Effects of Chemical and Mechanical Lysis on Microbial DNA Yield, Integrity, and Downstream Amplicon Sequencing of Rumen Bacteria and Protozoa

Abstract Understanding the development of the calf rumen microbiome is important in developing manipulation strategies to improve efficiency as the animal ages. We hypothesized that the cow maternal microbiome would influence the colonization of the calf rumen microbiome. Our objective was to relate the microbiomes of the cow rumen fluid (RFC) to the calf meconium (M) and calf rumen fluid (RFN) at twenty-eight days of age. Mature, multiparous Angus crossbred cows (n = 10) from the University of Wyoming beef herd were used in this study. Rumen fluid was collected from the cows prior to parturition. Immediately following parturition, meconium was collected from the calf and at 28 days post-parturition, rumen fluid was collected from the calves. Microbial DNA was isolated using a lysis buffer and mechanical bead-beating procedure and purified using the QIAamp DNA Stool Mini Kit (Qiagen). Amplicon sequencing of the 16S rRNA V4 region was completed on the MiSeq and analyzed with QIIME2. Both alpha and beta diversity were evaluated by sample type and day. Richness and evenness differed by sample type. The greatest richness and evenness was in RFC (q < 0.01) followed by RFN and M, which did not differ from each other (q ³ 0.5). Bray-Curtis and Jaccard beta diversity differed by each sample type (q < 0.01). These data indicate that the M and RFN do not differ in number and distribution of features, but the samples are compositionally different. Additionally, the RFC differed in both alpha and beta diversity from both calf samples. These profiles can be used to develop hypotheses for the pathway of colonization in the early gut yet still reflect the vast differences in the developmental stage between the cow rumen microbiome and the early calf gastrointestinal microbiome.

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DNA extraction approaches substantially influence the assessment of the human breast milk microbiome

Scientific Reports ◽

10.1038/s41598-019-55568-y ◽

2020 ◽

Vol 10 (1) ◽

Cited By ~ 10

Author(s):

Chloe A. Douglas ◽

Kerry L. Ivey ◽

Lito E. Papanicolas ◽

Karen P. Best ◽

Beverly S. Muhlhausler ◽

...

Keyword(s):

Breast Milk ◽

Dna Extraction ◽

Infant Development ◽

Amplicon Sequencing ◽

Rrna Gene ◽

Human Breast Milk ◽

Human Breast ◽

Dna Yield ◽

Commensal Microbiota ◽

Milk Samples

AbstractIn addition to providing nutritional and bioactive factors necessary for infant development, human breast milk contains bacteria that contribute to the establishment of commensal microbiota in the infant. However, the composition of this bacterial community differs considerably between studies. We hypothesised that bacterial DNA extraction methodology from breast milk samples are a substantial contributor to these inter-study differences. We tested this hypothesis by applying five widely employed methodologies to a mock breast milk sample and four individual human breast milk samples. Significant differences in DNA yield and purity were observed between methods (P < 0.05). Microbiota composition, assessed by 16S rRNA gene amplicon sequencing, also differed significantly with extraction methodology (P < 0.05), including in the contribution of contaminant signal. Concerningly, many of the bacterial taxa identified here as contaminants have been reported as components of the breast milk microbiome in other studies. These findings highlight the importance of using stringent, well-validated, DNA extraction methodologies for analysis of the breast milk microbiome, and exercising caution interpreting microbiota data from low-biomass contexts.

