Deep Pyrosequencing Research Articles

Detection of Nudibranch Chromodoris quadricolor associated microbiome using two DNA extraction methods

DNA extraction with high quality is critical to all molecular genetic analyses. However, obtaining DNA from microbes associated with animals is challenging. Despite the availability of various DNA extraction kits in the markets, no studies were conducted to date to evaluate their potential for the invertebrates such as nudibranch, one of Mollusca. This study compared the Quick—DNA Fungal/Bacterial Miniprep Kit and CTAB methods and tested them across four samples of Chromodoris quadricolor gut and skin tissues. The universal bacterial primers 331f and 797r and the animal-specific primers LCO1490-JJ and HCO2198-JJ were used to amplify the 16S rRNA gene and partial mitochondrial cytochrome oxidase I gene using extracted DNA as a template. The DNA and PCR products’ quality and concentration were verified with agarose gel and Nanodrop, respectively. The two methods' quality assessed using the deep pyrosequencing of the 16S rRNA gene in capturing a more diverse microbiome. The highest yield and purity (over 1000 ng / µL) were obtained with the CTAB method, while it was not exceeded 260 ng/µL with Quick DNA kit and display high purity. Also, 16S rRNA community amplicon sequencing revealed that the CTAB way could catch more diverse bacterial groups. The most efficient method of DNA extraction was CTAB, as it achieved both high concentration and purity

Large scale screening for benzimidazole resistance mutations in Nematodirus battus, using both pyrosequence genotyping and deep amplicon sequencing, indicates the early emergence of resistance on UK sheep farms

Benzimidazoles (BZ) have been the anthelmintic of choice for controlling Nematodirus battus infections since their release in the 1950s. Despite heavy reliance on this single anthelmintic drug class, resistance was not identified in this nematode until 2010 (Mitchell et al., 2011). The study aimed to explore the prevalence of BZ-resistance mutations in N. battus from UK sheep flocks using deep amplicon sequencing and pyrosequencing platforms. Based on evidence from other gastrointestinal nematodes, resistance in N. battus is likely to be conferred by single nucleotide polymorphisms (SNP) within the β-tubulin isotype 1 locus at codons 167, 198 and 200. Pyrosequencing and deep amplicon sequencing assays were designed to identify the F167Y (TTC to TAC), E198A (GAA to GCA) and F200Y (TTC to TAC) SNPs. Nematodirus battus populations from 253 independent farms were analysed by pyrosequencing; 174 farm populations were included in deep amplicon sequencing and 170 were analysed using both technologies. F200Y was the most prevalent SNP identified throughout the UK, in 12–27% of the populations tested depending on assay, at a low overall individual frequency of 2.2 ± 0.6% (mean ± SEM, based on pyrosequencing results). Four out of the five populations with high frequencies (>20%) of the F200Y mutation were located in NW England. The F167Y SNP was identified, for the first time in this species, in four of the populations tested at a low frequency (1.2% ± 0.01), indicating the early emergence of the mutation. E198A or E198L were not identified in any of the isolates. Results obtained were comparable between both techniques for F200Y (Lins’ CCC, rc = 0.96) with discrepancies being limited to populations with low frequencies. The recent emergence of resistance in this species will provide a unique opportunity to study the early stages of anthelmintic resistance within a natural setting and track its progress in the future.

Assembly of root-associated microbial community of typical rice cultivars in different soil types

Root-associated microbial communities have been widely studied in the model plant Arabidopsis thaliana, but have been much less explored in agronomically important crops. Here, we used deep pyrosequencing of bacterial 16S rRNA to identify and characterize the root-associated microbial community of three traditional rice cultivars (representing indica and japonica subspecies and a hybrid) cultivated in typical paddy soils from China. We separated the root microbiota into endosphere and rhizosphere compartments, which were found to be the major determinant of variation in the total root microbial community. The microbial communities in each rhizocompartment were strongly influenced by soil type, while rice genotype exhibited a small but significant influence on both endosphere and rhizosphere microbiota. Pairwise comparisons showed that the endophytic bacterial community of indica rice differed significantly from that of japonica rice, while no remarkable differences were noted when comparing the community of indica vs. hybrid or japonica vs. hybrid. A core functional rice endophytic microbiota was identified, which accounted for relative abundances of up to 72.5% of the total microbial community. The 88 core root operational taxonomic units (OTUs) mainly belonged to the phylum Proteobacteria specifically Alphaproteobacteria, Betaproteobacteria, and Deltaproteobacteria. These results clarify the rice root-associated microbial community assemblage and facilitate the construction of an artificial core root microbiota to promote plant growth and resistance.

