Comparative Microbiome Study of Mummified Peach Fruits by Metagenomics and Metatranscriptomics

The dried peach fruits clinging to peach trees or lying on the ground nearby are known as mummified peach fruits. Here, we examined the microbiome communities of three different mummified peach fruits from the nectarine cultivar “Hahong” by DNA- and RNA-sequencing. We found the dominance of Monilinia fructigena followed by Sclerotinia borealis, S. sclerotiorum, and Botrytis cinerea in the mummified peach fruits. Moreover, we found a high number of Proteobacteria, including Frateuria aurantia, Neoasaia chiangmaiensis, Robbsia andropogonis, and Ewingella Americana. Furthermore, we identified several viruses and viroids. Bacteriophages were identified by DNA- and RNA-sequencing, while viruses and viroids with RNA genomes were identified by only RNA-sequencing. Moreover, we identified a novel mycovirus referred to as Monilinia umbra-like virus 1 (MULV1) from M. fructigena. Our results revealed the co-inhabitance of fungi and bacteria in the mummified peach fruits, although dominant microorganisms were present. RNA-sequencing revealed that several fungal and bacterial genes were actively transcribed. Comparative analyses suggested that RNA-sequencing provides more detailed information on microbial communities; however, combining DNA- and RNA-sequencing results increased the diversity of microorganisms, suggesting the importance of databases and analysis tools for microbiome studies. Taken together, our study provides a comprehensive overview of microbial communities in mummified peach fruits by DNA shotgun sequencing and RNA-sequencing.

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Two-target quantitative PCR to predict library composition for shallow shotgun sequencing

10.1101/2020.09.21.304006 ◽

2020 ◽

Author(s):

Matthew Y. Cho ◽

Marc Oliva ◽

Anna Spreafico ◽

Bo Chen ◽

Xu Wei ◽

...

Keyword(s):

High Resolution ◽

Microbial Communities ◽

Quantitative Pcr ◽

Shotgun Sequencing ◽

Accurate Prediction ◽

Missing Information ◽

Bacterial Genes

AbstractShotgun sequencing enables retrieving high resolution information 40 from complex microbial communities. However, the technique is limited by missing information about host-to-microbe ratios observed in different sample types. This makes it challenging to plan sequencing experiments, especially in the context of high sample multiplexing and/or limited sequencing output. We evaluated a qPCR-based assay to predict host-to microbe ratio prior to sequencing. Using a two-target assay aimed at conserved human and bacterial genes, we predicted human-to-microbe ratios in two sample types and validated it on independently collected samples. The assay enabled accurate prediction for a broad range of sample compositions.

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Comparing total RNA sequencing and metagenomics pipelines for multi-domain taxonomic profiling: implications for ecological assessments

ARPHA Conference Abstracts ◽

10.3897/aca.4.e64996 ◽

2021 ◽

Vol 4 ◽

Author(s):

Christopher Hempel ◽

Julia Harvie ◽

Jose Hleap Lozano ◽

Natalie Wright ◽

Sarah Adamowicz ◽

...

Keyword(s):

Microbial Communities ◽

Rna Sequencing ◽

Sample Type ◽

Rna Seq ◽

Total Rna ◽

Taxonomic Profiling ◽

Dna And Rna ◽

Accuracy And Precision ◽

The Status ◽

Unicellular Organisms

Ecological assessments are necessary to evaluate the status of our deteriorating ecosystems, however, assessment methods traditionally omit most microbes because unicellular organisms are challenging to identify. This omission is not ideal, as microbes might be better indicators for changes in environmental conditions than taxa traditionally used. DNA- and RNA-based techniques are increasingly applied for ecological assessments to overcome this challenge but require more testing and optimization. In this study, we compare metagenomics and total RNA sequencing (total RNA-Seq) for their taxonomic profiling performance for microbial communities. We applied both techniques on two sample sets, 1) a commercially available microbial mock community consisting of eight bacterial and two eukaryotic species, and 2) a display tank water sample. We processed the data using 1,532 bioinformatics pipelines and evaluated each workflow, i.e., the combination of sample type (metagenomics or total RNA-Seq) and pipeline, in terms of their accuracy and precision. This talk will showcase preliminary results and highlight differences in workflow performances. A recommended workflow to maximize taxonomic profiling accuracy of microbial communities will also be presented.

