PowerSoil Kit Research Articles

O-281 Characterization of Vaginal microbiota and reproductive outcomes associated with assisted reproductive technologies through next-generation sequencing, from Indian population

Abstract Study question Is there any association between the composition of the vaginal microbiota and the reproductive outcomes in infertile women’s undergoing for assisted reproductive treatments (ART)? Summary answer Higher microbial abundance in non-pregnant patients compared to pregnant patients. These pathogens could be used as a possible marker for a higher IVF failure rate. What is known already Various next- generation sequencing methods have been used to study bacterial populations in infertile Women. Vaginal microbiota could have an influence on success rates of fertility treatments. The bacterial microbiota varies by the population. There is no direct evidence on this subject to guide clinical decision making or its influence on the hierarchy of fertility treatments. While the presence of pathogenic bacteria non-Lactobacilli dominated microbiota is associated with poor reproductive outcomes in assisted reproductive treatments (ART). Additional studies, particularly those using metagenomics approaches, may be useful. Study design, size, duration A total of 240 infertile women’s were included in the study. The study population consists of patients attending to the ART unit of a tertiary medical center Udaipur, India, between November 2021 to October 2023. Depending on the biochemical pregnancy results, they were divided into two groups. Group 1 (n = 128) was pregnancy (P), and Group 2 (n = 112) non- pregnancy (NP) group, Patients were aged between (20-45 years). The ethical approval was obtained from the institute. Participants/materials, setting, methods DNA was extracted from vaginal swab samples collected at the time of oocyte pick up using the commercially available ( DNeasy Power Soil kit). The Vaginal microbiota of samples were analyzed using 16S ribosomal RNA gene amplification (MinION) Oxford Nanopore Ltd. Bioinformatics analysis was performed using QIIME2 and Microbiome Analyst packages. Alpha, beta diversity and taxonomic characterization were compared for microbial composition. The findings of the principle coordinate analysis were shown using R version 4.0. Main results and the role of chance Our results show that higher Shannon and Simpson index were obtained in the non- pregnancy (NP) group compared to the pregnancy (P) group. In addition, the partial least squares discriminant analysis (PLS-DA) revealed a difference in microbial composition between pregnancies (P) and non- pregnancy (NP) groups, as well as a higher microbial abundance in non-pregnant patients compared to pregnant patients. At the phylum level Firmicutes, Actinobacteria, Proteobacteria, Fusobacteria, and Bacteroidetes are the most common predominant in the pregnancy (P) group (80.77, 9.52, 9.18, 0.04, and 0.40%, respectively) and the non- pregnancy (NP) group (72, 61, 12.41, 8.01, and 3.75%). A higher abundance of Firmicutes (80.77 vs. 72.61%) and Proteobacteria (9.18 vs. 8.01%), and a lower abundance of Actinobacteria (9.52 vs. 12.41%, p = 0.03), Fusobacteria (0.04 vs. 8.01%, p = 0.02), and Bacteroidetes (0.40 vs. 3.75%, p = 0.03) were observed in pregnancy (P) group. At the genus level, pregnancy group (P) increased the abundance of probiotics lactic acid bacteria (74.61 vs 63.09%) and decreased the abundance of the pathogen Gardnerella (6.03 vs 7.24%, p = 0.03), Atopobium (0.84 vs 4.14%, p = 0.02), Sneathia (0.03 vs. 3.75%, p = 0.02), and Prevotella (0.38 vs. 3, 02%, p = 0.04). Limitations, reasons for caution The limited sample size is the primary constraint of this study. Larger studies with a larger sample size are needed to confirm the distinct microbiome patterns identified in female reproductive tracts samples in connection to infertility and live birth rate (LBR) outcomes. Wider implications of the findings Vaginal microbiota composition associated to different clinical outcomes in patients undergoing ART might have profound implications. This may lead to failed fertility treatments and reduced live birth rate (LBR). More studies are needed to clarify the relative effectiveness of treatments for infertility. Trial registration number NA

A Comparison of Different Protocols for the Extraction of Microbial DNA Inhabiting Synthetic Mars Simulant Soil.

Compared with typical Earth soil, Martian soil and Mars simulant soils have distinct properties, including pH > 8.0 and high contents of silicates, iron-rich minerals, sulfates, and metal oxides. This unique soil matrix poses a major challenge for extracting microbial DNA. In particular, mineral adsorption and the generation of destructive hydroxyl radicals through cationic redox cycling may interfere with DNA extraction. This study evaluated different protocols for extracting microbial DNA from Mars Global Simulant (MGS-1), a Mars simulant soil. Two commercial kits were tested: the FastDNA SPIN Kit for soil ("MP kit") and the DNeasy PowerSoil Pro Kit ("PowerSoil kit"). MGS-1 was incubated with living soil for five weeks, and DNA was extracted from aliquots using the kits. After extraction, the DNA was quantified with a NanoDrop spectrophotometer and used as the template for 16S rRNA gene amplicon sequencing and qPCR. The MP kit was the most efficient, yielding approximately four times more DNA than the PowerSoil kit. DNA extracted using the MP kit with 0.5 g soil resulted in 28,642-37,805 16S rRNA gene sequence reads and 30,380-42,070 16S rRNA gene copies, whereas the 16S rRNA gene could not be amplified from DNA extracted using the PowerSoil kit. We suggest that the FastDNA SPIN Kit is the best option for studying microbial communities in Mars simulant soils.