Analysis of hepatitis B virus-mixed genotype infection by ultra deep pyrosequencing in Sudanese patients, 2015-2016.

The frequency of detection of HBV co-infection with multiple HBV genotypes is influenced by the detection method; usually co-infections are detected by multiplex PCR or hybridization assays, and are rarely confirmed by sequencing and conventional cloning. The objective of this study was to confirm by ultra-deep pyrosequencing (UDPS) mixed HBV infections, previously detected by multiplex-nested PCR. Sixteen samples from HBV co-infected Sudanese patients detected by multiplex-nested PCR, were amplified targeting the P/S region and sequenced by UDPS. The only genotypes identified using UDPS were D and E, while A, B, C and F genotypes, previously detected by multiplex-nested PCR, were not detected. Specifically, 10 samples were shown to be mono-infected (D or E); in 3 out of 10 mono-infected D patients, a subtype combination was observed: D1 + D7 in 2 cases and D2 + D6 in 1 case. The remaining 6 subjects were D + E co-infected (harboring different mixtures of D subtypes). Overall, UDPS is more effective than multiplex-nested PCR for identifying multiple HBV genotypes and subtypes infections.

Hepatitis C virus intrinsic molecular determinants may contribute to the development of cholestatic hepatitis after liver transplantation.

Cholestatic hepatitis C (CHC) is a severe form of hepatitis C virus (HCV) infection recurrence that leads to high graft loss rates early after liver transplantation (LT). To investigate the pathogenic mechanisms of CHC, we analysed HCV quasispecies in CHC patients compared to a control group (mild hepatitis C recurrence) by deep pyrosequencing. At the time of LT, NS5B quasispecies complexity was similar between the two groups but, after LT, it decreased more sharply in CHC patients than in the control group. Interestingly, the major variant before LT propagated efficiently and remained as the dominant sequence after LT in 62 % of CHC patients versus 11 % of controls (P=0.031). Sequence analysis of the complete non-structural region in a limited number of patients revealed a potential 12 aa signature specific to the CHC group. These data suggest that intrinsic molecular determinants in the circulating HCV quasispecies may provide a fitness advantage, contributing to the development of CHC.

Assessment of Transmitted HIV-1 Drug Resistance Mutations Using Ultra- Deep Pyrosequencing in a Turkish Cohort.

Antiretroviral treatment (ART) reduces morbidity and mortality caused by human immunodeficiency virus (HIV) infection; however, the emergence of drug-resistant strains poses an important obstacle to treatment success. Using conventional sequencing methods to determine antiretroviral resistance, mutations present in ≥20% of quasispecies can be identified, but drug-resistant minority variants can lead to virologic failure. We aimed to assess transmitted drug resistance mutations (TDRMs) within minority variants using ultra-deep pyrosequencing (UDPS). Treatment-naive adult patients were included in this observational study. Surveillance TDRMs were classified as ≥20% or at minority variant level (≥2% - <20%). Genotypic sensitivity score calculated by using all pre-treatment drug resistance mutations (PDRMs) was also evaluated. Thirty-six patients were analyzed. Any TDRM at ≥20% level was detected in 8.3% of the patients (n=3). This prevalence increased to 30.6% (n=11) with the inclusion of minority variants. All non-nucleoside reverse transcriptase inhibitor and protease inhibitor-related TDRMs were within minority variants. The genotypic sensitivity score of rilpivirine-based regimens was considerably diminished when minority variants were included in the PDRM analysis. UDPS was used for the first time to assess TDRM in a Turkish HIV cohort and uncovered several mutations hidden within minority variants. UDPS may be preferred to detect PDRMs for avoiding virologic failure with rilpivirine-based ART regimens.