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CaptureSeq: Hybridization-Based Enrichment of cpn60 Gene Fragments Reveals the Community Structures of Synthetic and Natural Microbial Ecosystems

Microorganisms ◽

10.3390/microorganisms9040816 ◽

2021 ◽

Vol 9 (4) ◽

pp. 816

Author(s):

Matthew G. Links ◽

Tim J. Dumonceaux ◽

E. Luke McCarthy ◽

Sean M. Hemmingsen ◽

Edward Topp ◽

...

Keyword(s):

Microbial Community ◽

Microbial Communities ◽

Dna Sequences ◽

Pcr Amplification ◽

Shotgun Sequencing ◽

Natural Ecosystems ◽

Gene Markers ◽

Microbial Profile ◽

Microbial Ecosystems ◽

Pcr Targeting

Background. The molecular profiling of complex microbial communities has become the basis for examining the relationship between the microbiome composition, structure and metabolic functions of those communities. Microbial community structure can be partially assessed with “universal” PCR targeting taxonomic or functional gene markers. Increasingly, shotgun metagenomic DNA sequencing is providing more quantitative insight into microbiomes. However, both amplicon-based and shotgun sequencing approaches have shortcomings that limit the ability to study microbiome dynamics. Methods. We present a novel, amplicon-free, hybridization-based method (CaptureSeq) for profiling complex microbial communities using probes based on the chaperonin-60 gene. Molecular profiles of a commercially available synthetic microbial community standard were compared using CaptureSeq, whole metagenome sequencing, and 16S universal target amplification. Profiles were also generated for natural ecosystems including antibiotic-amended soils, manure storage tanks, and an agricultural reservoir. Results. The CaptureSeq method generated a microbial profile that encompassed all of the bacteria and eukaryotes in the panel with greater reproducibility and more accurate representation of high G/C content microorganisms compared to 16S amplification. In the natural ecosystems, CaptureSeq provided a much greater depth of coverage and sensitivity of detection compared to shotgun sequencing without prior selection. The resulting community profiles provided quantitatively reliable information about all three domains of life (Bacteria, Archaea, and Eukarya) in the different ecosystems. The applications of CaptureSeq will facilitate accurate studies of host-microbiome interactions for environmental, crop, animal and human health. Conclusions: cpn60-based hybridization enriched for taxonomically informative DNA sequences from complex mixtures. In synthetic and natural microbial ecosystems, CaptureSeq provided sequences from prokaryotes and eukaryotes simultaneously, with quantitatively reliable read abundances. CaptureSeq provides an alternative to PCR amplification of taxonomic markers with deep community coverage while minimizing amplification biases.

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Small RNA-Sequencing: Approaches and Considerations for miRNA Analysis

Diagnostics ◽

10.3390/diagnostics11060964 ◽

2021 ◽

Vol 11 (6) ◽

pp. 964

Author(s):

Sarka Benesova ◽

Mikael Kubista ◽

Lukas Valihrach

Keyword(s):