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Performance comparison of different microbial DNA extraction methods on bird feces

Avian Research ◽

10.1186/s40657-021-00254-9 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Xian Hou ◽

Shengkai Pan ◽

Zhenzhen Lin ◽

Jiliang Xu ◽

Xiangjiang Zhan

Keyword(s):

Dna Extraction ◽

Relative Abundance ◽

Alpha Diversity ◽

Cell Lysis ◽

Extraction Methods ◽

Dna Integrity ◽

Microbial Composition ◽

Dna Yield ◽

Dna Extraction Methods ◽

Microbial Dna

Abstract Background As an important player during food digestion, gut microbiota has attracted much attention in diet adaptation studies in birds. Microbiota extracted from feces has been widely used as a proxy for gut microbiota. Although several methods have been developed for microbial DNA extraction, their performances in the bird feces have not been systematacially evaluated yet. Methods In this study, we applied three DNA extraction methods (Qiagen, MoBio and Bead) to extract DNA from feces of three avian dietary guilds (granivore, omnivore and carnivore), sequenced V4 region of 16S rRNA gene for each extract and evaluated the performances of DNA yield, DNA integrity, microbial composition, cell lysis capacity and alpha diversity for the three methods on each dietary guild. Results Bead method was the best on the performance of both DNA yield and DNA integrity regardless of dietary guild. In granivore, microbial relative abundance at both species and phylum levels, alpha diversity and cell lysis capacity were comparable among all methods. In omnivore, Qiagen had the best performance on alpha diversity, followed by Bead and MoBio. There were small variations on microbial relative abundance at both species and phylum levels among different extraction methods. MoBio exhibited the best performance on cell lysis capacity. In carnivore, considerable variations were found on microbial relative abundance at both species and phylum levels. Qiagen had the best performance on alpha diversity, followed by MoBio and Bead. MoBio had the highest cell lysis capacity. Conclusions DNA yield and integrity have no obvious impact on microbial composition, alpha diversity or cell lysis capacity. The microbiota results (e.g., microbial composition, cell lysis capacity, alpha diversity) obtained from different methods are comparable in granivorous avian species but not in omnivorous or carnivorous birds. Either method could be used in granivore microbiota studies. For omnivores and carnivores, we recommend Qiagen method when the research purpose is microbial diversity and MoBio when gram-positive bacteria is the research target.

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Ultra-deep amplicon sequencing to identify actionable mutations in matched plasma/tumor specimens from 44 patients with colorectal cancer of UICC stage III and IV.

Journal of Clinical Oncology ◽

10.1200/jco.2013.31.15_suppl.3634 ◽

2013 ◽

Vol 31 (15_suppl) ◽

pp. 3634-3634

Author(s):

Paulina Pechanska ◽

Matthias Pross ◽

Cornelia Radke ◽

Frank Marusch ◽

Hartmut Lobeck ◽

...

Keyword(s):

Colorectal Cancer ◽

Primary Tumor ◽

Stage Iv ◽

Amplicon Sequencing ◽

Stage Iii ◽

Plasma Samples ◽

High Quality ◽

Primary Tumors ◽

Dna Yield ◽

Matched Primary Tumor

3634 Background: Circulating cell-free DNA (cf-DNA) isolated from plasma samples of cancer patients (pts) is a promising source for noninvasive examination of tumor-specific mutation patterns. We examined the efficiency of ultra-deep amplicon sequencing (UDAS) of cf-DNA isolated from plasma and tumor DNA isolated from matched primary tumor tissue of pts with colorectal cancer (CRC). Methods: Blood was drawn prior to surgery from 44 pts: 20 male, 24 female; 44-79 years of age (median: 67.5); 11 stage III, 33 stage IV; 36 colon, 8 rectum tumors; no neo-adjuvant therapy. Cf-DNA was isolated from 2 ml of EDTA plasma. UDAS was applied to 72 DNA samples (44 plasma, 28 matched snap-frozen primary tumors) using the MiSeq platform (Illumina). A panel of 49 highly multiplexed amplicons was designed (Life Technologies) representing 9 cancer genes frequently mutated in CRC. For 16 primary tumors the mutation profile was determined using the TruSeq Amplicon Cancer Panel (Illumina). Results: Median cf-DNA yield was 310 ng / 2 ml plasma (1ng – 6.600 ng). There was no significant relation between cf-DNA yield and any clinical characteristic. Median amplicon coverage was 20.162 reads per bp (2.913 - 115.782). 33/49 amplicons (67%) had a coverage of > 10.000 reads. Altogether 61 high quality COSMIC-cited mutations were confirmed in plasma of 29/44 (66%) pts: 24/33 (73%) pts in stage IV, 5/11 (45%) pts in stage III. Confirmed mutations are: APC-17; BRAF-2, FBXW7-1, KRAS-15, NRAS-1, PIK3CA-4, SMAD4-2, and TP53-19 mutations. No high quality mutations were found in CTNNB1 and HRAS. Moreover two pts exhibited four high quality plasma mutations which were not detected in the matched primary tumor: APC (R216X), KRAS (Q61K), and SMAD4 (D355E); PIK3CA (E545K). Conclusions: Ultra deep amplicon sequencing is suitable to detect mutations in plasma samples of CRC pts with a high concordance to matched primary tumors. The concordance rate can be further increased by extending the spectrum of analyzed mutations or by the enrichment of cf-DNA tumor copies. This method could be applied to detect and monitor metastasis thus opening a new paradigm for the selection of pts for targeted therapies.