SYK expression in monomorphic epitheliotropic intestinal T-cell lymphoma

Monomorphic epitheliotropic intestinal T-cell lymphoma (MEITL), formerly known as type II enteropathy associated T-cell lymphoma (type II EATL), is a rare, aggressive primary intestinal T-cell lymphoma with a poor prognosis and an incompletely understood pathogenesis. We collected 40 cases of MEITL and 27 cases of EATL, formerly known as type I EATL, and comparatively investigated the T-cell receptor (TCR) itself and associated signaling molecules using immunohistochemistry, amplicon deep sequencing and bisulfite pyrosequencing. The TCR showed both an αβ-T-cell origin (30%) and a γδ-T-cell derivation (55%) resulting in a predominant positive TCR phenotype in MEITL compared with the mainly silent TCR phenotype in EATL (65%). The immunohistochemical expression of the spleen tyrosine kinase (SYK) turned out to be a distinctive feature of MEITL (95%) compared with EATL (0%). Aberrant SYK overexpression in MEITL is likely caused by hypomethylation of the SYK promoter, while no common mutations in the SYK gene or in its promoter could be detected. Using amplicon deep sequencing, mutations in DNMT3A, IDH2, and TET2 were infrequent events in MEITL and EATL. Immunohistochemical expression of linker for activation of T-cells (LAT) subdivided MEITL into a LAT expressing subset (33%) and a LAT silent subset (67%) with a potentially earlier disease onset in LAT-positive MEITL.

Prenatal Exposure to Bisphenol A Disrupts Naturally Occurring Bimodal DNA Methylation at Proximal Promoter of fggy, an Obesity-Relevant Gene Encoding a Carbohydrate Kinase, in Gonadal White Adipose Tissues of CD-1 Mice

Exposure of mammalian fetuses to endocrine disruptors can increase the risk of adult-onset diseases. We previously showed that exposure of mouse fetuses to bisphenol A (BPA) caused adult-onset obesity. To examine roles of epigenetic changes in this delayed toxicity, we determined the effects of fetal mouse exposure to BPA on genome-wide DNA methylation and messenger RNA (mRNA) expression in gonadal white adipose tissues (WATs) by deep sequencing, bisulfite pyrosequencing, and real-time quantitative polymerase chain reaction. Pregnant CD-1 mice (F0) were dosed daily with 0, 5, or 500 μg/kg/d BPA during gestational days 9 to 18, and the weaned F1 animals were fed ad libitum with standard chow until they were euthanized at 19 weeks old. In the vehicle-exposed F1 animals, fggy promoter showed a clear bimodal pattern of very strong (55% to 95%) or very weak (5% to 30%) DNA methylation occurring at nearly equal incidence with no intermediate strength. Promoter hypermethylation completely suppressed mRNA expression. BPA exposure eliminated this naturally occurring dichotomy, shifting fggy promoter toward the hypomethylation state to release transcriptional suppression. The strength of Fggy mRNA expression significantly correlated with increased whole body weight and gonadal fat weight of males but not females. Bioinformatics studies showed that expression of Fggy mRNA is stronger in mouse WATs than in brown adipose tissues and enhanced in gonadal fat by diet-induced obesity. These observations suggest that prenatal exposure to BPA may disrupt the physiological bimodal nature of epigenetic regulation of fggy in mouse WATs, possibly contributing to the adult-onset obesity phenotype.

Insights into the Regulation of Rhizosphere Bacterial Communities by Application of Bio-organic Fertilizer in Pseudostellaria heterophylla Monoculture Regime.