An experimental design for obtaining DNA of a target species and its diet from a single non-invasive genetic protocol.

Next-generation sequencing technology has enabled accurate insights into the diet of wildlife species. The protocols for faecal sample collection and DNA extraction for diet analysis have differed from those focusing on target species, even in most studies combining questions on both aspects. We designed an experiment to evaluate two protocols using 11 parameters and select a single one that will generate both target species (Asiatic wild ass, Equus hemionus, in Israel) and diet DNA, as an effective strategy to minimise time, effort, and cost without hampering efficiency. In Protocol A, we swabbed the outer surface of faecal boluses and extracted DNA using a Stool Kit, while for Protocol B, we homogenised faecal matter from inside the bolus followed by extraction using a Powersoil Kit. Protocol A performed significantly better for four parameters, which included, for the target species, microsatellite amplification success and the quantity of the GAPDH gene; and for its diet, the number of exact sequence variants (ESVs) obtained at genus level and plant genus richness. However, there was no significant difference in the amplification success of sex-linked and plant markers, total reads at genus level, number of genera obtained and plant genus composition. Although we chose Protocol A, both protocols yielded results for the target species and its diet, demonstrating that one single protocol can be used for both purposes, although a pilot study is recommended to optimise the protocol for specific systems. This strategy may also be useful for studies combining target species and their gut microbiome and parasitic load.

Comparison of nine extraction methods for bacterial identification using the ONT MinION sequencer.

The Anthrax mailings bioterrorism attack in 2001 revealed the need for universal and rapid microbial forensic analyses on unknown biological evidence. However, the gold standard for bacterial identification includes culturing isolates, which is laborious. Molecular approaches for bacterial identification revolve around 16S ribosomal gene sequencing using Sanger or next generation sequencing (NGS) platforms, but these techniques are laboratory-based and can also be time-consuming. The Oxford Nanopore Technologies (ONT) MinION sequencer can generate long read lengths that span the entire bacterial 16S rRNA gene and accurately identify the species level. This platform can be used in the field, allowing on-site evidence analysis. However, it requires higher quantities of pure DNA compared to other sequencing platforms; thus, the extraction method for bacterial DNA is critical for downstream analysis, which to date are tailored toward a priori knowledge of the species' taxonomic grouping. During an attack, the investigative team may not know what species they are handling; therefore, identifying an extraction method that can handle all bacterial groups and generate clean DNA for the MinION is useful for microbial forensic analysis. The purpose of this study was to identify a "universal" extraction method that can be coupled with ONT MinION sequencing for use in forensic situations for rapid identification. It also evaluated the cloud-based data analysis software provided by ONT, EPI2ME. No "universal" extraction method was identified as optimal for downstream MinION sequencing. However, the DNeasy PowerSoil Kit and Noda et al. Chelex-100 method gave comparable sequencing results and could be used as rapid extraction techniques. This study showed that the ONT 16S Barcoding Kit 1-24 coupled with the 16S FASTQ workflow might not be the best for use in forensic situations where species-level identification needs to be obtained, as most alignments were approximately 89% accurate. In all seven test organisms and nine extraction methods, accurate species identification was onlyobtained in 63% of the cases.

Unravelling bird nest arthropod community structure using metabarcoding

Bird nests are fascinating microcosms harboring a wide range of arthropods parasitizing the nesting birds or feeding on prey remains, feces, and the nest material. Studies of these communities have been entirely based on emergence traps which collect live organisms out of the nests. The analysis of nest contents and environmental DNA (eDNA) via metabarcoding could expand our knowledge and identify prey, exuviae, and other animal remains in bird nests. Here, we investigated the potential of arthropod remains, nest dust, and feathers to better describe arthropod diversity accumulated in 20 bird nests collected in Guelph (Canada). We used subsampling strategies and tested two extraction approaches to investigate the distribution of DNA in nests, account for low-quality DNA, and the presence of inhibitory substances. In total, 103 taxa were detected via metabarcoding. Arthropod remains delivered the highest number of taxa (n = 67), followed by nest dust (n = 29). Extractions with the PowerSoil kit outperformed DNeasy extractions coupled with PowerClean Pro inhibitor removal. Per nest, on average 5.5% taxonomic overlap between arthropod remains of different size classes was detected and subsamples of nest dust extracted with the PowerSoil kit showed 47.3% taxonomic overlap indicating a heterogeneous eDNA distribution in nests. Most detected species were either feeding in the nest, i.e., herbivorous / predatory, or bird food. We also detected molecular traces of 25 bird species, whose feathers were likely used as nest material. Consequently, the metabarcoding of bird nest materials provides a more complete picture of nest communities, which can enable future studies on functional diversity and better comparisons between nesting species.

