scholarly journals Quantifying and Understanding Well-to-Well Contamination in Microbiome Research

mSystems ◽  
2019 ◽  
Vol 4 (4) ◽  
Author(s):  
Jeremiah J. Minich ◽  
Jon G. Sanders ◽  
Amnon Amir ◽  
Greg Humphrey ◽  
Jack A. Gilbert ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Extraction Methods ◽  
Data Set ◽  
Single Tube ◽  
Alpha And Beta Diversity ◽  
Sample Contamination ◽  
Microbiome Research ◽  
Dna Extraction Methods ◽  
Hybrid Plate ◽  
Negative Controls

ABSTRACT Microbial sequences inferred as belonging to one sample may not have originated from that sample. Such contamination may arise from laboratory or reagent sources or from physical exchange between samples. This study seeks to rigorously assess the behavior of this often-neglected between-sample contamination. Using unique bacteria, each assigned a particular well in a plate, we assess the frequency at which sequences from each source appear in other wells. We evaluate the effects of different DNA extraction methods performed in two laboratories using a consistent plate layout, including blanks and low-biomass and high-biomass samples. Well-to-well contamination occurred primarily during DNA extraction and, to a lesser extent, in library preparation, while barcode leakage was negligible. Laboratories differed in the levels of contamination. Extraction methods differed in their occurrences and levels of well-to-well contamination, with plate methods having more well-to-well contamination and single-tube methods having higher levels of background contaminants. Well-to-well contamination occurred primarily in neighboring samples, with rare events up to 10 wells apart. This effect was greatest in samples with lower biomass and negatively impacted metrics of alpha and beta diversity. Our work emphasizes that sample contamination is a combination of cross talk from nearby wells and background contaminants. To reduce well-to-well effects, samples should be randomized across plates, samples of similar biomasses should be processed together, and manual single-tube extractions or hybrid plate-based cleanups should be employed. Researchers should avoid simplistic removals of taxa or operational taxonomic units (OTUs) appearing in negative controls, as many will be microbes from other samples rather than reagent contaminants. IMPORTANCE Microbiome research has uncovered magnificent biological and chemical stories across nearly all areas of life science, at times creating controversy when findings reveal fantastic descriptions of microbes living and even thriving in what were once thought to be sterile environments. Scientists have refuted many of these claims because of contamination, which has led to robust requirements, including the use of controls, for validating accurate portrayals of microbial communities. In this study, we describe a previously undocumented form of contamination, well-to-well contamination, and show that this sort of contamination primarily occurs during DNA extraction rather than PCR, is highest with plate-based methods compared to single-tube extraction, and occurs at a higher frequency in low-biomass samples. This finding has profound importance in the field, as many current techniques to “decontaminate” a data set simply rely on an assumption that microbial reads found in blanks are contaminants from “outside,” namely, the reagents or consumables.

scholarly journals Quantifying and understanding well-to-well contamination in microbiome research

2019 ◽  
Author(s):  
Jeremiah J Minich ◽  
Jon G Sanders ◽  
Amnon Amir ◽  
Greg Humphrey ◽  
Jack Gilbert ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Life Science ◽  
Extraction Methods ◽  
Alpha And Beta Diversity ◽  
Sample Contamination ◽  
Microbiome Research ◽  
Manual Methods ◽  
Dna Extraction Methods ◽  
Negative Controls ◽  
Lower Biomass

AbstractMicrobial sequences inferred as belonging to one sample may not have originated from that sample. Such contamination may arise from laboratory or reagent sources or from physical exchange between samples. This study seeks to rigorously assess the behavior of this often-neglected between-sample contamination. Using unique bacteria each assigned a particular well in a plate, we assess the frequency at which sequences from each source appears in other wells. We evaluate the effects of different DNA extraction methods performed in two labs using a consistent plate layout including blanks, low biomass, and high biomass samples. Well-to-well contamination occurred primarily during DNA extraction, and to a lesser extent in library preparation, while barcode leakage was negligible. Labs differed in the levels of contamination. DNA extraction methods differed in their occurrences and levels of well-to-well contamination, with robotic methods having more well-to-well contamination while manual methods having higher background contaminants. Well-to-well contamination was observed to occur primarily in neighboring samples, with rare events up to 10 wells apart. The effect of well-to-well was greatest in samples with lower biomass, and negatively impacted metrics of alpha and beta diversity. Our work emphasizes that sample contamination is a combination of crosstalk from nearby wells and background contaminants. To reduce well-to-well effects, samples should be randomized across plates, and samples of similar biomass processed together. Researchers should evaluate well-to-well contamination in study design and avoid removal of taxa or OTUs appearing in negative controls, as many will be microbes from other samples rather than reagent contaminants.ImportanceMicrobiome research has uncovered magnificent biological and chemical stories across nearly all areas of life science, at times creating controversy when findings reveal fantastic descriptions of microbes living and even thriving in once thought to be sterile environments. Scientists have refuted many of these claims because of contamination, which has led to robust requirements including use of controls for validating accurate portrayals of microbial communities. In this study, we describe a previously undocumented form of contamination, well-to-well contamination and show that contamination primarily occurs during DNA extraction rather than PCR, is highest in plate-based methods as compared to single tube extraction, and occurs in higher frequency in low biomass samples. This finding has profound importance on the field as many current techniques to ‘decontaminate’ a dataset simply relies on an assumption that microbial reads found in blanks are contaminants from ‘outside’ namely the reagents or consumables.