The biomass and quality of Pseudostellariae heterophylla suffers a significant decline under monoculture. Since rhizosphere miobiome plays crucial roles in soil health, deep pyrosequencing combined with qPCR was applied to characterize the composition and structure of soil bacterial community under monoculture and different amendments. The results showed compared with the 1st-year planted (FP), 2nd-year monoculture of P. heterophylla (SP) led to a significant decline in yield and resulted in a significant increase in Fusarium oxysporum but a decline in Burkholderia spp. Bio-organic fertilizer (MT) formulated by combining antagonistic bacteria with organic matter could significantly promote the yield by regulating rhizosphere bacterial community. However, organic fertilizer (MO) without antagonistic bacteria could not suppress Fusarium wilt. Multivariate statistics analysis showed a distinct separation between the healthy samples (FP and MT) and the unhealthy samples (SP and MO), suggesting a strong relationship between soil microbial community and plant performance. Furthermore, we found the application of bio-organic fertilizer MT could significantly increase the bacterial community diversity and restructure microbial community with relatively fewer pathogenic F. oxysporum and more beneficial Burkholderia spp. In conclusion, the application of novel bio-organic fertilizer could effectively suppress Fusarium wilt by enriching the antagonistic bacteria and enhancing the bacterial diversity.

Incorporating Cutaneous and Wound Bacterial Bioburden Biomarkers into Clinical Research: A Review of Best Practices.

To provide information about initiating interdisciplinary research related to microbiomes and their role in human immunity, disease, and metabolic processes. This continuing education activity is intended for physicians and nurses with an interest in skin and wound care. After participating in this educational activity, the participant should be better able to:1. Describe techniques to identify and characterize bacterial bioburden.2. Identify optimal collection, transport, and storage of samples. The purpose of this review is to provide a roadmap for clinical scientists interested in integrating bacterial bioburden (BB) biomarkers into the next generation of cutaneous or wound disease research studies. Complex relationships exist between humans and their microbiome. Until now, clinical scientists have been limited in fully characterizing relationships between humans and their microbiome. Recent technological innovations, such as next-generation DNA sequencing, also known as deep sequencing or pyrosequencing, have enhanced clinicians' capacity to identify, characterize, and elucidate the role of BB (ie, bacterial load, diversity, pathogenicity) in human immunity, disease, and metabolic processes. The understanding of common terminology, intervening variables that influence BB, limitations of next-generation DNA sequencing, and specimen selection, collection, transport, and storage practices are needed to support interdisciplinary communication, research design, and integrity of the specimen. This review serves as a primer for building foundational knowledge in microbiome research, which will aid clinical scientists with initiating interdisciplinary communication necessary for scientific team building.

Probing the Rare Biosphere of the North-West Mediterranean Sea: An Experiment with High Sequencing Effort.

High-throughput sequencing (HTS) techniques have suggested the existence of a wealth of species with very low relative abundance: the rare biosphere. We attempted to exhaustively map this rare biosphere in two water samples by performing an exceptionally deep pyrosequencing analysis (~500,000 final reads per sample). Species data were derived by a 97% identity criterion and various parametric distributions were fitted to the observed counts. Using the best-fitting Sichel distribution we estimate a total species richness of 1,568–1,669 (95% Credible Interval) and 5,027–5,196 for surface and deep water samples respectively, implying that 84–89% of the total richness in those two samples was sequenced, and we predict that a quadrupling of the present sequencing effort would suffice to observe 90% of the total richness in both samples. Comparing the HTS results with a culturing approach we found that most of the cultured taxa were not obtained by HTS, despite the high sequencing effort. Culturing therefore remains a useful tool for uncovering marine bacterial diversity, in addition to its other uses for studying the ecology of marine bacteria.

Effects of consecutive monoculture of Pseudostellaria heterophylla on soil fungal community as determined by pyrosequencing.

Under consecutive monoculture, the biomass and quality of Pseudostellaria heterophylla declines significantly. In this study, a three-year field experiment was conducted to identify typical growth inhibition effects caused by extended monoculturing of P. heterophylla. Deep pyrosequencing was used to examine changes in the structure and composition of soil fungal community along a three-year gradient of monoculture. The results revealed a distinct separation between the newly planted plot and the two-year, three-year monocultured plots. The Shannon and Simpson diversity indices were significantly higher in the two-year and three-year monoculture soils than in the newly planted soil. Consecutive monoculture of this plant led to a significant increase in relative abundance of Fusarium, Trichocladium and Myrothecium and Simplicillium, etc., but a significant decrease in the relative abundance of Penicillium. Quantitative PCR analysis confirmed a significant increase in Fusarium oxysporum, an agent known to cause wilt and rot disease of P. heterophylla. Furthermore, phenolic acid mixture at a ratio similar to that found in the rhizosphere could promote mycelial growth of pathogenic F. oxysporum. Overall, this study demonstrated that consecutive monoculture of P. heterophylla can alter the fungal community in the rhizosphere, including enrichment of host-specific pathogenic fungi at the expense of plant-beneficial fungi.