Ganoderma zonatum causes butt rot of areca (Dypsis lutescens) and robellini (Phoenix roebelenii) palms in Florida.

Ganoderma butt rot is a lethal disease of palms (Arecaceae) prevalent in palm-growing regions in the US that infects at least 58 species of palms (Elliott and Broschat 2001). Early symptoms appear as wilting of older fronds in the lower part of the canopy, and as disease progresses, wilting advances to younger leaves higher in the canopy towards the unopened spear leaf eventually killing the palm. A characteristic sign of the disease is the appearance of fruiting bodies (basidiomata) at the base of the palm trunk close to soil line. Ganoderma butt rot disease was detected on clustering palm species, areca palms, with 9 (82%) clusters showing Ganoderma basidiocarps and dead stumps, and mortality was observed in 5 (45%) clusters. A sterile scalpel was used to transfer the context tissue from Ganoderma basidiomata to full-strength potato dextrose agar selective media supplemented with streptomycin (100 mg/l), lactic acid (2 ml/l) and benomyl (4 mg/l). The pure culture for isolate GAN-33 was grown at 28°C in complete darkness for 10 days. The fungal colony was ivory white in color that grew radially as a dense mycelial mat without any sporulation. To establish the identity of the fungus, DNA was extracted using the Qiagen DNeasy PowerSoil kit (Cat. #12888). Three barcoding genes, nuclear ribosomal DNA internal transcribed spacer (ITS) region, RNA polymerase II subunit 2 (rpb2) and translation elongation factor 1α (tef1α) were amplified using primers ITS1/ITS4 (White et al 1990), bRPB2-6f/bRPB2-b7.1R (Matheny et al 2007) and EF1-983F/EF1-2212R (Matheny et al 2007), respectively. The sequences were deposited in GenBank, accession numbers KX853442, KX853466 and KX853491 for ITS, rpb2 and tef1α, respectively (Elliott et al 2018). Comparison to the NCBI nucleotide sequence database identified isolate GAN-33 as Ganoderma zonatum based on 100, 99 and 99% similarity to ITS, rpb2 and tef1α sequences, respectively. Pathogenicity of G. zonatum isolate GAN-33 was determined on 1-year old seedlings of areca palm (Dypsis lutescens) and pygmy date palm (Phoenix roebelenii). Ganoderma zonatum inoculum was prepared by transferring two-week old cultures to autoclaved wheat berries and allowed to colonize for two weeks. Seedlings were gently removed from the pot and the roots were trimmed before placing them back in the pot ensuring that the roots were in contact with the G. zonatum colonized wheat berries. The inoculated and control seedlings were maintained in a growth chamber at 28°C 60% RH (daytime) and 24°C 50% RH (night time), 12h:8h light:dark period, and watered twice a week. Initial wilting symptoms started appearing approximately one month after inoculation and mortality was observed for a total of four seedlings at three months after inoculation i.e., 2 out of 3 G. zonatum inoculated seedlings died for both areca and robellini palms, whereas the non-inoculated areca and robellini palm control seedlings remained healthy and alive. The pathogen was re-isolated from inoculated roots, and its identity was confirmed by colony morphology and PCR using G. zonatum specific primers (Chakrabarti et al 2022). To the best of our knowledge this is the first report establishing G. zonatum as the pathogen responsible for Ganoderma butt rot of palms.

Comparison of DNA concentration and bacterial pathogen PCR detection when using two DNA extraction kits for nasopharyngeal/oropharyngeal samples.

Several important human pathogens that cause life-threatening infections are asymptomatically carried in the Nasopharynx/Oropharynx (NP/OP). DNA extraction is a prerequisite for most culture-independent techniques used to identify pathogens in the NP/OP. However, components of DNA extraction kits differ thereby giving rise to differences in performance. We compared the DNA concentration and the detection of three pathogens in the NP/OP using the discontinued DNeasy PowerSoil Kit (Kit DP) and the DNeasy PowerLyzer PowerSoil Kit (Kit DPP). DNA was extracted from the same set of 103 NP/OP samples using the two kits. DNA concentration was measured using the Qubit 2.0 Fluorometer. Real-time Polymerase Chain reaction (RT-PCR) was done using the QuantStudio 7-flex system to detect three pathogens: S. pneumoniae, H. influenzae, and N. meningitidis. Bland-Altman statistics and plots were used to determine the threshold cycle (Ct) value agreement for the two kits. The average DNA concentration from kit DPP was higher than Kit DP; 1235.6 ng/ml (SD = 1368.3) vs 884.9 ng/ml (SD = 1095.3), p = 0.002. Using a Ct value cutoff of 40 for positivity, the concordance for the presence of S. pneumoniae was 82% (84/102); 94%(96/103) for N. meningitidis and 92%(95/103) for H. influenzae. Kit DP proportionately resulted in higher Ct values than Kit DPP for all pathogens. The Ct value bias of measurement for S. pneumoniae was +2.4 (95% CI, 1.9-3.0), +1.4 (95% CI, 0.9-1.9) for N. meningitidis and +1.4 (95% CI, 0.2-2.5) for H. influenzae. The higher DNA concentration obtained using kit DPP could increase the chances of recovering low abundant bacteria. The PCR results were reproducible for more than 90% of the samples for the gram-negative H. influenzae and N. meningitidis. Ct value variations of the kits must be taken into consideration when comparing studies that have used the two kits.

