High-throughput Gene Sequencing Research Articles

BRCA mutation status and pathological characterization of breast cancer in Zhoushan Islands, China.

To investigate BRCA1/2 gene mutations and their relationship with clinicopathological features in patients with breast cancer in Zhoushan Islands. High-throughput whole-exome gene sequencing was used to detect BRCA1/2 mutations in 776 breast cancer patients in Zhoushan Islands. The BRCA1/2 mutation rate of breast cancer patients in Zhoushan Islands was 4.38% (34/776). BRCA1 mutations were significantly correlated with age, molecular type, and family history of breast and ovarian cancers. BRCA2 mutations were most commonly found in invasive lobular carcinoma. Moreover, the BRCA2 mutation rate of cancers with molecular type luminal B (receptor protein-tyrosine kinase [HER2]-negative) was also relatively high. The rate of BRCA1/2 mutations in breast cancer patients from Zhoushan Islands is approximately 4.38%, and BRCA1 mutation is related to age, molecular type, and family history of breast and ovarian cancers.

Analysis of the correlation between cervical HPV infection, cervical lesions and vaginal microecology.

Vaginal microbiota is involved in human papillomavirus (HPV) infection and cervical cancer (CC) progression, and the specific changes in vaginal microbial composition during this process remains uncertain. This study aimed to observe the changes in the specific composition of vaginal microorganisms in different cervical lesions and identify biomarkers at different stages of lesions. In this study we used the illumina high-throughput gene sequencing technology to determine the V4 region of 16SrRNA and observed the vaginal microbial composition in different cervical lesions. The vaginal microbiota of patients with high-risk HPV infection and cervical lesions is significantly different from that of the normal population, but there is no significant difference in the richness of vaginal microbes. The diversity of vaginal species in CC patients is higher than that in high-risk HPV infection or CIN patients. The main manifestation is an increase in the diversity of vaginal microbes, a decrease in the relative abundance of cyanobacteria and Lactobacillus, and an increase in the relative abundance of dialister, peptonephila and other miscellaneous bacteria. There are characteristic vaginal biomarker in normal women, high risk HPV patients and CC patients. In detail, the biomarker in the normal group was varibaculum, the biomarker in the high-risk HPV group was saccharopolyspora, the biomarker of the CC group was the Proteobacteria, Corynebacterium, Coprococcus, Peptococcus and Ruminococcus. The study indicated that the compositions of vaginal microbes in different cervical lesions is different. The vaginal microbial composition has a certain diagnostic effect on healthy women, patients with high-risk HPV infection and cervical lesions. These microbes may serve as potential biomarkers for CC. It also provided an effective way for the treatment of HPV infections and cervical lesions.

Seasonal dynamics of free-living (FL) and particle-attached (PA) bacterial communities in a plateau reservoir.

In terms of lifestyle, bacterioplankton can be classified as free-living (FL) and particle-attached (PA) forms, and both play essential roles in biogeochemical cycling in aquatic ecosystems. Structure, distribution, and community assembly of FL and PA bacteria in plateau riverine waterbodies are largely unknown. Therefore, we explored the seasonal dynamics of FLand PA bacterial communities in the Wujiangdu reservoir, Yungui Plateau using 16S rRNA gene high-throughput sequencing. Results revealed there was a significant environmental heterogeneity in Wujiangdu reservoir seasonally. The dominant phylum was Actinomycetota for FL and Pseudomonadota for PA bacteria. Species richness and diversity was higher in autumn and winter compared to spring and summer. In general, PA diversity was greater than FL, but with some temporal variations. Species turnover was the major contributor to β-diversity of both FL and PA lifestyles, and significant differences were noticed between FL and PA bacterial community composition. Distinct co-occurrence network patterns implied that more connections exist between FL bacteria, while more complex PA networks were in parallel to their greater diversity and stronger interactions in biofilms on particles. Dispersal limitation was the major driving force for both FL and PA bacterial community assembly. Deterministic processes were of relatively low importance, with homogeneous selection for FL and heterogeneous selection for PA bacteria. Temperature was the most important environmental driver of seasonal bacterial dynamics, followed by nitrate for FL and Secchi depth for PA bacteria. This study allows for a better understanding of the temporal variability of different bacteria lifestyles in reservoirs in the vulnerable and rapidly changing plateau environment, facilitating further microbial research related to global warming and eutrophication.