Comparison of Fungal Community in Black Pepper-Vanilla and Vanilla Monoculture Systems Associated with Vanilla Fusarium Wilt Disease.

Long-term vanilla monocropping often results in the occurrence of vanilla Fusarium wilt disease, seriously affecting its production all over the world. In the present study, vanilla exhibited significantly less Fusarium wilt disease in the soil of a long-term continuously cropped black pepper orchard. The entire fungal communities of bulk and rhizosphere soils between the black pepper-vanilla system (i.e., vanilla cropped in the soil of a continuously cropped black pepper orchard) and vanilla monoculture system were compared through the deep pyrosequencing. The results showed that the black pepper-vanilla system revealed a significantly higher fungal diversity than the vanilla monoculture system in both bulk and rhizosphere soils. The UniFrac-weighted PCoA analysis revealed significant differences in bulk soil fungal community structures between the two cropping systems, and fungal community structures were seriously affected by the vanilla root system. In summary, the black pepper-vanilla system harbored a lower abundance of Fusarium oxysporum in the vanilla rhizosphere soil and increased the putatively plant-beneficial fungal groups such as Trichoderma and Penicillium genus, which could explain the healthy growth of vanilla in the soil of the long-term continuously cropped black pepper field. Thus, cropping vanilla in the soil of continuously cropped black pepper fields for maintaining the vanilla industry is executable and meaningful as an agro-ecological system.

A Follow-Up of the Multicenter Collaborative Study on HIV-1 Drug Resistance and Tropism Testing Using 454 Ultra Deep Pyrosequencing.

BackgroundUltra deep sequencing is of increasing use not only in research but also in diagnostics. For implementation of ultra deep sequencing assays in clinical laboratories for routine diagnostics, intra- and inter-laboratory testing are of the utmost importance.MethodsA multicenter study was conducted to validate an updated assay design for 454 Life Sciences’ GS FLX Titanium system targeting protease/reverse transcriptase (RTP) and env (V3) regions to identify HIV-1 drug-resistance mutations and determine co-receptor use with high sensitivity. The study included 30 HIV-1 subtype B and 6 subtype non-B samples with viral titers (VT) of 3,940–447,400 copies/mL, two dilution series (52,129–1,340 and 25,130–734 copies/mL), and triplicate samples. Amplicons spanning PR codons 10–99, RT codons 1–251 and the entire V3 region were generated using barcoded primers. Analysis was performed using the GS Amplicon Variant Analyzer and geno2pheno for tropism. For comparison, population sequencing was performed using the ViroSeq HIV-1 genotyping system.ResultsThe median sequencing depth across the 11 sites was 1,829 reads per position for RTP (IQR 592–3,488) and 2,410 for V3 (IQR 786–3,695). 10 preselected drug resistant variants were measured across sites and showed high inter-laboratory correlation across all sites with data (P<0.001). The triplicate samples of a plasmid mixture confirmed the high inter-laboratory consistency (mean% ± stdev: 4.6 ±0.5, 4.8 ±0.4, 4.9 ±0.3) and revealed good intra-laboratory consistency (mean% range ± stdev range: 4.2–5.2 ± 0.04–0.65). In the two dilutions series, no variants >20% were missed, variants 2–10% were detected at most sites (even at low VT), and variants 1–2% were detected by some sites. All mutations detected by population sequencing were also detected by UDS.ConclusionsThis assay design results in an accurate and reproducible approach to analyze HIV-1 mutant spectra, even at variant frequencies well below those routinely detectable by population sequencing.