P-067 Characterization of seminal microbiota in patients with asthenozoospermic and normozoospermic using next-generation sequencing

Abstract Study question How are the semen microbiota compositions of patients with asthenozoospermic and normozoospermic different, According to data from 16S rRNA gene-specific Next-generation sequencing (NGS)? Summary answer Large numbers of microbes can be present in seminal fluid, and there are differences in the semen microbiota between asthenozoospermic and normozoospermic semen samples. What is known already Male factor is attributable in up to 50% of cases of infertility. Most studies on normal and abnormal sperm microbiota are based on 16S rRNA gene-specific next-generation sequencing (NGS). Microbiome analysis based on subunit 16S rRNA sequencing is a fast tool that can enable the identification of all the pathogenic microorganisms associated with semen in clinical pathology. Studies on the impact of semen micro-biomes in asthenozoospermic and normozoospermic samples could improve the results of assisted reproductive technologies. The major bacterial diversity in asthenozoospermic and normozoospermic samples belongs to the genera Lactobacillus, Prevotella, Staphylococcus, and Anaerococcus. Study design, size, duration Two eighty patients with their own semen samples were included in the study. The study population consists of patients attending to the “Pacific IVF Center” Pacific Medical University and Hospital (Udaipur, India) from November 2020 to October 2022. Depending on the spermiogram results, they were divided into two groups. Group 1 (n = 127) was asthenozoospermic, and Group 2 (n = 153) had normozoospermic semen samples, Patients were aged between (20-45 years). Ethical approval was obtained from the institute. Participants/materials, setting, methods Genomic DNA was extracted from samples using commercially available (Qiagen, DNeasy Power Soil kit). The amount of extracted DNA was measured using a Nanodrop spectrophotometer (Thermo Scientific). The microbiota of semen was analyzed using 16S ribosomal RNA (rRNA) gene amplification (MinION) Oxford Nanopore Ltd. Bioinformatics analysis was performed using QIIME2 and Microbiome Analyst packages. Alpha, beta diversity, and taxonomic characterization were compared for the seminal microbiome in asthenozoospermic and normozoospermic semen samples. Main results and the role of chance Different bacterial communities were detected when asthenozoospermic and normozoospermic semen samples were analyzed. In patients with asthenozoospermic parameters, a higher alpha diversity index tendency was found. In normozoospermic semen samples, a p-value of bacterial diversity was non-significant (p = 0.14 for the Shannon index), and significant differences in asthenozoospermic semen samples (p = 0.01 for the Shannon index). In the beta diversity analysis, no significant differences were observed between both groups Relative abundance analysis identified the bacterial communities, Lactobacillus, Streptococcus, and Ureaplasma in normozoospermic semen samples, and Anaerococcus and Gardnerella in asthenozoospermic samples, Comparison of asthenozoospermic and normozoospermic samples with alpha diversity (p = 0.06 for the Shannon index and p = 0.08 for the Simpson index) and beta diversity (p < 0.001) showed significant differences. Relative abundance in normozoospermic semen samples identified the bacterial communities, Lactobacillus (p = 3.70E-4), Prevotella (p = 8.11E-4), and in asthenozoospermic semen samples Anaerococcus (p = 0.004) and Gardnerella (p = 0.004). Limitations, reasons for caution The limited sample size is the primary constraint of this study. Larger studies with a larger sample size are needed to confirm the distinct microbiome patterns identified in asthenozoospermic and normozoospermic samples in connection to male infertility. Wider implications of the findings Further research could determine the detection rate of the described bacterial diversity in semen with other pathologies. Determining the relationship between sperm microbiota characteristics and infertility could enable the development of new algorithms for treating patients with reproductive disorders, An abnormal seminal microbiome appears to be strongly correlated with infertility. Trial registration number N/A

Abstract 5894: Changes in the gut microbiota associated with oncolytic virotherapy efficacy in a gastric cancer murine model