Rna Sequencing ◽

Small Rna ◽

High Sensitivity ◽

Small Rna Sequencing ◽

Rna Seq ◽

Liquid Biopsies ◽

Comprehensive Overview ◽

Rna Molecules ◽

Novel Mirna ◽

The Many

MicroRNAs (miRNAs) are a class of small RNA molecules that have an important regulatory role in multiple physiological and pathological processes. Their disease-specific profiles and presence in biofluids are properties that enable miRNAs to be employed as non-invasive biomarkers. In the past decades, several methods have been developed for miRNA analysis, including small RNA sequencing (RNA-seq). Small RNA-seq enables genome-wide profiling and analysis of known, as well as novel, miRNA variants. Moreover, its high sensitivity allows for profiling of low input samples such as liquid biopsies, which have now found applications in diagnostics and prognostics. Still, due to technical bias and the limited ability to capture the true miRNA representation, its potential remains unfulfilled. The introduction of many new small RNA-seq approaches that tried to minimize this bias, has led to the existence of the many small RNA-seq protocols seen today. Here, we review all current approaches to cDNA library construction used during the small RNA-seq workflow, with particular focus on their implementation in commercially available protocols. We provide an overview of each protocol and discuss their applicability. We also review recent benchmarking studies comparing each protocol’s performance and summarize the major conclusions that can be gathered from their usage. The result documents variable performance of the protocols and highlights their different applications in miRNA research. Taken together, our review provides a comprehensive overview of all the current small RNA-seq approaches, summarizes their strengths and weaknesses, and provides guidelines for their applications in miRNA research.

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HGG-34. DETECTION OF ONCOGENIC FUSION EVENTS IN SUPRATENTORIAL GLIOBLASTOMAS OF YOUNG CHILDREN

Neuro-Oncology ◽

10.1093/neuonc/noaa222.315 ◽

2020 ◽

Vol 22 (Supplement_3) ◽

pp. iii349-iii350

Author(s):

Torsten Pietsch ◽

Christian Vokuhl ◽

Gerrit H Gielen ◽

Andre O von Bueren ◽

Everlyn Dörner ◽

...

Keyword(s):

Tyrosine Kinase ◽

Rna Sequencing ◽

First Year ◽

Gene Fusions ◽

Older Children ◽

Kinase Gene ◽

Dna And Rna ◽

Infancy And Early Childhood ◽

Stable Genome ◽

First Year Of Life

Abstract INTRODUCTION Glioblastoma in infancy and early childhood is characterized by a more favorable outcome compared to older children, a stable genome, and the occurrence of tyrosine kinase gene fusions that may represent therapeutic targets. METHODS 50 glioblastomas (GBM) with supratentorial location occurring in children younger than four years were retrieved from the archives of the Brain Tumor Reference Center, Institute of Neuropathology, University of Bonn. DNA and RNA were extracted from FFPE tumor samples. Gene fusions were identified by FISH using break-apart probes for ALK, NTRK1, -2, -3, ROS1 and MET, Molecular Inversion Probe (MIP) methodology, and targeted RNA sequencing. RESULTS 37 supratentorial GBM occurred in the first year of life, 13 GBM between one and four years. 18 cases showed fusions of ALK to different fusion partners; all occurred in the first year of life (18/37 cases, 48.6%). Fusions of ROS1 were found in 5, MET in 3, NTRK1, -2, -3 in 10 cases. 12 cases showed no and two novel fusions. The different methods led to comparable results; targeted RNA sequencing was not successful in a fraction of cases. Break-apart FISH led to reliable results on the next day, MIP technology represented the most sensitive method for analysis of FFPE samples. CONCLUSIONS Gene fusions involving the tyrosine kinase genes ALK, MET, ROS1 and NTRK1, -2, -3 occurred in 72% of glioblastomas of children younger than four years; the most frequent were ALK fusions occurring in infant GBM. DNA based MIP technology represented the most robust and sensitive assay.

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Abstract 642: Genomes for Kids: Comprehensive DNA and RNA sequencing defining the scope of actionable mutations in pediatric cancer

10.1158/1538-7445.am2021-642 ◽

2021 ◽

Author(s):

David A. Wheeler ◽

Scott Newman ◽

Joy Nakitandwe ◽

Chimene A. Kesserwan ◽

Elizabeth M. Azzato ◽

...

Keyword(s):

Rna Sequencing ◽

Pediatric Cancer ◽

Dna And Rna

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Recent developments in nucleic acid based techniques for use in rumen manipulation

Revista Brasileira de Zootecnia ◽

10.1590/s1516-35982009001300034 ◽

2009 ◽

Vol 38 (spe) ◽

pp. 341-351 ◽

Cited By ~ 5

Author(s):

Christopher McSweeney ◽

Seungha Kang ◽

Emma Gagen ◽

Carl Davis ◽

Mark Morrison ◽

...