Soil Microbial Communities in Pseudotsuga sinensis Forests with Different Degrees of Rocky Desertification in the Karst Region, Southwest China

Rocky desertification (RD), a natural and human-induced process of land degradation in karst areas, has become the primary ecological disaster and one of the obstacles to sustainable ecological development in southwest China. Nevertheless, the variation of soil physical and chemical properties, bacterial and fungal communities, and their relationships in RD forests remains limited. Therefore, soil samples were collected from forests under four degrees of RD (NRD, non-RD; LRD, light RD; MRD, moderate RD; and SRD, severe RD) and subjected to high-throughput sequencing of 16S rRNA and ITS1 genes. The results showed a significant reduction in bacterial richness and diversity, while fungal richness and diversity decreased markedly and then showed a balanced trend with the increase in RD degree, indicating that bacteria and fungi did not present the same dynamics in response to the process of RD. The bacterial communities were dominated by Proteobacteria, Actinobacteria, Acidobacteria, and Chloroflexi, while the fungal communities were dominated by Basidiomycota, Ascomycota, and Mortierellomycota. The PCoA and NMDS demonstrated significant differences in microbial communities in study sites, among which the fungal communities in non-RD forest and LRD forest clustered together, suggesting that fungal communities were more stable than bacteria in RD forest. The db-RDA, Mantel test, and random forest model confirmed the important role of soil BD, pH, SOC, AN, and AP in driving microbial diversity and communities. The IndVal analysis suggested that Chloroflexi, Patescibacteria, Atheliales, and Cantharellales with high indicator values were identified as potential bio-indicators for RD forests. This study could not only improve our understanding of bacterial and fungal community dynamics across RD gradients, but also could provide useful information for the further use of microorganisms as indicators to reflect the environmental changes and ecosystem status during forest RD.

Succession of the bacterial community from a spacecraft assembly clean room when enriched in brines relevant to Mars

Abstract Interplanetary spacecraft are built in a spacecraft assembly facility (SAF), a clean room designed to reduce microbial contamination that could confound life detection missions or influence native ecosystems. The frigid hyperarid near-surface environment of Mars has ample hygroscopic Mg and Na salts of chloride, (per)chlorate and sulphate that may deliquesce to form dense brines, liquids with low water activity, and freezing points <0°C. The current study sought to define the climax microbial community after 6 mo of enrichment of SAF floor wipe samples in salt plains medium supplemented with 50% (w/v; ~2 M; aw = 0.94) MgSO4 or 20% (w/v; ~1.9 M; aw = 0.91) NaClO3. After 1 wk, 4 wk and 6 mo of incubation, metagenomic DNA extracts of the enriched SAF microbial community were used for high-throughput sequencing of 16S rRNA genes and subsequent phylogenetic analyses. Additionally, dozens of bacterial strains were isolated by repetitive streak-plating from the climax community after 6 mo of enrichment. Early in the enrichment, staphylococci greatly dominated and then remained abundant members of the community. However, actinobacteria succeeded the staphylococci as the dominant taxa as the cultures matured, including Arthrobacter, Brachybacterium and Brevibacterium. A diverse assemblage of bacilli was present, with Oceanobacillus being especially abundant. The SAF culture collection included representatives of Brachybacterium conglomeratum, Brevibacterium sediminis, Oceanobacillus picturae and Staphylococcus sciuri. These were characterized with biochemical and physiological tests, revealing their high salinotolerance. Shannon diversity indices were generally near 2, reflecting modest diversity at several levels of identity and the community structures were uneven throughout. However, minor members of the community seem capable of the ecosystem functions required for biogeochemical cycling. For instance, organisms capable of all the functions of the N cycle were detected. The microbial assemblage in SAFs is the most likely to be transported by spacecraft to another world. While individual microbial populations may exhibit the qualities needed for survival at the near-surface of Mars, certainly entire communities with the capacity for complete biogeochemical cycling, would have a greater chance of survival and proliferation.

Analysis of the microbial diversity in takin (Budorcas taxicolor) feces.

