Project description:High-throughput amplicon sequencing technology has been widely used in soil microbiome studies. Here, we estimated the bias of amplicon sequencing data affected by DNA extraction methods in a saline soil, and a non-saline normal soil was used as a control. Compared with the normal soil, several unique points were observed in the saline soil. The soil washing pretreatment can improve not only DNA quantity and quality but also microbial diversities in the saline soil; therefore, we recommend the soil washing pretreatment for saline soils especially hypersaline soils that cannot be achieved with detectable DNA amounts without the pretreatment. Also, evenness indices were more easily affected by DNA extraction methods than richness indices in the saline soil. Moreover, proportions of Gram-positive bacteria had significant positive correlations with the achieved microbial diversities within replicates of the saline soil. Though DNA extraction methods can bias the microbial diversity or community and relative abundances of some phyla/classes can vary by a factor of more than five, soil types were still the most important factor of the whole community. We confirmed good comparability in the whole community, but more attention should be paid when concentrating on an exact diversity value or the exact relative abundance of a certain taxon. Our study can provide references for the DNA extraction from saline and non-saline soils and comparing sequencing data across studies who may employ different DNA extraction methods.
Project description:Bias is a pervasive problem when characterizing microbial communities. An important source is the difference in lysis efficiencies of different populations, which vary depending on the extraction protocol used. To avoid such biases impacting comparisons between gene and transcript abundances in the environment, the use of one protocol that simultaneously extracts both types of nucleic acids from microbial community samples has gained popularity. However, knowledge regarding tradeoffs to combined nucleic acid extraction protocols is limited, particularly regarding yield and biases in the observed community composition. Here, we evaluated a commercially available protocol for simultaneous extraction of DNA and RNA, which we adapted for freshwater microbial community samples that were collected on filters. DNA and RNA yields were comparable to other commonly used, but independent DNA and RNA extraction protocols. RNA protection agents benefited RNA quality, but decreased DNA yields significantly. Choice of extraction protocol influenced the perceived bacterial community composition, with strong method-dependent biases observed for specific phyla such as the Verrucomicrobia. The combined DNA/RNA extraction protocol detected significantly higher levels of Verrucomicrobia than the other protocols, and those higher numbers were confirmed by microscopic analysis. Use of RNA protection agents as well as independent sequencing runs caused a significant shift in community composition as well, albeit smaller than the shift caused by using different extraction protocols. Despite methodological biases, sample origin was the strongest determinant of community composition. However, when the abundance of specific phylogenetic groups is of interest, researchers need to be aware of the biases their methods introduce. This is particularly relevant if different methods are used for DNA and RNA extraction, in addition to using RNA protection agents only for RNA samples.
Project description:The human gut is colonised by a vast array of microbes that include bacteria, viruses, fungi, and archaea. While interest in these microbial entities has largely focused on the bacterial constituents, recently the viral component has attracted more attention. Metagenomic advances, compared to classical isolation procedures, have greatly enhanced our understanding of the composition, diversity, and function of viruses in the human microbiome (virome). We highlight that viral extraction methodologies are crucial in terms of identifying and characterising communities of viruses infecting eukaryotes and bacteria. Different viral extraction protocols, including those used in some of the most significant human virome publications to date, have introduced biases affecting their a overall conclusions. It is important that protocol variations should be clearly highlighted across studies, with the ultimate goal of identifying and acknowledging biases associated with different protocols and, perhaps, the generation of an unbiased and standardised method for examining this portion of the human microbiome.
Project description:With the advent of molecular biological techniques, especially next-generation sequencing and metagenomics, the number of microbial biogeography studies is rapidly increasing. However, these studies involve the synthesis of data generated by different laboratories using different protocols, chemicals, etc., all with inherent biases. The aim of this study was to assess inter- as well as intralaboratory variations in microbial community composition when standardized protocols are applied to a single soil sample. Aliquots from a homogenized soil sample from a rice field in Italy were sent to five participating laboratories. DNA was extracted by two investigators per laboratory using an identical protocol. All DNA samples were sent to one laboratory to perform DNA quantification, quantitative PCR (QPCR), and microarray and denaturing gradient gel electrophoresis (DGGE) analyses of methanotrophic communities. Yields, as well as purity of DNA, were significantly different between laboratories but in some cases also between investigators within the same laboratory. The differences in yield and quality of the extracted DNA were reflected in QPCR, microarray, and DGGE analysis results. Diversity indices (Shannon-Wiener, evenness, and richness) differed significantly between laboratories. The observed differences have implications for every project in which microbial communities are compared in different habitats, even if assessed within the same laboratory. To be able to make sensible comparisons leading to valid conclusions, intralaboratory variation should be assessed. Standardization of DNA extraction protocols and possible use of internal standards in interlaboratory comparisons may help in rendering a "quantifiable" bias.