Abstract Gastric cancer is the third leading cause of cancer-related mortality worldwide, and current standard-of-care treatment allows for a 5-year survival rate of 20%. Newer treatments against solid tumors include an oncolytic adenovirus armed with T-cell activator OX40L, named Delta-24-RGDOX, which expresses the positive immune checkpoint OX-40L. While studies have revealed the interaction between the gut microbiome and immunotherapy agents, it’s still unknown how the gut microbiome might influence viroimmunotherapy efficacy. We hypothesized that the gut microbiota plays an important role in modulating the virus-driven anti-tumor response. To test this hypothesis, immunocompetent C57BL/6 mice were subcutaneously implanted M12Luc gastric cancer cells and were administered PBS (control) or Delta-24-RGDOX by intratumoral injections. Mice fecal pellets were collected at three different time points: (1) before tumor implantation, (2) after tumor establishment, and (3) 14 days after the first dose of the treatment. Genomic DNA was isolated with the QIAGEN DNeasy Powersoil Kit from fecal pellets of cancer-bearing mice (n=60), and the V4 region of the 16S ribosomal RNA gene (prokaryotes) was sequenced using the Illumina platform. Data was deposited in QIITA for quality assessment, followed by microbial community analysis using the taxonomic reference database SILVA and the downstream platforms QIIME2 and R with a rarefaction depth of 9,187 reads per sample. We found significant differences in the gut microbiome community structure of viroimmunotherapy-treated mice compared to controls (ANOSIM p-value=0.025). No differences in gut diversity were observed, although control animals tended to show higher richness (Kruskal-Wallis p-value>0.05). Of interest, the bacterial composition of responders to the viroimmunotherapy had a decrease in Actinobacteria and an increase of Patescibacteria. Additionally, responders had higher levels of butyrate-producer bacteria such as Ruminococcaceae and Clostridium sensu stricto. These findings suggest that butyrate producers may be important in regulating host gut homeostasis associated with the clinical efficacy of Delta-24-RGDOX, suggesting possible benefits of microbiota modulation in patient survival. This project was sponsored by a pilot award within The University of Puerto Rico/University of Texas MD Anderson Cancer Center (MDACC) Partnership for Excellence in Cancer Research Grant (2U54CA096297-16). Citation Format: Natalie M. Melendez-Vazquez, Sagar Sohoni, Arie Van Wieren, Juan Fueyo, Candelaria Gomez-Manzano, Filipa Godoy-Vitorino. Changes in the gut microbiota associated with oncolytic virotherapy efficacy in a gastric cancer murine model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5894.

Characterization of the eukaryotic microbial communities in the chicken ileum in cage-free and conventional commercial housing systems

The current shift from conventional cage (CC) hen housing facilities towards cage-free (CF) hen housing in the egg industry has left many questions regarding how level of fecal exposure and exposure to other hens may influence the hen intestinal microbiota. In a previous publication we reported differences in the bacterial ileal communities and ileal morphology between hens in CC and CF production environments at a single commercial site. Here, we present the first 18S rRNA gene amplicon sequencing-based characterization of the eukaryotic ileal microbiota of adult layer hens, and their associations with intestinal health parameters and the bacterial microbiota. DNA was extracted from the ileal digesta of hens (n=32 CC, n=48 CF) using the Qiagen Powerlyzer Powersoil kit, followed by amplification of the V9 region of the 18S rRNA gene. Paired end sequencing was performed with the Illumina MiSeq platform, and the resulting reads were processed according to the Mothur MiSeq protocol in Mothur v1.43.0. De novo operational taxonomic unit (OTU) clustering was performed in mothur with a 99% similarity threshold, and OTUs were taxonomically classified with the SILVA SSU v138 reference database. OTUs classified as vertebrate, plant, or arthropod were removed, resulting in 3,136,400 high quality reads and 1,370 OTUs. Associations between OTUs and intestinal parameters were calculated using PROC GLIMMIX. PERMANOVA over Bray-Curtis distances revealed differences between CC and CF eukaryotic ileal microbiota at the whole community level, but no OTUs were differentially abundant after correcting for false discovery (P > 0.05; q > 0.1). Kazachstania and Saccharomyces, closely related genera of yeast, represented 77.1% and 9.7% of sequences respectively. Two Kazachstania OTUs and 1 Saccharomycetaceae OTU were positively correlated with intestinal permeability (r2 ≥ 0.35). Eimeria accounted for 7.6% of sequences across all samples. Intriguingly, 15 OTUs classified as Eimeria were inversely associated with intestinal permeability (r2 ≤ -0.35), suggesting Eimeria may play a more complex role in the microbiota of healthy birds than has been observed in disease challenges.

Comparison of three DNA extraction methods for recovery of microbial DNA from Arctic permafrost.

Permafrost soils, which contain one of Earth's largest terrestrial carbon stocks, are vulnerable to thaw and microbial decomposition, exacerbating climate change. Advancements in sequencing technologies have facilitated the identification and functional profiling of microbial communities in permafrost, but DNA extraction from these soils is challenging due to their high microbial diversity and low biomass. This study assessed the effectiveness of the DNeasy PowerSoil Pro kit in extracting DNA from permafrost samples and found that it produced significantly different results than the discontinued DNeasy PowerSoil kit. The study highlights the importance of consistent DNA extraction methods in permafrost studies.