Application of Deep Pyrosequencing to the Analysis of Soil Microbial Communities in Different Lotus Fields

Fusarium oxyporum f. sp. nelumbicola is a very important plant pathogen that can cause lotus Fusarium wilt and lead to vast economic loss in Jiangxi Province, China. Sequencing data of 16S ribosomal RNA genes and internal transcribed spacer regions revealed that different bacterial and fungal communities were present in soil samples from healthy or Fusarium wilt-infected lotus fields with different years of continuous cropping, indicating that the long-term continuous cropping of lotus significantly altered the soil microbial communities. Fungal diversity indexes in the healthy soil samples increased with the years of monoculture, but decreased in the Fusarium wilt-infected soil samples, whereas bacterial community diversity in all the soil samples was relatively stable. A cluster analysis revealed that consecutive cropping time was a key factor for determining fungal community structure, but not for bacterial community structure. The relative abundances of Proteobacteria, Acidobacterial, Ascomycota, and Basidiomycota phyla decreased with the years of lotus monoculture. Compared with the healthy soil samples, the abundances of beneficial bacteria in the Fusarium wilt-infected soil samples significantly decreased with time. These observations indicate that soil weakness and lotus Fusarium wilt disease may be attributed to structural changes of soil microbial communities after long-term continuous cropping, and the changes include reduction of beneficial microorganisms and accumulation of fungal pathogens.

Application of Deep Pyrosequencing to the Analysis of Soil Microbial Communities in Different Lotus Fields

Application of Deep Pyrosequencing to the Analysis of Soil Microbial Communities in Different Lotus Fields

Implementation of Next-Generation Sequencing for Hepatitis B Virus Resistance Testing and Genotyping in a Clinical Microbiology Laboratory.

Sanger sequencing or DNA hybridization have been the primary modalities for hepatitis B (HBV) resistance testing and genotyping; however, there are limitations, such as low sensitivity and the inability to detect novel mutations. Next-generation sequencing (NGS) for HBV can overcome these limitations, but there is limited guidance for clinical microbiology laboratories to validate this novel technology. In this study, we describe an approach to implementing deep pyrosequencing for HBV resistance testing and genotyping in a clinical virology laboratory. A nested PCR targeting the pol region of HBV (codons 143 to 281) was developed, and the PCR product was sequenced by the 454 Junior (Roche). Interpretation was performed by ABL TherapyEdge based on European Association for the Study of the Liver (EASL) guidelines. Previously characterized HBV samples by INNO-LiPA (LiPA) were compared to NGS with discordant results arbitrated by Sanger sequencing. Genotyping of 105 distinct samples revealed a concordance of 95.2% (100/105), with Sanger sequencing confirming the NGS result. Resistance testing by NGS was concordant with LiPA in 85% (68/80) of previously characterized samples. Additional mutations were found in 8 samples, which related to the identification of low-level mutant subpopulations present at <10% (6/8). To balance the costs of testing for the validation study, reproducibility of the NGS was investigated through an analysis of sequence variants at loci not associated with resistance in a single patient sample. Our validation approach attempts to balance costs with efficient data acquisition.

Cloacal Microbiome Structure in a Long-Distance Migratory Bird Assessed Using Deep 16sRNA Pyrosequencing.

Effects of vertebrate-associated microbiota on physiology and health are of significant interest in current biological research. Most previous studies have focused on host-microbiota interactions in captive-bred mammalian models. These interactions and their outcomes are still relatively understudied, however, in wild populations and non-mammalian taxa. Using deep pyrosequencing, we described the cloacal microbiome (CM) composition in free living barn swallows Hirundo rustica, a long-distance migratory passerine bird. Barn swallow CM was dominated by bacteria of the Actinobacteria, Proteobacteria and Firmicutes phyla. Bacteroidetes, which represent an important proportion of the digestive tract microbiome in many vertebrate species, was relatively rare in barn swallow CM (< 5%). CM composition did not differ between males and females. A significant correlation of CM within breeding pair members is consistent with the hypothesis that cloacal contact during within-pair copulation may promote transfer of bacterial assemblages. This effect on CM composition had a relatively low effect size, however, possibly due to the species’ high level of sexual promiscuity.