The intestinal tract of animals is a complex and dynamic microecosystem that is inextricably linked to the health of the host organism. Takin (Budorcas taxicolor) is a threatened species, and its gut microbiome is poorly understood. Therefore, this study aimed to analyze the microbial community structure and potential pathogens of takin. Takin fecal samples were collected from five sites in a nature reserve to ensure the uniformity of sample collection, determine the effects of different geographical locations on gut microbes, and analyze the differences in microbial communities between sites. Subsequently, high-throughput 16S rDNA gene sequencing was performed to analyze the microbial diversity and potential pathogens in the gut; the findings were verified by isolating and culturing bacteria and metagenomic sequencing. The takin gut microflora consisted mainly of four phyla: Firmicutes (69.72%), Bacteroidota (13.55%), Proteobacteria (9.02%), and Verrucomicrobiota (3.77%), representing 96.07% of all microorganisms. The main genera were UCG-005 (20.25%), UCG-010_unclassified (12.35%), Firmicus_unclassified (4.03%), and Rumino coccsea_unclassified (3.49%), while the main species were assigned to Bacteria_unclassified. Potential pathogens were also detected, which could be used as a reference for the protection of takin. Pseudomonas presented the highest abundance at Shuichiping and may represent the main pathogen responsible for the death of takin at the site. This study provides an important reference for investigating the composition of the bacterial community in the intestine of takin.

Characteristics of bacterial and fungal communities and their associations with sugar compounds in atmospheric aerosols at a rural site in northern China

Abstract. Bioaerosols play significant roles in causing health and climate effects. Sugar compounds in air have been widely used to trace the source of bioaerosols. However, knowledge about the association of sugar molecules and the microbial community at taxonomic levels in atmospheric aerosols remains limited. Here, microbial community compositions and sugar molecules in total suspended particles collected from a typical rural site, Gucheng, in the North China Plain were investigated by gas chromatography–mass spectrometry and high-throughput gene sequencing, respectively. Results show that fungal community structure exhibited distinct diurnal variation with largely enhanced contribution of Basidiomycota at night, while bacterial community structure showed no obvious difference between daytime and night. SourceTracker analysis revealed that fungi and bacteria were mainly from plant leaves and unresolved sources (presumably human-related emissions and/or long-distance transport). All the detected anhydrosugars and sugar alcohols and trehalose showed diurnal variations with lower concentrations in the daytime and higher concentrations at night, which may be affected by enhanced fungal emissions at night, while primary sugars (except trehalose) showed an opposite trend. Mantel's test showed that more sugar compounds exhibited significant associations with fungal community structure than bacterial community structure. Co-occurrence analysis revealed the strong associations between sugar compounds and a few saprophytic fungal genera with low relative abundances, e.g., Hannaella, Lectera, Peniophora, Hydnophlebia, Sporobolomyces and Cyphellophora. This study suggests that the entire fungal community, rather than specific fungal taxa, likely greatly contributes to sugar compounds in rural aerosols, while the contribution of bacteria is limited.

The Response of Soil Bacterial Communities to Cropping Systems in Saline–Alkaline Soil in the Songnen Plain

The high salt content in saline–alkaline land leads to insufficient nutrients, thereby reducing agricultural productivity. This has sparked widespread interest in improving saline–alkaline soil. In this investigation, 16S rRNA gene high-throughput sequencing was employed to examine the impacts of three cropping systems (monoculture, rotation, and mixture) on soil bacterial communities. It was found that cropping rotations and mixtures significantly increased soil bacterial α-diversity. Random forest analysis showed a significant linear relationship between AK and EC and bacterial α-diversity. In addition, principal coordinates analysis (PCoA) further confirmed the significant differences in β-diversity between different soil layers. Through co-occurrence network analysis, it was found that cropping rotations and mixtures increased the stability and complexity of co-occurrence networks. By calculating NST to analyze the assembly process of soil bacterial communities in different cropping systems, it was found that the assembly process of soil bacterial communities was dominated by a stochastic process. Functional prediction results showed that a large number of C, N, and S cycling microbes appeared in soil bacterial communities. Our study aims to establish a fresh perspective on the improvement and recovery of saline–alkaline soil.

Effects of straw addition on biological nitrogen fixation under continuous and intermittent flooding irrigation in a rice cropping system

Both straw addition and water management affect biological nitrogen fixation (BNF), but it is still unclear how straw addition impacts BNF under continuous or intermittent flooding irrigation in a rice cropping system. A 15N2-labelling chamber system placed in a rice field was used to evaluate BNF under straw addition with continuous or intermittent flooding irrigation for 90 days. The nitrogenase reductase (nifH) gene and reverse transcription gene (cDNA) in the soil were amplified using Real-time polymerase chain reaction (qPCR), and nifH gene high-throughput sequencing was applied. The BNF in the straw addition treatment (14.0 g straw added to 1.40 kg soil) under continuous flooding (14.3 kg ha-1) was 116% higher than that under intermittent flooding (6.62 kg ha-1). Straw addition and continuous flooding showed significant synergistic effects on BNF and the gene copy numbers of nifH DNA and nifH cDNA. The increase in BNF was mainly due to the increase in the abundance of heterotrophic diazotrophs such as Desulfovibrio, Azonexus, and Azotobacter. These results indicate that straw addition under continuous flooding stimulates BNF relative to intermittent flooding, which may ultimately lead to a rapid enhancement of soil fertility.