Keyword(s):

Microbial Community ◽

Nucleic Acid ◽

Microbial Communities ◽

Marker Gene ◽

Molecular Ecology ◽

Functional Gene ◽

Rumen Microorganisms ◽

Dna And Rna ◽

Conventional Culture ◽

Reference Strains

Nucleic acid-based techniques which can be used to characterise complex microbial communities without incubation are now being employed regularly in ruminant nutrition studies. Conventional culture-based methods for enumerating rumen microorganisms (bacteria, archaea, protozoa, and fungi) have been superseded and are now used mainly to obtain pure isolates of novel organisms and reference strains that are required for the development and validation of the nucleic acid approaches. These reference strains are also essential for physiological studies of the lifestyle of the organisms as well as sources of genomic DNA and RNA that can be analysed for functional gene activity. The foundation of the molecular ecology techniques is 16S/18S rDNA sequence analysis which has provided a phylogenetically based classification scheme for enumeration and identification of microbial community members. The use of this marker gene in assays involving the use of single nucleic acid probes or primer sets is rapidly evolving to high throughput approaches such as microarray analysis and new generation sequencing technologies. While these analyses are very informative for determining the composition of the microbial community and monitoring changes in population size, they can only infer function based on these observations. The focus of nucleic acid research is now shifting to the functional analysis of the ecosystem which involves the measurement of functional genes and their expression in the predominant or specific members of the rumen microbial community. Functional gene studies are less developed than 16S rDNA-based analysis of community structure. Also for gene expression studies there are inherent problems involved in extracting high quality RNA from digesta, and priming cDNA synthesis from bacterial mRNA. This paper reviews nucleic acid based molecular methods which have recently been developed for studying the structure and function of rumen microbial communities.

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Identification of allelic expression imbalance genes in human hepatocellular carcinoma through massively parallel DNA and RNA sequencing

Medical Oncology ◽

10.1007/s12032-016-0751-y ◽

2016 ◽

Vol 33 (4) ◽

Cited By ~ 4

Author(s):

Qiudao Wang ◽

Yan An ◽

Qing Yuan ◽

Yao Qi ◽

Ying Ou ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Rna Sequencing ◽

Human Hepatocellular Carcinoma ◽

Allelic Expression ◽

Massively Parallel ◽

Allelic Expression Imbalance ◽

Dna And Rna

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Acid-Based Decalcification Methods Compromise Genomic Profiling from DNA and RNA

Blood ◽

10.1182/blood-2019-131362 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 4659-4659

Author(s):

Ian Duncan ◽

Natalie Danziger ◽

Daniel Duncan ◽

Amanda Hemmerich ◽

Claire Edgerly ◽

...

Keyword(s):