Rhizosphere microbial community manipulated by 2 years of consecutive biofertilizer application associated with banana Fusarium wilt disease suppression

In our previous work, applying biofertilizer containing Bacillus amyloliquefaciens strain NJN-6 to a banana orchard infected by a serious Fusarium wilt disease over two consecutive years effectively controlled this soil-borne disease. In this study, deep pyrosequencing of 16S ribosomal RNA (rRNA) genes and internal transcribed spacer (ITS) sequences was performed to investigate how the composition of rhizosphere microbial community responded to the application of biofertilizer (BIO), pig manure compost (PM), and chemical fertilizer (CF) and to explore the potential correlation between the microbial community composition and the Fusarium wilt disease. A total of 104,201 bacterial 16S rRNA genes and 154,953 fungal ITS sequence reads were obtained after basic quality control, and Acidobacteria, Actinobacteria, Bacteroidetes, Firmicutes, Proteobacteria, and Ascomycota were the most abundant bacterial and fungal phyla across all samples. Compared with the PM and CF control, the alpha diversity of bacteria significantly (P < 0.05) increased, whereas the value of the fungi was significantly (P < 0.05) reduced following two consecutive years of biofertilizer application. Moreover, the abundance of Acidobacteria (Gp1 and Gp3), Firmicutes, Leptosphaeria, and Phaeosphaeriopsis was significantly (P < 0.05) increased, while the abundance of Proteobacteria and Ascomycota was significantly (P < 0.05) decreased in the BIO treatment. Furthermore, the abundance of Fusarium, a causal pathogen for Fusarium wilt disease, was significantly (P < 0.05) reduced in the BIO treatment compared with the CF control and was slightly reduced (not significant) compared with the PM control. Interestingly, the disease incidence was negatively correlated with the enriched taxa of Acidobacteria (Gp1 and Gp3) and Firmicutes, Leptosphaeria, and Phaeosphaeriopsis but positively correlated with abundance of Proteobacteria, Ascomycota, Fusarium, Cylindrocarpon, Gymnascella, Monographella, Pochonia, and Sakaguchia taxa. The results from this study suggest that 2 years of biofertilizer application manipulated the composition of rhizosphere microbial community and induced the Fusarium suppression by increasing bacterial diversity and potentially stimulating microbial consortia taxa, such as Acidobacteria (Gp1 and Gp3), Firmicutes, Leptosphaeria, and Phaeosphaeriopsis.

Dynamics of HCV genotype 4 resistance-associated variants during virologic escape with pIFN/RBV + daclatasvir: A case study using ultra deep pyrosequencing

BackgroundDaclatasvir (DCV) is an approved NS5A inhibitor with potent anti-HCV activity and broad genotype coverage. DCV resistance-associated variants (RAVs) have been described for patients infected with genotype (GT) 1, but increased GT4 prevalence in European countries as a result of immigration has boosted interest in this genotype. ObjectivesEstablishment of NS5A variability in treatment-naive patients with HCV genotype 4 infection and a case study of the dynamics of resistance-associated variants in a virologic failure receiving pIFN/RBV+DCV, as assessed by ultra-deep sequencing. Study designFive treatment-naïve GT4 patients (GT4a [n=1], GT4d [n=3], GT4o [n=1]) were evaluated for inclusion in the COMMAND-4 study and treatment with pIFN/RBV±DCV. ResultsPatient (Pt) 1 received pIFN/RBV; Pts2–4 received pIFN/RBV + DCV; Pt5 was a screening failure. Pt1 relapsed; Pt2 experienced breakthrough at Wk4; Pts3 and 4 achieved a sustained virologic response. No substitutions associated with DCV-resistance were detected at baseline. In terms of viremic time points for Pts1 and 2, the extent of NS5A diversity pre-treatment was not significantly related to viral load (r = −0568; p = 0.035). In Pt2, multiple substitutions associated with DCV-resistance were observed after breakthrough at NS5A amino acid positions 28, 31 and 93. These substitutions were frequently observed on the same haplotype (L28S + M31I = 55.52, 82.50, and 99.36% at Wk4, 8 and 9; L28S + M31I + Y93H = 11.77, 5.01 and <0.6% at Wk4, 8 and 9). ConclusionsThis is the first report to describe DCV-resistance in patients infected with GT4d, supporting a possible role for a recently described RAV (L28S), and presenting the dynamics of HCV quasispecies during therapy failure, with indications of changes of diversity and association of mutations.