Bacterial diversity of stingless bee honey in Yunnan, China: isolation and genome sequencing of a novel acid-resistant Lactobacillus pentosus (SYBC-MI) with probiotic and L. tryptophan producing potential via millet fermentation.

Stingless bee (Hymenoptera, Apidae, and Trigona) honey is a remarkable "miracle liquid" with a wide range of medical benefits for conditions including gastroenteritis, cataracts, and wound healing. Our study aimed to isolate, identify, and characterize acid-resistant Lactobacillus spp. from sour honey distributed in Yunnan, China. To assess the safety of an entirely novel Lactobacillus pentosus strain, S4 (OM618128), based on probiotic property evaluation and whole-genome sequencing analysis. A 16S rRNA gene high-throughput sequencing analysis showed that Lactobacillus was abundant at the genus level in sour honey. Seven Lactobacillus strains (viz. S1-7) were isolated from sour honey using a multiple-anaerobic culture enrichment method. One potential acid-resistant isolate, Lactobacillus sp. S4, was obtained after screening the seven Lactobacillus isolates, and it had the highest lactic acid production (17.62g/L), followed by Lactobacillus sp. S3 (17.07g/L). Phylogenetic and comparative analyses of conserved sequence regions have shown that all seven strains are phylogenetically located in the Lactobacillus pentosus sub-cluster. In L. pentosus SYBC-MI, there is a circular chromosome (3288615 bps) and 11,466 bps plasmids. GC content is 44.03%. The number of predicted genes is 3,129, with 16 rRNAs and 74 tRNAs present. During the fermentation of foxtail millet by seven Lactobacillus pentosus (S1-7) strains isolated from sour honey, a potential tryptophan accumulating isolate, Lactobacillus pentosus S4, was obtained, which could reach a maximum tryptophan content of 238.43mgL-1 that is 1.80 times the initial tryptophan content in the fermentation broth. This strain has strong acid tolerance, salt tolerance, and fermentation acid production abilities. This strain degrades nitrite at a rate of over 99%, and it has high probiotic potential as well. This project has established a solid foundation for further exploring the excellent lactic acid bacteria in sour honey. It is also investigating the key taxa and their role in the environment. According to the results of our studies, these LAB isolates provide a lot of potential for use in the future, as a source of probiotics for human, animals, and starter cultures for food applications.

Diversity of heterotrophic picoeukaryotes in the ice of the Kandalaksha Gulf (White Sea, Russia) based on rRNA gene high-throughput sequencing

Diversity of heterotrophic picoeukaryotes in the ice of the Kandalaksha Gulf (White Sea, Russia) based on rRNA gene high-throughput sequencing

Assessing the structure and diversity of fungal community in plant soil under different climatic and vegetation conditions

IntroductionUnderstanding microbial communities in diverse ecosystems is crucial for unraveling the intricate relationships among microorganisms, their environment, and ecosystem processes. In this study, we investigated differences in the fungal community structure and diversity in soils from two contrasting climatic and vegetation conditions: the Xinjiang western China plateau and the Fujian southeastern coastal province.MethodsA total of 36 soil samples collected from two climatic regions were subjected to high-throughput ITS gene sequencing for fungal community analysis. In conjunction soil physicochemical properties were assessed and compared. Analyses included an examination of the relationship of fungal community structure to environmental factors and functional profiling of the community structure was using the FUNGuild pipeline.ResultsOur data revealed rich fungal diversity, with a total of 11 fungal phyla, 31 classes, 86 orders, 200 families, 388 genera, and 515 species identified in the soil samples. Distinct variations in the physicochemical properties of the soil and fungal community structure were seen in relation to climate and surface vegetation. Notably, despite a colder climate, the rhizosphere soil of Xinjiang exhibited higher fungal (α-)diversity compared to the rhizosphere soil of Fujian. β-diversity analyses indicated that soil heterogeneity and differences in fungal community structure were primarily influenced by spatial distance limitations and vegetation type. Furthermore, we identified dominant fungal phyla with significant roles in energy cycling and organic matter degradation, including members of the Sordariomycetes, Leotiomycetes, Archaeosporomycetes, and Agaricomycetes. Functional analyses of soil fungal communities highlighted distinct microbial ecological functions in Xinjiang and Fujian soils. Xinjiang soil was characterized by a focus on wood and plant saprotrophy, and endophytes, whereas in Fujian soil the fungal community was mainly associated with ectomycorrhizal interactions, fungal parasitism, and wood saprotrophy.DiscussionOur findings suggest fungal communities in different climatic conditions adapt along distinct patterns with, plants to cope with environmental stress and contribute significantly to energy metabolism and material cycling within soil-plant systems. This study provides valuable insights into the ecological diversity of fungal communities driven by geological and environmental factors.