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Hidden components of microbiome diversity revealed by cell wall lysis with Adaptive Focused Acoustics

10.1101/2020.05.21.109736 ◽

2020 ◽

Author(s):

Giselle F. Wallace ◽

Sonia Ahluwalia ◽

Vishal Thovarai ◽

James Laugharn ◽

Hamid Khoja ◽

...

Keyword(s):

Cell Wall ◽

Wall Thickness ◽

Energy Levels ◽

Functional Characterization ◽

Cell Wall Thickness ◽

16S Sequencing ◽

Alpha And Beta Diversity ◽

Dna Yield ◽

Mechanical Lysis

AbstractWithin the rapidly evolving field of microbiome sequencing, a primary need exists for experimentally capturing microbiota in a manner as close as possible to their in vivo composition. During microbiome profiling, the first step necessarily involves lysis of the cell wall, releasing nucleic acids for next-generation sequencing. Microbial cell wall thicknesses can vary between 5nm to 80nm; while some species are quite easy to lyse, others are particularly resistant to lysis. Despite this, current chemical/mechanical lysis protocols ignore the possibility that species with different cell wall thicknesses are lysed at differential rates. This creates noise in species compositions and possibly skews current microbiome results in ways that are not currently understood. To develop a cell wall thickness-agnostic lysis protocol, we used Adaptive Focused Acoustics (AFA), a tunable acoustic methodology for processing of biological samples. Using identical aliquots of mouse stool homogenate as the lysis substrate, we compared AFA with chemical/mechanical lysis methodology routinely used in microbiome studies and found that AFA-mediated lysis substantially increases both microbial DNA yield as well as alpha and beta diversity. By starting with lower AFA energy levels, sequentially removing aliquots at each step, and subjecting the remainder to progressively stronger AFA treatment, we developed a sequential lysis method that accounts for differences in cell wall thickness. This method revealed even greater levels of diversity than single-timepoint AFA treatment. 16S sequencing results from the above experiments were verified by shotgun metagenome sequencing of a subset of the AFA samples. We found that lysis-induced noise affects not just species compositaions, but also functional characterization of shotgun metagenome data. AFA samples also showed a higher detection of eukaryotic and fungal DNA. We suggest that AFA-mediated lysis produces a truer representation of the native microbiota, and that this method deserves consideration as a potential addition to microbiome lysis protocols.

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Optimisation and Benchmarking of Targeted Amplicon Sequencing for Mycobiome Analysis of Respiratory Specimens

International Journal of Molecular Sciences ◽

10.3390/ijms20204991 ◽

2019 ◽

Vol 20 (20) ◽

pp. 4991 ◽

Cited By ~ 5

Author(s):

Ali ◽

Mac Aogáin ◽

Morales ◽

Tiew ◽

Chotirmall

Keyword(s):