Assessing the lysis of diverse pollen from bulk environmental samples for DNA metabarcoding

Pollen is ubiquitous year-round in bulk environmental samples and can provide useful information on previous and current plant communities. Characterization of pollen has traditionally been completed based on morphology, requiring significant time and expertise. DNA metabarcoding is a promising approach for characterizing pollen from bulk environmental samples, but accuracy hinges on successful lysis of pollen grains to free template DNA. In this study, we assessed the lysis of morphologically and taxonomically diverse pollen from one of the most common bulk environmental sample types for DNA metabarcoding, surface soil. To achieve this, a four species artificial pollen mixture was spiked into surface soils collected from Colorado, North Carolina, and Pennsylvania, and subsequently subjected to DNA extraction using both the PowerSoil and PowerSoil Pro Kits (Qiagen) with a heated incubation (either 65 °C or 90 °C). Amplification and Illumina sequencing of the internal transcribed spacer subunit 2 (ITS2) was completed in duplicate for each sample (total n, 76), and the resulting sequencing reads taxonomically identified using GenBank. The PowerSoil Pro Kit statistically outperformed the PowerSoil Kit for total DNA yield. When using either kit, incubation temperature (65 °C or 90 °C) used had no impact on the recovery of DNA, plant amplicon sequence variants (ASVs), or total plant ITS2 reads. This study highlighted that lysis of pollen in bulk environmental samples is feasible using commercially available kits, and downstream DNA metabarcoding can be used to accurately characterize pollen DNA from such sample types.

Truth or Lie: Does the DNA Extraction Procedure Really Affect the Insight in Composition and Diversity of Microbial Communities in Saffron Cultivated Soils?

The aim of this study was to evaluate the performance of two of the most commonly used commercial kits for soil DNA extraction regarding the values of the taxonomic diversity of prokaryotes and community composition of saffron (Crocus sativus) cultivated fields. The impact of the QIAGEN-DNeasy PowerSoil Kit (MO) and Macherey-Nagel™ NucleoSpin™ Soil (MN) kit was tested on the soil of an Italian western alpine experimental site located in Saint Christophe (Aosta Valley, AO). Nine biological replicas of bulk soil were collected and analyzed independently with the two kits. 16S rRNA metabarcoding was applied to characterize soil microbial communities. We first noticed that both DNA extraction kits yielded nearly the same number of OTUs: 1284 and 1268 for MN and MO, respectively. Both kits did not differ in the alpha diversity of the samples, while they had an influence on the beta diversity. The comparative analysis of the microbial community composition displayed differences in microbial community structure depending on which kit was used. These differences were especially highlighted at Phylum and Class levels. On the other hand, the fact that, from a functional point of view, our approach did not highlight any differences allows us to state that the results obtained with the two extraction kits are comparable and interchangeable. Based on these results and those in the literature, we could undoubtedly recommend both commercial kits, especially if the soil target microorganisms are prokaryotes and the study focuses on agricultural sites.

The Effects of DNA Extraction Kits and Primers on Prokaryotic and Eukaryotic Microbial Community in Freshwater Sediments.

DNA based sequencing technology has revolutionized the field of microbial ecology and environmental studies. However, biases can be introduced at all experimental steps and, thus, affect the interpretation of microbial community. So far, previous studies on the biases introduced from the key steps of DNA extraction and primer sets mainly focused on the bacterial communities in soil or sediment samples, while little is known about the effect on the eukaryotic microbial communities. Here, we studied the effects of three different DNA extraction kits on both prokaryotic and micro-eukaryotic communities by 16S and 18S rRNA gene amplicon sequencing, and further disentangled the influence of primer choice on the micro-eukaryotic communities. Our results showed that the FastDNA SPIN Kit for Soil and DNeasy PowerSoil Kit produced much higher DNA yield with good reproducibility, and observed more eukaryotic OTUs compared to the MinkaGene DNA extraction kit, but all three kits exhibited comparable ability in recovering bacterial alpha diversity. Of the two primer sets, both targeting the V4 region of the 18S rRNA gene, the TAR primer set detected higher number of unique OTUs than the EK primer set, while the EK primer set resulted in longer amplicons and better reproducibility between replicates. Based on our findings, we recommend using the DNeasy PowerSoil Kit with the EK primer set to capture the abundant micro-eukaryotic taxa from freshwater sediment samples. If a more complete picture of the eukaryotic microbial community is desired, the TAR primer set in combination with the FastDNA SPIN Kit is more efficient in this study.

Influence of DNA extraction kits on freshwater fungal DNA metabarcoding.

BackgroundEnvironmental DNA (eDNA) metabarcoding is a common technique for efficient biodiversity monitoring, especially of microbes. Recently, the usefulness of aquatic eDNA in monitoring the diversity of both terrestrial and aquatic fungi has been suggested. In eDNA studies, different experimental factors, such as DNA extraction kits or methods, can affect the subsequent analyses and the results of DNA metabarcoding. However, few methodological studies have been carried out on eDNA of fungi, and little is known about how experimental procedures can affect the results of biodiversity analysis. In this study, we focused on the effect of DNA extraction method on fungal DNA metabarcoding using freshwater samples obtained from rivers and lakes.MethodsDNA was extracted from freshwater samples using the DNeasy PowerSoil kit, which is mainly used to extractmicrobial DNA from soil, and the DNeasy Blood & Tissue kit, which is commonly used for eDNA studies on animals. We then compared PCR inhibition and fungal DNA metabarcoding results; i.e., operational taxonomic unit (OTU) number and composition of the extracted samples.ResultsNo PCR inhibition was detected in any of the samples, and no significant differences in the number of OTUs and OTU compositions were detected between the samples processed using different kits. These results indicate that both DNA extraction kits may provide similar diversity results for the river and lake samples evaluated in this study. Therefore, it may be possible to evaluate the diversity of fungi using a unified experimental method, even with samples obtained for diversity studies on other taxa such as those of animals.