Deciphering the Structural Variants in B Cell Acute Lymphoblastic Leukemia By Optical Genome Mapping

Introduction Structural variants (SVs) of the genome play a key role in the leukemogenesis and development of B cell lymphoblastic leukemia (B-ALL), including gene deletion, amplification, translocation, and other forms. Methods for analyzing SVs and CNVs include karyotype analysis, fluorescence in situ hybridization, gene chips, and high-throughput gene sequencing technologies. Optical genome mapping (OGM) is a novel ultra-long single-molecule DNA detection based high throughput SVs analysis technology, which has the characteristics of whole genome coverage, high detection incidence and sensitivity. There were few publications presenting technical concordance of SVs detection beween karyotyping and OGM, and there are no systematic studies comparing the concordance of OGM and RNA-seq in detecting gene fusion. This study aimed to compare the concordance of OGM and RNA-seq in the detection of fusion genes in B-ALL cases, and the additional findings using OGM technology. Methods Bone marrow aspirates of 70 patients with newly diagnosed or relapsed/refractory B-ALL were recruited into the study. All samples were conducted with both OGM and transcriptome sequencing (RNA-seq). Among these cases, 42 males and 28 females, 30 pediatric (< 18 years) and 40 adults (≥ 18 years) cases, ages ranged 4-60 years (median 16 years). Results Totally 50 gene fusions were reported in 48 cases, with 2 cases each carrying two fusion genes ( BCR::ABL1/ PAX5::ELK3 and P2RY8::CRLF2/ PAX5::GSE1, respectively) (Figure 1A). OGM and RNA-seq have complete concordance in reporting gene fusions, and OGM provides further insight into the genomic events that lead to gene fusion. A subtype of B-ALL characterized by UBTF microdeletion leading to UBTF::ATXN7L3 fusion and PAN3 downstream deletion-induced CDX2 aberrant expression has been reported recently, and has been accepted by the International Consensus Classification as a novel molecular subtype of UBTF/CDX2 of B-ALL. We performed the OGM investigation on 6 UBTF/CDX2 cases which were identified by RNA-seq. OGM identified both microdeletion events in all 6 cases, and further reported 1q amplification in 3 of them (Figure 1B, C). B-lymphocyte differentiation-associated transcription factor gene defects are common and important genetic abnormalities in B-ALL, often manifested as complete or partial deletion of genes. We focused on a panel including 30 genes in the OGM data and determined IKZF1 deletion in 35 patients, CDKN2A/B deletion in 32 patients, and PAX5 deletion in 25 patients (Figure 1 A). Follow-up data showed that patients with CDKN2A/B deletion had a worse prognosis than those without CDKN2A/B deletion ( P = 0.025). Conclusion This is the first B-ALL cohort to systematically compare OGM and RNA-seq in gene fusion detection, and the result showed complete consistency. RNA-seq has significant advantages in analyzing gene fusion transcripts, while OGM has technical advantages in analyzing SVs at the genome level. Combining the two technologies can accurately analyze gene fusion and other SVs in B-ALL, significantly improve the detection rate of SVs with important pathological/diagnostic classification significance, and may bring new discoveries on SVs and pathogenic mechanisms.