Dna Extraction ◽

Its Region ◽

Amplicon Sequencing ◽

Extraction Methods ◽

Sample Type ◽

Enzymatic Lysis ◽

Dna Extraction Methods ◽

Mechanical Lysis ◽

Respiratory Specimens

(1) Background: Firm consensus has yet to be established in relation to taxonomic classification and primer choice in targeted amplicon sequencing of the mycobiome. While the nuclear ribosomal internal transcribed spacer (ITS) region are recognized as the formal fungal taxonomic barcode, appraisal of different ITS sub-regions and the influence of DNA extraction methods have not been comprehensively undertaken using human respiratory specimens. (2) Methods: We performed ITS analysis of respiratory (sputum) samples by assessing (a) the effect of alternate DNA extraction techniques and (b) an evaluation of four different ITS primer pairs (ITS1F and ITS2; ITS1-30F and ITS1-217R; gITS7ngs and ITS4ng; and Fseq and Rseq) on the mycobiome profiles generated for mock fungal communities and their respective clinical (airway) specimens. (3) Results: Primer pairs varied in their resulting ITS mycobiome profiles, suggesting that particular pairs may be more relevant for analysis of respiratory samples compared to others. Assessment of DNA extraction methods highlighted lower final DNA concentrations achieved by mechanical disruption compared to enzymatic lysis. However, despite lower yields, DNA liberated by mechanical lysis more readily yielded ITS bands with highest success in combination with the Fseq and Rseq primers. (4) Conclusion: Choice of extraction method, primers used, and sequencing approach are all important considerations in sequencing the mycobiome and should be tailored to sample type. A standardization of approach to mycobiome studies using respiratory specimens will permit more reliable comparisons between studies and improve our understanding of the role of fungi in the human airway.

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Hidden Components of Microbiome Diversity Revealed by Cell Wall Lysis with Adaptive Focused Acoustics

10.21203/rs.3.rs-73121/v1 ◽

2020 ◽

Author(s):

Shurjo Kumar Sen ◽

Giselle F. Wallace ◽

Sonia Ahluwalia ◽

Vishal Thovarai ◽

James Laugharn ◽

...

Keyword(s):

Cell Wall ◽

Wall Thickness ◽

Energy Levels ◽

Functional Characterization ◽

Cell Wall Thickness ◽

16S Sequencing ◽

Alpha And Beta Diversity ◽

Dna Yield ◽

Mechanical Lysis

Abstract Within the rapidly evolving field of microbiome sequencing, a primary need exists for experimentally capturing microbiota in a manner as close as possible to their in vivo composition. During microbiome profiling, the first step necessarily involves lysis of the cell wall, releasing nucleic acids for next-generation sequencing. Microbial cell wall thicknesses can vary between 5nm to 80nm; while some species are quite easy to lyse, others are particularly resistant to lysis. Despite this, current chemical/mechanical lysis protocols ignore the possibility that species with different cell wall thicknesses are lysed at differential rates. This creates noise in species compositions and possibly skews current microbiome results in ways that are not currently understood. To develop a cell wall thickness-agnostic lysis protocol, we used Adaptive Focused Acoustics (AFA), a tunable acoustic methodology for processing of biological samples. Using identical aliquots of mouse stool homogenate as the lysis substrate, we compared AFA with chemical/mechanical lysis methodology routinely used in microbiome studies and found that AFA-mediated lysis substantially increases both microbial DNA yield as well as alpha and beta diversity. By starting with lower AFA energy levels, sequentially removing aliquots at each step, and subjecting the remainder to progressively stronger AFA treatment, we developed a sequential lysis method that accounts for differences in cell wall thickness. This method revealed even greater levels of diversity than single-timepoint AFA treatment. 16S sequencing results from the above experiments were verified by shotgun metagenome sequencing of a subset of the AFA samples. We found that lysis-induced noise affects not just species compositions, but also functional characterization of shotgun metagenome data. AFA samples also showed a higher detection of eukaryotic and fungal DNA. We suggest that AFA-mediated lysis produces a truer representation of the native microbiota, and that this method deserves consideration as a potential addition to microbiome lysis protocols.

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