scholarly journals Toward standards in clinical microbiome studies: comparison of three DNA extraction methods and two bioinformatic pipelines

2019 ◽  
Author(s):  
Q.R. Ducarmon ◽  
B.V.H. Hornung ◽  
A.R. Geelen ◽  
E.J. Kuijper ◽  
R.D. Zwittink
Keyword(s):  
Dna Extraction ◽  
Extraction Method ◽  
Bacterial Biomass ◽  
Extraction Methods ◽  
Rrna Gene ◽  
Method Choice ◽  
Advantages And Disadvantages ◽  
Dna Extraction Method ◽  
Dna Extraction Methods ◽  
Negative Controls

ABSTRACTWhen studying the microbiome using next generation sequencing, DNA extraction method, sequencing procedures and bioinformatic processing are crucial to obtain reliable data. Method choice has been demonstrated to strongly affect the final biological interpretation. We assessed the performance of three DNA extraction methods and two bioinformatic pipelines for bacterial microbiota profiling through 16S rRNA gene amplicon sequencing, using positive and negative controls for DNA extraction and sequencing, and eight different types of high- or low-biomass samples. Performance was evaluated based on quality control passing, DNA yield, richness, diversity and compositional profiles. All DNA extraction methods retrieved the theoretical relative bacterial abundance with maximum three-fold change, although differences were seen between methods, and library preparation and sequencing induced little variation. Bioinformatic pipelines showed different results for estimating richness, but diversity and compositional profiles were comparable. DNA extraction methods were successful for feces and oral swabs and variation induced by DNA extraction methods was lower than inter-subject (biological) variation. For low-biomass samples, a mixture of genera present in negative controls and sample-specific genera, possibly representing biological signal, were observed. We conclude that the tested bioinformatic pipelines perform equally with pipeline-specific advantages and disadvantages. Two out of three extraction methods performed equally well, while one method was less accurate regarding retrieval of compositional profiles. Lastly, we demonstrate the importance of including negative controls when analyzing low bacterial biomass samples.IMPORTANCEMethod choice throughout the workflow of a microbiome study, from sample collection to DNA extraction and sequencing procedures, can greatly affect results. This study evaluated three different DNA extraction methods and two bioinformatic pipelines by including positive and negative controls, and various biological specimens. By identifying an optimal combination of DNA extraction method and bioinformatic pipeline use, we hope to contribute to increased methodological consistency in microbiome studies. Our methods were not only applied to commonly studied samples for microbiota analysis, e.g. feces, but also for more rarely studied, low-biomass samples. Microbiota composition profiles of low-biomass samples (e.g. urine and tumor biopsies) were not always distinguishable from negative controls, or showed partial overlap, confirming the importance of including negative controls in microbiome studies, especially when low bacterial biomass is expected.

scholarly journals Choice of Commercial DNA Extraction Method Does Not Affect 16S Sequencing Outcomes in Cloacal Swabs

Animals ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 1372
Author(s):  
Emily Van Syoc ◽  
Natália Carrillo Gaeta ◽  
Erika Ganda
Keyword(s):  
16S Rrna ◽  
Dna Extraction ◽  
Amplicon Sequencing ◽  
Extraction Methods ◽  
Negative Control ◽  
Careful Consideration ◽  
16S Sequencing ◽  
16S Rrna Amplicon Sequencing ◽  
Microbiome Research ◽  
Negative Controls