Detection of DNA from Toxoplasma gondii oocysts in water for reuse.

The absence of a standardized method for detecting oocysts in water samples makes it difficult to characterize them, including in water for reuse. This study aimed to detect Toxoplasma gondii oocysts using two extraction methods. Using method 1693/2014 USEPA, 30 L of water for reuse from two wastewater treatment plants (WWTPs) in the city of São Paulo, Brazil, was concentrated, totaling 20 samples. The supernatant generated from the immunomagnetic separation (IMS) step was collected for detection of T. gondii oocysts. For DNA extraction, two techniques were used: the commercial kit DNeasy PowerSoil Kit® optimized with the enzyme Zymolyase® and with freeze-thaw steps. DNA quantification was performed with the target sequence of gene B1. From 16 samples submitted to enzymatic extraction, four were positive. In freeze-thaw extraction, no DNA was detected. DNA extraction was the essential step for oocyst detection given the resistant nature of their wall.

Optimization of microbial DNA extraction from human skin samples for CRISPR typing

The human skin microbiome, a complex microbial community harbored by the human skin surface, has been proposed as a new forensic genetic marker for human identification to complement short tandem repeat typing. A recent study utilized Streptococcus clustered regularly interspaced short palindromic repeat (CRISPR) polymorphism of the skin microbiome to characterize individuality of the skin microbiome; however, further validation is required for forensic purposes. Optimization of the DNA extraction protocol is critical for both forensic human DNA and microbiome analyses. Previous reports have demonstrated that DNA extraction protocols may alter the results of microbiome analysis, suggesting careful selection of DNA extraction protocols. In this study, we evaluated protocols for DNA extraction from skin swab samples by comparing two DNA extraction protocols, EZ1 DNA Investigator Kit and DNeasy PowerSoil Kit, in order to optimize the metagenomic CRISPR typing method. Quantification of 16S rRNA gene copy numbers and qualification of CRISPR diversities suggested that microbial DNA yield and composition of the CRISPRs were not altered by the two DNA extraction methods. Furthermore, the accuracy of the individual prediction models built using each sample set was comparable. Thus, our results support that the EZ1 platform, an automated DNA extraction protocol used in forensic laboratories, can also be used for metagenomic CRISPR typing intended for forensic human identification.

Altered Fecal Microbiota Signatures in Patients With Anxiety and Depression in the Gastrointestinal Cancer Screening: A Case-Control Study.

Background: Increasing attention has been devoted to cancer screening and microbiota in recent decades, but currently there is less focus on microbiota characterization among screeners and its relationship to anxiety and depression.Methods: We characterized the microbial communities of fecal samples collected through the FOBT card from anxiety and depression screeners and paired controls in Henan, China (1:2, N = 69). DNA was extracted using the MOBIO PowerSoil kit. The V4 region of the 16S rRNA gene was sequenced using MiniSeq and processed using QIIME1. LEfSe was used to identify differentially abundant microbes, the Wilcoxon rank-sum test was used to test alpha diversity differences, and permutational multivariate analysis of variance was used to test for differences in beta diversity.Results: Similar fecal microbiota signatures in composition were found among screeners. The intestinal microbial environments by phylum were all composed primarily of Firmicutes, Bacteroidetes, and Proteobacteria, and the corresponding top genera were Faecalibacterium, Roseburia, and Prevotella. Compared with controls, the ranking of the top five genera in the anxiety and depression group changed, and the dominant genus was Prevotella in the anxiety and depression group and Faecalibacterium in the control group. There was a lower relative abundance of Gemmiger (1.4 vs. 2.3%, P = 0.025), Ruminococcus (0.6 vs. 0.8%, P = 0.037), and Veillonella (0.6 vs. 1.3%, P = 0.020). This may be linked to the lower alpha diversity in participants with anxiety and depression (Observed OTUs: 122.35 vs. 143.24; Chao1: 127.35 vs. 149.98), although no significant differences were observed. Distinct clustering in microbial composition between the two groups was detected for the Jaccard distance (P = 0.011).Conclusions: Our study showed differing microbial characterization among participants with anxiety and depression in the endoscopic screening of upper gastrointestinal cancer. Gemmiger, Ruminococcus, and Veillonella were informative and have potential clinical implications, which need to be confirmed by large-scale, prospective cohort studies and biological mechanism research.

