiota Composition

AbstractThe aim of this study was to characterise the gut microbiota composition of piglets fed bovine colostrum (BC), milk replacer (MR) or sow milk (SM) in the post-weaning period. Piglets (n36), 23-d old, were randomly allocated to the three diets. Faecal samples were collected at 23, 25, 27 and 30 d of age. Digesta from the stomach, ileum, caecum and mid-colon was collected at 30 d of age. Bacterial DNA from all samples was subjected to amplicon sequencing of the 16S rRNA gene. Bacterial enumerations by culture and SCFA analysis were conducted as well. BC-piglets had the highest abundance ofLactococcusin the stomach (P<0·0001) and ileal (P<0·0001) digesta, whereas SM-piglets had the highest abundance ofLactobacillusin the stomach digesta (P<0·0001). MR-piglets had a high abundance of Enterobacteriaceae in the ileal digesta (P<0·0001) and a higher number of haemolytic bacteria in ileal (P=0·0002) and mid-colon (P=0·001) digesta than SM-piglets. BC-piglets showed the highest colonic concentration of iso-butyric and iso-valeric acid (P=0·02). Sequencing and culture showed that MR-piglets were colonised by a higher number of Enterobacteriaceae, whereas the gut microbiota of BC-piglets was characterised by a change in lactic acid bacteria genera when compared with SM-piglets. We conclude that especially the ileal microbiota of BC-piglets had a closer resemblance to that of SM-piglets in regard to the abundance of potential enteric pathogens than did MR-piglets. The results indicate that BC may be a useful substitute for regular milk replacers, and as a feeding supplement in the immediate post-weaning period.

scholarly journals Shaping of the Present-Day Deep Biosphere at Chicxulub by the Impact Catastrophe That Ended the Cretaceous

2021 ◽  
Vol 12 ◽  
Author(s):  
Charles S. Cockell ◽  
Bettina Schaefer ◽  
Cornelia Wuchter ◽  
Marco J. L. Coolen ◽  
Kliti Grice ◽  
...  
Keyword(s):  
Microbial Communities ◽  
Amplicon Sequencing ◽  
Granitic Rocks ◽  
Rrna Gene ◽  
Deep Biosphere ◽  
Deep Subsurface ◽  
Cell Biomass ◽  
Colonization Potential ◽  
The Impact

We report on the effect of the end-Cretaceous impact event on the present-day deep microbial biosphere at the impact site. IODP-ICDP Expedition 364 drilled into the peak ring of the Chicxulub crater, México, allowing us to investigate the microbial communities within this structure. Increased cell biomass was found in the impact suevite, which was deposited within the first few hours of the Cenozoic, demonstrating that the impact produced a new lithological horizon that caused a long-term improvement in deep subsurface colonization potential. In the biologically impoverished granitic rocks, we observed increased cell abundances at impact-induced geological interfaces, that can be attributed to the nutritionally diverse substrates and/or elevated fluid flow. 16S rRNA gene amplicon sequencing revealed taxonomically distinct microbial communities in each crater lithology. These observations show that the impact caused geological deformation that continues to shape the deep subsurface biosphere at Chicxulub in the present day.

scholarly journals The Influence of DNA Extraction and Lipid Removal on Human Milk Bacterial Profiles

2020 ◽  
Vol 3 (2) ◽  
pp. 39 ◽  
Author(s):  
Anna Ojo-Okunola ◽  
Shantelle Claassen-Weitz ◽  
Kilaza S. Mwaikono ◽  
Sugnet Gardner-Lubbe ◽  
Heather J. Zar ◽  
...  
Keyword(s):  
Human Milk ◽  
Dna Extraction ◽  
Bacterial Population ◽  
Illumina Miseq ◽  
Molecular Techniques ◽  
Rrna Gene ◽  
Bacterial Dna ◽  
Dna Recovery ◽  
Culture Independent ◽  
Zymo Research

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.

scholarly journals Gut Microbiota Analysis Results Are Highly Dependent on the 16S rRNA Gene Target Region, Whereas the Impact of DNA Extraction Is Minor