As the applications of microbiome science in agriculture expand, laboratory methods should be constantly evaluated to ensure optimization and reliability of downstream results. Most animal microbiome research uses fecal samples or rectal swabs for profiling the gut bacterial community; however, in birds, this is difficult given the unique anatomy of the cloaca where the fecal, urinary, and reproductive tracts converge into one orifice. Therefore, avian gut microbiomes are usually sampled from cloacal swabs, creating a need to evaluate sample preparation methods to optimize 16S sequencing. We compared four different DNA extraction methods from two commercially available kits on cloacal swabs from 10 adult commercial laying hens and included mock communities and negative controls, which were then subjected to 16S rRNA amplicon sequencing. Extracted DNA yield and quality, diversity analyses, and contaminants were assessed. Differences in DNA quality and quantity were observed, and all methods needed further purification for optimal sequencing, suggesting contaminants due to cloacal contents, method reagents, and/or environmental factors. However, no differences were observed in alpha or beta diversity between methods. Importantly, multiple bacterial contaminants were detected in each mock community and negative control, indicating the prevalence of laboratory and handling contamination as well as method-specific reagent contamination. We found that although the extraction methods resulted in different extraction quality and yield, overall sequencing results were not affected, and we did not identify any method that would be an inappropriate choice in extracting DNA from cloacal swabs for 16S rRNA sequencing. Overall, our results highlight the need for careful consideration of positive and negative controls in addition to DNA isolation method and lend guidance to future microbiome research in poultry.

scholarly journals Toward Standards in Clinical Microbiota Studies: Comparison of Three DNA Extraction Methods and Two Bioinformatic Pipelines

mSystems ◽  
2020 ◽  
Vol 5 (1) ◽  
Author(s):  
Q. R. Ducarmon ◽  
B. V. H. Hornung ◽  
A. R. Geelen ◽  
E. J. Kuijper ◽  
R. D. Zwittink
Keyword(s):  
Dna Extraction ◽  
Extraction Method ◽  
Bacterial Biomass ◽  
Extraction Methods ◽  
Rrna Gene ◽  
Method Choice ◽  
Advantages And Disadvantages ◽  
Dna Extraction Method ◽  
Dna Extraction Methods ◽  
Negative Controls

ABSTRACT When studying the microbiome using next-generation sequencing, the DNA extraction method, sequencing procedures, and bioinformatic processing are crucial to obtain reliable data. Method choice has been demonstrated to strongly affect the final biological interpretation. We assessed the performance of three DNA extraction methods and two bioinformatic pipelines for bacterial microbiota profiling through 16S rRNA gene amplicon sequencing, using positive and negative controls for DNA extraction and sequencing and eight different types of high- or low-biomass samples. Performance was evaluated based on quality control passing, DNA yield, richness, diversity, and compositional profiles. All DNA extraction methods retrieved the theoretical relative bacterial abundance with a maximum 3-fold change, although differences were seen between methods, and library preparation and sequencing induced little variation. Bioinformatic pipelines showed different results for observed richness, but diversity and compositional profiles were comparable. DNA extraction methods were successful for feces and oral swabs, and variation induced by DNA extraction methods was lower than intersubject (biological) variation. For low-biomass samples, a mixture of genera present in negative controls and sample-specific genera, possibly representing biological signal, were observed. We conclude that the tested bioinformatic pipelines perform equally, with pipeline-specific advantages and disadvantages. Two out of three extraction methods performed equally well, while one method was less accurate regarding retrieval of compositional profiles. Lastly, we again demonstrate the importance of including negative controls when analyzing low-bacterial-biomass samples. IMPORTANCE Method choice throughout the workflow of a microbiome study, from sample collection to DNA extraction and sequencing procedures, can greatly affect results. This study evaluated three different DNA extraction methods and two bioinformatic pipelines by including positive and negative controls and various biological specimens. By identifying an optimal combination of DNA extraction method and bioinformatic pipeline use, we hope to contribute to increased methodological consistency in microbiota studies. Our methods were applied not only to commonly studied samples for microbiota analysis, e.g., feces, but also to more rarely studied, low-biomass samples. Microbiota composition profiles of low-biomass samples (e.g., urine and tumor biopsy specimens) were not always distinguishable from negative controls, or showed partial overlap, confirming the importance of including negative controls in microbiota studies, especially when low bacterial biomass is expected.

OPTIMIZATION OF DNA EXTRACTION METHODS FROM BIOLOGICAL MATERIALS OF HONEY BEES IN A DIFFERENT METAMOTPHOSIS PHASE

2016 ◽  
Vol 52 ◽  
pp. 171-176
Author(s):  
M. Palkina ◽  
O. Metlitska
Keyword(s):  
Ion Exchange ◽  
Dna Extraction ◽  
Honey Bees ◽  
Biological Material ◽  
Exchange Resin ◽  
Ion Exchange Resin ◽  
Extraction Methods ◽  
Chelex 100 ◽  
Drone Brood ◽  
Dna Extraction Methods