Differences in Fecal Microbiota in Long-Term Adolscent/Young Adult Hodgkin Lymphoma Survivors and Their Unaffected Twins

Background: Hodgkin lymphoma is one of the most common malignancies among adolescents and young adults (AYA) in economically developed settings. Although the survival rate is very high (85%), severe late effects are common. Immune dysregulation is clinically and pathologically associated with HL at diagnosis and alterations in inflammatory cytokine levels are evident years after cure. Gut bacteria play a role in modulating immune responses and inflammation. In a pilot study of 13 AYA HL-discordant twin pairs, we saw significantly lower fecal microbial diversity in the HL survivor compared to their unaffected twin. We conducted a follow-up study in a larger set of twins to determine whether there are differences in the relative abundance of specific fecal microbiota in long-term AYAHL survivors compared to their unaffected twins. Twin pairs are an ideal study population because they are partially matched on genetic and early life factors, both of which influence the composition of the gut microbiome.Methods: The subjects are members of identical twin pairs enrolled in the USC International Twin Study, in whom one twin was diagnosed with histologically verified AYA HL diagnosed at 15-50 years old, at least 10 years ago (mean interval since diagnosis = 22 years ago), and the other twin has remained unaffected. 24 AYA HL survivors and their unaffected twins provided stool samples and detailed answers to an online questionnaire on timing of life milestones, quality of life and medical conditions. A single unaffected co-twin also participated; her twin (the AYA HL survivor) was ineligible because of recent antibiotic use. Fresh stool samples were collected, frozen at -800, shipped to our laboratory and maintained at that temperature until use. DNA extraction was performed using bead beating in conjunction with the MO BIO Powersoil kit. Sequencing of the 253 base pair V4 region of 16S ribosomal DNA was performed using an Illumina HiSeq 2500. Raw sequence data was processed in QIIME and 97% OTUs picked using the Silva database. Taxa present in less than 10% of samples were excluded. Alpha diversity metrics including Chao1 index, Shannon index, and Faith's phylogenetic diversity were calculated for each sample and differences tested with a paired t-test. Sequences were rarefied with 1,000 samplings of the lowest number of reads to standardize the sample and an average was taken to calculate alpha diversity. Differential genera between AYA HL survivors and their unaffected twins were determined using DESeq2, based upon negative binomial models with twin pair as a covariate. P-values were adjusted for multiple comparisons.Results: Sequencing yielded an average of 219,373 reads, with a minimum of 64,742 and a maximum of 391,120. There were no significant differences between AYA HL survivors and their unaffected twins in the various alpha diversity metrics. However for each measure, the unaffected twin had a higher diversity score compared with their HL case twin survivor. There were no differences in beta-diversity. Significant differences in relative abundance of the following genera were observed between AYA HL survivors and their unaffected twins: Streptococcus (p=9.7x 10-5), Sellimonas (p= 0.005), Candidatus Stoquefichus (p=0.020), unclassified S24-7 (p=0.035), Faeclitalea (p=0.035), Veillonella (p=0.040), Faecalibacterium (p=0.026) and [Eubacterium] oxidoreducens group (p=0.028). The last two taxa were more abundant in survivors compared with unaffected twin and the remaining taxa were more abundant in unaffected twin controls compared with survivors.Conclusions: Even after decades, AYA HL survivors harbor fecal microbiota that differ from that of their unaffected twins who never experienced the lymphoma or its treatment. Further studies are necessary to determine the biological significance of these differences and whether they affect survivorship quality of life. DisclosuresNo relevant conflicts of interest to declare.

Gut microbiome in adolescent depression

ObjectivesTo examine the association of major depressive disorder (MDD) and selective serotonin reuptake inhibitor (SSRI) use with gut microbiome in older adolescents and younger adults. MethodsFifteen to 20-year-old participants within a month of starting an SSRI and unmedicated controls were enrolled in a longitudinal study. They underwent a diagnostic evaluation comprising self-completed and rater-administered questionnaires and clinical interview. They also provided a stool sample, which was stored at -80°C until DNA extraction. Microbial DNA was extracted with the MoBio PowerSoil kit, and the V4 region of the 16S rRNA was amplified and sequenced. Raw sequence data was processed with the LotuS pipeline. Only samples with no antibiotic exposure in the last 6 months and with >1000 quality filtered reads were included in the analysis. Results160 participants (57.5% female, mean age 20.0±1.9 years, 29% taking SSRIs) were enrolled, comprising 110 MDD patients (60% in acute episode), 27 healthy controls, and 23 psychiatric controls. No significant group differences were observed in bacterial richness or alpha and beta diversity. Differential abundance analysis of bacterial taxa found no significant group differences at the phylum and genus levels. Neither being in a major depressive episode vs. remission nor using SSRIs was associated with differential bacterial composition. ConclusionsIn this sizeable sample of older adolescents, neither MDD nor SSRI use was associated with differences in gut bacterial microbiome. In this age group, the bi-directional interaction between the gut bacteria and brain may be more nuanced than in adults, requiring further investigation.