2017 ◽  
Vol 28 (1) ◽  
pp. 19-30 ◽  
Author(s):  
Anniina Rintala ◽  
Sami Pietilä ◽  
Eveliina Munukka ◽  
Erkki Eerola ◽  
Juha-Pekka Pursiheimo ◽  
...  
Keyword(s):  
16S Rrna ◽  
Gut Microbiota ◽  
16S Rrna Gene ◽  
Dna Extraction ◽  
Rrna Gene ◽  
Gene Target ◽  
Target Region ◽  
Microbiota Analysis ◽  
The Impact ◽  
The 16S Rrna Gene

scholarly journals Optimisation of sample storage and DNA extraction for human gut microbiota studies​

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Jekaterina Kazantseva ◽  
Esther Malv ◽  
Aleksei Kaleda ◽  
Aili Kallastu ◽  
Anne Meikas
Keyword(s):  
Gut Microbiota ◽  
Dna Extraction ◽  
Personalised Medicine ◽  
Amplicon Sequencing ◽  
Rrna Gene ◽  
Human Gut ◽  
Human Gut Microbiota ◽  
Sequencing Technologies ◽  
Different Temperatures ◽  
Reliable Solution

Abstract Background New developments in next-generation sequencing technologies and massive data received from this approach open wide prospects for personalised medicine and nutrition studies. Metagenomic analysis of the gut microbiota is paramount for the characterization of human health and wellbeing. Despite the intensive research, there is a huge gap and inconsistency between different studies due to the non-standardised and biased pipeline. Methodical and systemic understanding of every stage in the process is necessary to overcome all bottlenecks and grey zones of gut microbiota studies, where all details and interactions between processes are important. Results Here we show that an inexpensive, but reliable iSeq 100 platform is an excellent tool to perform the analysis of the human gut microbiota by amplicon sequencing of the 16 S rRNA gene. Two commercial DNA extraction kits and different starting materials performed similarly regarding the taxonomic distribution of identified bacteria. DNA/RNA Shield reagent proved to be a reliable solution for stool samples collection, preservation, and storage, as the storage of faecal material in DNA/RNA Shield for three weeks at different temperatures and thawing cycles had a low impact on the bacterial distribution. Conclusions Altogether, a thoroughly elaborated pipeline with close attention to details ensures high reproducibility with significant biological but not technical variations.

Comparison of the utility of five commercial kits for extraction of DNA from Aspergillus fumigatus spores.

2010 ◽  
Vol 57 (4) ◽  
Author(s):  
Urszula Nawrot ◽  
Katarzyna Wlodarczyk ◽  
Magdalena Wrobel ◽  
Anita Wasik ◽  
Tadeusz Dobosz
Keyword(s):  
Dna Extraction ◽  
Enzymatic Digestion ◽  
Cell Disruption ◽  
Clinical Samples ◽  
Rrna Gene ◽  
28S Rrna ◽  
Bacterial Dna ◽  
Blood Samples ◽  
Dna Yield ◽  
Commercial Kits

The aim of this study was to compare the efficiency of DNA extraction from water as well as from blood samples spiked with A. fumigatus spores, using selected commercial kits. Extraction of DNA according to manufacturer's protocols was preceded by blood cells lysis and disruption of fungal cells by enzymatic digestion or bead beating. The efficiency of DNA extraction was measured by PCR using Aspergillus-specific primers and SYBR Green I dye or TaqMan probes targeting 28S rRNA gene. All methods allowed the detection of Aspergillus at the lowest tested density of water suspensions of spores (10¹ cells/ml). The highest DNA yield was obtained using the ZR Fungal/Bacterial DNA kit, YeastStar Genomic DNA kit, and QIAamp DNA Mini kit with mechanical cell disruption. The ZR Fungal/Bacterial DNA and YeastStar kits showed the highest sensitivity in examination of blood samples spiked with Aspergillus (100 % for the detection of 10² spores and 75 % for 10¹ spores). Recently, the enzymatic method ceased to be recommended for examination of blood samples for Aspergillus, thus ZR Fungal/Bacterial DNA kit and QIAamp DNA Mini kit with mechanical cell disruption could be used for extraction of Aspergillus DNA from clinical samples.

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