The aim of the research – adaptation, optimization and using of existing DNA extraction methods from bees’ biological material with the reagent «Chelex-100" under complex economic conditions of native laboratories, which will optimize labour costs and improve the economic performance of DNA extraction protocol. Materials and methods. In order to conduct the research the samples of honey bees’ biological material: queen pupae exuviae, larvae of drone brood, some adult bees’ bodies (head and thorax) were selected. Bowl and drone brood were obtained from the experimental bee hives of Institute of Apiculture nd. a. P. I. Prokopovich of NAAS. DNA extraction from biosamples of Apis mellifera ssp. was carried out using «Chelex-100®» ion exchange resin in different concentrations and combinations. Before setting tests for determination of quantitative and quality indexes, dilution of DNA samples of the probed object was conducted in ratio 1:40. The degree of contamination with protein and polysaccharide fractions (OD 260/230), quantitative content of DNA (OD 260/280) in the extracted tests were conducted using spectrophotometer of «Biospec – nano» at the terms of sample volume in 2 µl and length of optical way in 0,7 mm [7]. Verification of DNA samples from biological material of bees, isolated by «Chelex-100®», was conducted after cold keeping during 24 hours at 20°C using PСR with primaries to the fragment of gene of quantitative trait locus (QTL) Sting-2 of next structure [8]:  3' – CTC GAC GAG ACG ACC AAC TTG – 5’; 3' – AAC CAG AGT ATC GCG AGT GTT AC – 5’ Program of amplification: 94 °C – 5 minutes – 1 cycle; 94 °C – 1 minute, 57°C – 1 minute, 72 °C – 2 minutes – 30 cycles; elongation after 72°C during 2 minutes – 1 cycle. The division of obtained amplicons was conducted by gel electrophoresis at a low current – 7 µÀ, in 1,5 % agarose gel (Sigma ®) in TAE buffer [7]. The results. At the time of optimization of DNA isolation methods, according to existing methods of foreign experts, it was found optimal volume of ion exchange resin solution was in the proposed concentration: instead of 60 µl of solution used 120 µl of «Chelex-100®», time of incubation was also amended from 30 minutes to 180 minutes [9]. The use of the author's combination of method «Chelex-100®» with lysis enzymes, proteinase K and detergents (1M dithiothreitol), as time of incubation was also amended, which was reduced to 180 minutes instead of the proposed 12 hours [10]. Changes in quality characteristics of obtained DNA in samples after reduction in incubation time were not found. Conclusions. The most economical method of DNA isolation from bees’ biological material is 20% solution of «Chelex-100» ion exchange resin with the duration of the incubation period of 180 minutes. It should also be noted that the best results can be obtained from exuviae, selected immediately after the queen’s exit from bowl, that reduces the likelihood of DNA molecules destruction under the influence of nucleases activation, but not later than 12 hours from release using the technology of isolated obtain of queens.

scholarly journals Impact of Collection Volume and DNA Extraction Method on the Detection of Biomarkers and HPV DNA in First-Void Urine

Molecules ◽  
2021 ◽  
Vol 26 (7) ◽  
pp. 1989
Author(s):  
Laura Téblick ◽  
Severien Van Keer ◽  
Annemie De Smet ◽  
Pierre Van Damme ◽  
Michelle Laeremans ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Internal Control ◽  
Extraction Methods ◽  
Urine Collection ◽  
Hpv Dna ◽  
Non Invasive ◽  
Herpesvirus 1 ◽  
Dna Extraction Methods ◽  
Detection Of Biomarkers ◽  
High Risk Hpv

The potential of first-void (FV) urine as a non-invasive liquid biopsy for detection of human papillomavirus (HPV) DNA and other biomarkers has been increasingly recognized over the past decade. In this study, we investigated whether the volume of this initial urine stream has an impact on the analytical performance of biomarkers. In parallel, we evaluated different DNA extraction protocols and introduced an internal control in the urine preservative. Twenty-five women, diagnosed with high-risk HPV, provided three home-collected FV urine samples using three FV urine collection devices (Colli-Pee) with collector tubes that differ in volume (4, 10, 20 mL). Each collector tube was prefilled with Urine Conservation Medium spiked with phocine herpesvirus 1 (PhHV-1) DNA as internal control. Five different DNA extraction protocols were compared, followed by PCR for GAPDH and PhHV-1 (qPCR), HPV DNA, and HBB (HPV-Risk Assay), and ACTB (methylation-specific qPCR). Results showed limited effects of collection volume on human and HPV DNA endpoints. In contrast, significant variations in yield for human endpoints were observed for different DNA extraction methods (p < 0.05). Additionally, the potential of PhHV-1 as internal control to monitor FV urine collection, storage, and processing was demonstrated.

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