Natural grass coverage enriches arbuscular mycorrhizal fungal communities in subtropical citrus orchards through the regulation of Glomus on a regional scale

Arbuscular mycorrhizal (AM) fungi are ubiquitous soil fungi in orchard ecosystem, conferring a diverse array of benefits to citrus plants. However, how natural grass coverage, a common phenomenon in citrus orchards in southern China, shifts AM fungal communities in subtropical citrus orchards is far from being understood, especially on a regional scale, which impedes the understanding of the indigenous AM fungal adaptations. In this study, natural grass coverage rate, soil chemical properties, and soil AM fungal community in 80 sampling sites spanning 179, 700 km2 area in subtropical citrus orchards were investigated. The result of 18S rRNA gene high-throughput sequencing revealed that a total of eight AM fungal genera were identified and Glomus (90.15 %) was dominant genus in the community in citrus orchards. Natural grass coverage significantly increased soil organic matter (SOM), soil available phosphorus, and AM fungal alpha diversity, especially Glomus. Variation partitioning analysis revealed that grass coverage rate explained more variation in AM fungal alpha diversity than SOM. All the contrasting AM fungal species between different grass coverage rates and all the core AM fungal species belonged to Glomus. Null model analysis revealed that determinism played a slightly larger role in the assembly process of AM fungal communities than stochasticity with a distance-decay relationship. Moreover, natural grass coverage increased the relative importance of stochasticity in the community assembly. Taken together, these results suggest that maintaining natural grass coverage (naturally growing grass) is an effective nature-based solution for enriching AM fungal communities, which is essential for the sustainability of citrus production.

Effects of environmental factors on vertical distribution of the eukaryotic plankton community in early summer in Danjiangkou Reservoir, China

IntroductionEukaryotic plankton plays crucial roles in ecosystem processes, impacting aquatic ecosystem stability. This study focuses on Danjiangkou Reservoir, a canyon lake in central China, that acts as the water source of the Mid-route of the South-to-North Water Diversion Project.MethodsIn this study, high-throughput 18S rDNA gene sequencing was employed to investigate eukaryotic plankton community at four water depths (0.5 m, 5 m, 10 m, and 20 m). The environmental factors including pH, water temperature (WT), nitrate nitrogen (NO3−-N), ammonia nitrogen (NH4+-N), total nitrogen (TN), conductivity (Cond), and dissolved oxygen (DO) in reservoir areas were measured, and their correlations with abundance and diversity of eukaryotic plankton were analyzed.ResultsThe results showed the presence of 122 genera of eukaryotic plankton from 38 phyla. Eukaryotic plankton communities were mainly composed of Eurytemora, Thermocyclops, Sinocalanus, Mesocyclops, and Cryptomonas. In particular, significant differences in the diversity of eukaryotic plankton communities were found in vertical distribution. The diversity and abundance of eukaryotic plankton communities in 7 sampling sites decreased with the increase of depth from 0.5 to 10 m, while the diversity and abundance of plankton communities increased at 20 m. RDA analysis indicated that pH, depth, WT, NH4+-N, DO, Cond, and NO3−-N could influence the vertical distribution of the eukaryotic plankton community in the Danjiangkou Reservoir. Among these eukaryotic plankton, Eurytemora, Thermocyclops, and Volvox were negatively correlated with pH and WT and positively correlated with depth.DiscussionThis study revealed a novel perspective on the distribution of the eukaryotic plankton community in Danjiangkou Reservoir, particularly in terms of vertical variation, which will be helpful to comprehensively understand ecological processes and to further ensure the water quality safety in this canyon-style reservoir.

Detection of TP53 gene mutation in blood of breast cancer patients based on circulating tumor DNA and its application in prognosis.

This experiment was carried out to explore the application value of high throughput gene sequencing technology in detecting TP53 gene mutations in the blood of patients with breast cancer by detecting ctDNA gene mutations, and exploring the relationship between TP53 mutations and clinicopathological characteristics and prognosis of patients. The gene mutation of peripheral blood ctDNA and tissue paraffin DNA (tDNA) of 50 patients was detected by high-throughput sequencing technology. The basic data of 50 cases of Medium to high-risk breast cancer diagnosed and were retrospectively collected, and the clinicopathological characteristics and survival results of TP53 mutant and wild-type patients were compared and analyzed according to the ctDNA detection results and relevant follow-up data. Analyze the impact of TP53 mutations on overall survival and progression-free survival using univariate and multivariate Cox regression models. Among 50 patients, there were 29 cases of 7 kinds of gene mutations detected by ctDNA, and 37 cases of 9 kinds of gene mutations detected by tDNA. Using the gene mutation results detected by tDNA as the gold standard, the sensitivity and specificity of peripheral blood ctDNA in diagnosing TP53 gene mutations are 75% to 100%, 92.31% to 100%, and the overall coincidence rate with tDNA results was 83.33% to 100%. Exon 5 was the most prone to mutation, with a frequency of 24.14% (7/29). The most common type of mutation was the missense mutation of 37.93% (11/29). There was no significant correlation between TP53 mutation and PFS (HR=0.67, 95% CI: 0.41-1.08, P=0.102), while TP53 mutation was a protective factor for OS (HR=0.49, 95% CI: 0.27-0.90, P=0.022). The detection of ctDNA in peripheral blood of breast cancer patients by high-throughput gene sequencing technology can replace tumor tissue sections to understand gene mutation. The TP53 mutation in breast cancer patients is related to tumor size, lymph node metastasis and vascular tumor thrombus, but the prognosis of TP53 mutant patients is similar to that of wild-type patients.