Project description:With the emergence of large-scale epidemiologic human microbiome studies, there is a need to understand the reproducibility of microbial DNA sequencing and the impact of specimen collection and processing methods on measures of microbial community composition and structure, with reproducibility studies in infants and young children particularly lacking. Here, we examined batch-to-batch variability and reliability of collection, handling, and processing protocols, testing replicate stool samples from infants and young children using Illumina MiSeq sequencing of the bacterial 16S rRNA gene V4-V5 hypervariable region, evaluating 33 conditions with different protocols and extraction methods. We detected no evidence of batch effects in replicate DNA samples or extractions from the same stool sample. Variability in DNA yield and alpha diversity was observed between the different collection, handling, and processing protocols. However, across all protocols, subject variability was the dominant contributor to microbiome structure, with comparatively little impact of the protocol used. While collection method and DNA extraction kit may affect DNA yield, and correspondingly alpha diversity, our findings suggest that characterization of the structure and composition of the fecal microbiome of infants and young children are reliably measurable by standardized collection, handling, and processing protocols and DNA extraction methods within an individual longitudinal study.
Project description:<h4>Premise</h4>Within a broader study on leaf fossilization in freshwater environments, a long-term study on the development and microbiome composition of biofilms on the foliage of aquatic plants has been initiated to understand how microbes and biofilms contribute to leaf decay and preservation. Here, water lily leaves are employed as a study model to investigate the relationship between bacterial microbiomes, biodegradation, and fossilization. We compare four DNA extraction kits to reduce biases in interpretation and to identify the most suitable kit for the extraction of DNA from bacteria associated with biofilms on decaying water lily leaves for 16S rRNA amplicon analysis.<h4>Methods</h4>We extracted surface-associated DNA from <i>Nymphaea</i> leaves in early stages of decay at two water depth levels using four commercially available kits to identify the most suitable protocol for bacterial extraction, applying a mock microbial community standard to enable a reliable comparison of the kits.<h4>Results</h4>Kit 4, the FastDNA Spin Kit for Soil, resulted in high DNA concentrations with better quality and yielded the most accurate depiction of the mock community. Comparison of the leaves at two water depths showed no significant differences in community composition.<h4>Discussion</h4>The success of Kit 4 may be attributed to its use of bead beating with a homogenizer, which was more efficient in the lysis of Gram-positive bacteria than the manual vortexing protocols used by the other kits. Our results show that microbial composition on leaves during early decay remains comparable and may change only in later stages of decomposition.
Project description:Different DNA extraction protocols were evaluated on a reference soil. A wide difference was found in the total extractable DNA as derived from different extraction protocols. Concerning the DNA yield phenol-chloroform-isomyl alcohol extraction resulted in high DNA yield but also in a remarkable co-extraction of contaminants making PCR from undiluted DNA extracts impossible. By comparison of two different extraction kits, the Macherey&Nagel SoilExtract II kit resulted in the highest DNA yields when buffer SL1 and the enhancer solution were applied. The enhancer solution not only significantly increased the DNA yield but also the amount of co-extracted contaminates, whereas additional disintegration strategies did not. Although a three times repeated DNA extraction increased the total amount of extracted DNA, microbial fingerprints were merely affected. However, with the 5th extraction this changed. A reduction of total DGGE band numbers was observed for archaea and fungi, whereas for bacteria the diversity increased. The application of ethidium monoazide (EMA) or propidium monoazide (PMA) treatment aiming on the selective removal of soil DNA derived from cells lacking cell wall integrity resulted in a significant reduction of total extracted DNA, however, the hypothesized effect on microbial fingerprints failed to appear indicating the need for further investigations.
Project description:The advent of high-throughput sequencing techniques has recently provided an astonishing insight into the composition and function of the human microbiome. Next-generation sequencing (NGS) has become the gold standard for advanced microbiome analysis; however, 3rd generation real-time sequencing, such as Oxford Nanopore Technologies (ONT), enables rapid sequencing from several kilobases to >2 Mb with high resolution. Despite the wide availability and the enormous potential for clinical and translational applications, ONT is poorly standardized in terms of sampling and storage conditions, DNA extraction, library creation, and bioinformatic classification. Here, we present a comprehensive analysis pipeline with sampling, storage, DNA extraction, library preparation, and bioinformatic evaluation for complex microbiomes sequenced with ONT. Our findings from buccal and rectal swabs and DNA extraction experiments indicate that methods that were approved for NGS microbiome analysis cannot be simply adapted to ONT. We recommend using swabs and DNA extractions protocols with extended washing steps. Both 16S rRNA and metagenomic sequencing achieved reliable and reproducible results. Our benchmarking experiments reveal thresholds for analysis parameters that achieved excellent precision, recall, and area under the precision recall values and is superior to existing classifiers (Kraken2, Kaiju, and MetaMaps). Hence, our workflow provides an experimental and bioinformatic pipeline to perform a highly accurate analysis of complex microbial structures from buccal and rectal swabs. <b>IMPORTANCE</b> Advanced microbiome analysis relies on sequencing of short DNA fragments from microorganisms like bacteria, fungi, and viruses. More recently, long fragment DNA sequencing of 3rd generation sequencing has gained increasing importance and can be rapidly conducted within a few hours due to its potential real-time sequencing. However, the analysis and correct identification of the microbiome relies on a multitude of factors, such as the method of sampling, DNA extraction, sequencing, and bioinformatic analysis. Scientists have used different protocols in the past that do not allow us to compare results across different studies and research fields. Here, we provide a comprehensive workflow from DNA extraction, sequencing, and bioinformatic workflow that allows rapid and accurate analysis of human buccal and rectal swabs with reproducible protocols. This workflow can be readily applied by many scientists from various research fields that aim to use long-fragment microbiome sequencing.
Project description:Soils impact global carbon cycling and their resident microbes are critical to their biogeochemical processing and ecosystem outputs. Based on studies in marine systems, viruses infecting soil microbes likely modulate host activities via mortality, horizontal gene transfer, and metabolic control. However, their roles remain largely unexplored due to technical challenges with separating, isolating, and extracting DNA from viruses in soils. Some of these challenges have been overcome by using whole genome amplification methods and while these have allowed insights into the identities of soil viruses and their genomes, their inherit biases have prevented meaningful ecological interpretations. Here we experimentally optimized steps for generating quantitatively-amplified viral metagenomes to better capture both ssDNA and dsDNA viruses across three distinct soil habitats along a permafrost thaw gradient. First, we assessed differing DNA extraction methods (PowerSoil, Wizard mini columns, and cetyl trimethylammonium bromide) for quantity and quality of viral DNA. This established PowerSoil as best for yield and quality of DNA from our samples, though ?1/3 of the viral populations captured by each extraction kit were unique, suggesting appreciable differential biases among DNA extraction kits. Second, we evaluated the impact of purifying viral particles after resuspension (by cesium chloride gradients; CsCl) and of viral lysis method (heat vs bead-beating) on the resultant viromes. DNA yields after CsCl particle-purification were largely non-detectable, while unpurified samples yielded 1-2-fold more DNA after lysis by heat than by bead-beating. Virome quality was assessed by the number and size of metagenome-assembled viral contigs, which showed no increase after CsCl-purification, but did from heat lysis relative to bead-beating. We also evaluated sample preparation protocols for ssDNA virus recovery. In both CsCl-purified and non-purified samples, ssDNA viruses were successfully recovered by using the Accel-NGS 1S Plus Library Kit. While ssDNA viruses were identified in all three soil types, none were identified in the samples that used bead-beating, suggesting this lysis method may impact recovery. Further, 13 ssDNA vOTUs were identified compared to 582 dsDNA vOTUs, and the ssDNA vOTUs only accounted for ?4% of the assembled reads, implying dsDNA viruses were dominant in these samples. This optimized approach was combined with the previously published viral resuspension protocol into a sample-to-virome protocol for soils now available at protocols.io, where community feedback creates 'living' protocols. This collective approach will be particularly valuable given the high physicochemical variability of soils, which will may require considerable soil type-specific optimization. This optimized protocol provides a starting place for developing quantitatively-amplified viromic datasets and will help enable viral ecogenomic studies on organic-rich soils.
Project description:In recent years, 16S rRNA gene amplicon sequencing has been widely adopted for analyzing the microbial communities in drinking water (DW). However, no comprehensive attempts have been made to illuminate the inherent method biases specifically relating to DW communities. In this study, we investigated the impact of DNA extraction and primer choice on the observed microbial community, and furthermore estimated the detection limit of the 16S rRNA gene amplicon sequencing in these experimental settings. Of the two DNA extraction kits investigated, the PowerWater DNA Isolation Kit resulted in higher yield, better reproducibility and more OTUs identified compared to the FastDNA SPIN Kit for Soil, which is also commonly used within DW microbiome research. The use of three separate primer-sets targeting the V1-3, V3-4, and V4 region of the 16S rRNA gene revealed large differences in OTU abundances, with some of the primers unable to detect entire phyla. Estimations of the detection limit were based on bacteria-free water samples (1 L) spiked with Escherichia coli cells in different concentrations [101-106 cells/ml]. E.coli could be detected in all samples, however, samples with ?101 cells/ml had several contaminating OTUs constituting approximately 8% of the read abundances. Based on our findings, we recommend using the PowerWater DNA Isolation Kit for DNA extraction in combination with PCR amplification of the V3-4 or V4 region for DW samples if a broad overview of the microbial community is to be obtained.