Early-life gut microbiota in food allergic children and its impact on the development of allergic disease

BackgroundThe prevalence of food allergies (FA) has been steadily increasing over 2 to 3 decades, showing diverse symptoms and rising severity. These long-term outcomes affect children’s growth and development, possibly linking to inflammatory bowel disease. However, the cause remains unclear. Previous studies reveal that early infancy significantly impacts FA development through gut microbiota. Yet, a consistent view on dysbiosis characteristics and its connection to future allergies is lacking. We explored how early-life gut microbiota composition relates to long-term clinical signs in children with FA through longitudinal research.MethodsWe employed high-throughput 16S rDNA gene sequencing to assess gut microbiota composition in early-life FA children in southern Zhejiang. Follow-up of clinical manifestations over 2 years allowed us to analyze the impact of early-life gut microbiota dysbiosis on later outcomes.ResultsWhile the diversity of gut microbiota in FA children remained stable, there were shifts in microbiota abundance. Abundant Akkermansia, Parabacteroides, Blautia, and Escherichia-Shigella increased, while Bifidobacterium and Clostridium decreased. After 2 years, two of ten FA children still showed symptoms. These two cases exhibited increased Escherichia-Shigella and reduced Bifidobacterium during early childhood. The other eight cases experienced symptom remission.ConclusionsOur study suggests that FA and its prognosis might not correlate with early-life gut microbiota diversity. Further experiments are needed due to the small sample size, to confirm these findings.

Phylogenetic variation in raw cow milk microbiota and the impact of forage combinations and use of silage inoculants.

The microbiota of bulk tank raw milk is known to be closely related to that of microbial niches of the on-farm environment. Preserved forage types are partof this ecosystem and previous studies have shown variations in their microbial ecology. However, little is known of the microbiota of forage ration combinations and the transfer rates of associated species to milk. We identified raw milk bacteria that may originate from forage rations encompassing either hay (H) or grass/legume silage uninoculated (GL) as the only forage type, or a combination of GL and corn silage uninoculated (GLC), or grass/legume and corn silage both inoculated (GLICI). Forage and milk samples collected in the fall and spring from 24 dairy farms were analyzed using 16S rRNA gene high-throughput sequencing following a treatment with propidium monoazide to account for viable cells. Three community types separating H, GL, and GLICI forage were identified. While the H community was co-dominated by Enterobacteriaceae, Microbacteriaceae, Beijerinckiaceae, and Sphingomonadaceae, the GL and GLICI communities showed high proportions of Leuconostocaceae and Acetobacteraceae, respectively. Most of the GLC and GLICI rations were similar, suggesting that in the mixed forage rations involving grass/legume and corn silage, the addition of inoculant in one or both types of feed does not considerably change the microbiota. Raw milk samples were not grouped in the same way, as the GLC milk was phylogenetically different from that of GLICI across sampling periods. Raw milk communities, including the GLICI group for which cows were fed inoculated forage, were differentiated by Enterobacteriaceae and other Proteobacteria, instead of by lactic acid bacteria. Of the 113 amplicon sequence variants (ASVs) shared between forage rations and corresponding raw milk, bacterial transfer rates were estimated at 18 to 31%. Silage-based forage rations, particularly those including corn, share more ASVs with raw milk produced on corresponding farms compared to that observed in the milk from cows fed hay. These results show the relevance of cow forage rations as sources of bacteria that contaminate milk and serve to advance our knowledge of on-farm raw milk contamination.

PSXI-9 Investigation to Characterize the Swine Gut Microbiota at Different Growth Stages Using 16s Rrna Gene High-Throughput Sequencing Data

Abstract The swine gut microbiota is populated by large and diverse bacterial community that play an essential role in pig intestinal development. Therefore, it is important to characterize the composition of swine gut microbiota and determine which factor shape the composition of the gut microbiota. Our objective was to characterize the swine gut microbiota at the different growth stages using 16S rRNA gene high-throughput sequencing data from gastrointestinal (GI) samples. First, the samples were collected from different GI tract sections (duodenum, jejunum, ileum, cecum, colon, fecal) from a total of 36 piglets at early growth [day (d) 0, 21, 42] stages and then stored in liquid nitrogen for further microbiome analysis. DNA was extracted and subjected to sequencing with an Illumina Miseq sequencer targeting the V3-V4 region of the 16S rRNA gene. Sequences were processed using the QIIME2 software package. Linear discriminant analysis (LDA) effect size (LEfSe) was used to identify growth stage-associated bacterial genera that were enriched within the total GI tract samples. An LDA score (log10) of 4.0 was used as the cutoff for identifying differentially abundant genera. LEfSe analysis revealed total 18 bacterial features that are stage-specific. Among the more notable genera, Lactobacillus and Prevotella was more abundant at the d 0, Romboutsia, Bacteroides, and Ruminococcus at the d 21, Escherichia_shigella, Staphlylococcus, and Turicibacter at the d 42 stage. Our study may provide a foundation for future studies aimed to manipulate the swine gut microbiota for potential health benefits.

Deciphering the Structural Variants in Acute Myeloid Leukemia and Myelodysplastic Neoplasms By Optical Genome Mapping

Introduction Acute myeloid leukemia (AML) and myelodysplastic neoplasm (MDS) are common hematological malignancies, and the majority of patients carry genetic mutations, structural variants (SVs) and copy number variants (CNVs). Identifying these variants is important for diagnosis and prognosis, and in some cases can alter treatment management and outcomes. Methods for analyzing SVs and CNVs include karyotype analysis, fluorescence in situ hybridization, gene chips, and high-throughput gene sequencing technologies. Optical genome mapping (OGM) is a novel ultra-long single-molecule DNA detection based high throughput SVs analysis technology, which has the characteristics of whole genome coverage, high detection incidence and sensitivity. There were few publications presenting technical concordance of SVs detection between karyotyping and OGM, and there are no systematic studies comparing the concordance of OGM versus transcriptome sequencing (RNA-seq) in detecting gene fusion. This study aimed to compare the concordance of OGM and RNA-seq in the detection of fusion genes in AML and MDS cases, and the additional findings using OGM technology. Methods Bone marrow aspirates of 52 newly diagnosed or relapsed/refractory AML/MDS patients were recruited for OGM, RNA-seq, and leukemia-related gene DNA panel sequencing. Among these cases, 36 males and 16 were females, 15 pediatric (< 18 years) and 37 adults (≥18 years), with a median age of 31 years. Results Based on the RNA-seq results, 29/52 (55.8%) cases were detected with pathogenic gene fusions, including RUNX1::RUNX1T1, CBFB::MYH11, KMT2Aretc. (Figure 1). All those fusions were detected by OGM. For SVs with VAF>5%, the concordance was 98.1% (51/52 cases) when comparing OGM with karyotyping. OGM missed one case whose translocation happened close to the centromere. OGM reported more pathological variants with clinically significant in 5/52 cases (9.62%) and helped revise karyotype results in 1 case with clarifying marker chromosome. According to the ELN-2022 risk stratification mainly based on karyotype results, OGM's report results helped revise the risk stratification of 3 patients from intermediate to high risk. Fifty of 52 (96.2%) patients carried the gene mutation detected by the gene sequencing panel (Figure 1). In the samples of 4 patients who relapsed after receiving allogeneic hematopoietic stem cell transplantation (allo-HSCT) with donors and patients of different genders, we can infer the proportion of male and female DNA based on the copy number of chr Y detected by OGM, and then determine the proportion of donor cells. Conclusion This is the first AML cohort study to systematically compare the performance of OGM and RNA-seq, and the reported results of fusion genes are completely consistent. This is also the first report of applying the OGM technology in patients who relapsed after allo-HSCT and for chimerism analysis. Combined using OGM and targeted sequencing to fully characterize the CNV, SV, and mutation can be used for accurate stratification risk of AML/MDS. In conclusion, combining OGM and NGS can significantly improve the detection rate of genetic abnormalities with important pathological/diagnostic significance, and may bring new discoveries on SVs and pathogenic mechanisms.