Bone Marrow ◽

Soft Tissue ◽

Rna Sequencing ◽

Pathology Report ◽

Genomic Profiling ◽

Failure Rates ◽

Dna And Rna ◽

Equity Ownership ◽

Generation Sequencing ◽

Better Than

BACKGROUND: Comprehensive genomic profiling (CGP) performed by next-generation sequencing of DNA detects genomic alterations including point mutations, insertions/deletions, copy number variations, and select gene rearrangements. When RNA sequencing is included in CGP, it allows for expanded detection of gene fusions, which are common in hematologic malignancies and sarcomas. When such tumors involve bone, a decalcification step is frequently employed to soften tissues prior to processing and sectioning. While commonly used acid-based decalcification methods work quickly, the resulting nucleic acid damage can be profound. In this study, we examine the effects of decalcification on DNA and RNA sequencing in the clinical setting. DESIGN: 1711 consecutive formalin-fixed paraffin embedded samples were evaluated by CGP during routine clinical care via DNA and RNA sequencing, using a hybrid-capture next-generation sequencing assay (FoundationOne®Heme). Specimen site [e.g. bone/ bone marrow or soft tissue] and decalcification status were extracted from pathology reports and H&E review. Samples were considered decalcified if reported as such in the pathology report or if visible decalcified bone was present on the H&E. Samples documented to be processed with fixatives other than formalin were excluded. Sequencing failures were defined as samples that failed DNA extraction (DNAx), RNA extraction (RNAx), or library construction (LC) due to insufficient nucleic acid to advance into sequencing. Samples were only evaluated for RNA if DNAx was successful (1594 cases). RESULTS: Specimen site was a strong predictor of sequencing failure, with a significant increase in failure rate from bone/bone marrow samples (n=619) compared to samples from soft tissue sites (n=1092) for both DNA (13.4% vs 4.6%, p=4.7E-9) and RNA (42.5% vs 13.5%, p<2.2E-16). Of the bone/bone marrow samples, 237 of 619 samples were decalcified. Decalcification was associated with significantly higher failure rates than non-decalcified samples for both DNA (29.1% vs 3.7%) and RNA (67.4% vs 30.8%) (Table 2). One method of avoiding decalcification for bone marrow samples is utilization of clot preparations, where aspirates are processed as an FFPE block. Clot preparations fail sequencing significantly less often than decalcified core biopsies (DNA: 3.3% vs 18.8%, p=9.2E-06; RNA: 39.2% vs 70.4%, p=2.5E-03) (Table 3). CONCLUSIONS: CGP of samples acquired from bone and bone marrow sites is challenging, with a lower success rate for DNA and RNA sequencing than soft tissue sites. The higher overall failure rate correlates with use of decalcification agents leading to degradation of nucleic acids and impacts RNA sequencing significantly more than DNA (67.4% vs 30.8% failed). Clot preparations of bone marrow samples performed better than core biopsies for both DNA and RNA. The higher overall RNA sequencing failure rates still observed in in non-decalcified bone/bone marrow are predominantly due to RNA failure of non-decalcified clot preparations. These samples likely have increased failure rates secondary the use of non-standard fixatives (e.g. B+, Bouin's, AZF, etc.) not documented in the pathology report and the frequency of hypocellular clot preparations in conjunction with higher requirements for RNA yield compared to DNA yield. To increase CGP success rates, decalcification should be avoided when possible. Peripheral blood and bone marrow aspirate samples rarely fail sequencing (<1%, data not shown) and are preferable to decalcified samples if adequate tumor is present. Bone marrow clot preparations perform better than bone marrow core biopsies and clot preparations should be fixed with 10% neutral buffered formalin. If decalcification is required for processing, EDTA based decalcification methods and/or minimizing decalcification times is recommended. Disclosures Duncan: Foundation Medicine, Inc.: Employment. Danziger:Foundation Medicine, Inc.: Employment; F. Hoffman La Roche, Ltd.: Equity Ownership. Duncan:Foundation Medicine, Inc.: Employment; F. Hoffman La Roche, Ltd.: Equity Ownership. Hemmerich:F. Hoffman La Roche, Ltd.: Equity Ownership; Foundation Medicine, Inc.: Employment. Edgerly:F. Hoffman La Roche, Ltd.: Equity Ownership; Foundation Medicine, Inc: Employment. Huang:F. Hoffman La Roche, Ltd.: Equity Ownership; Foundation Medicine, Inc.: Employment. Vergilio:Foundation Medicine, Inc.: Employment; F. Hoffman La Roche, Ltd.: Equity Ownership. Elvin:Foundation Medicine, Inc.: Employment; F. Hoffman La Roche, Ltd.: Equity Ownership. He:Foundation Medicine, Inc.: Employment; F. Hoffman La Roche, Ltd.: Equity Ownership. Britt:Foundation Medicine, Inc: Employment. Reddy:F. Hoffman La Roche, Ltd.: Equity Ownership; Foundation Medicine, Inc: Employment. Sathyan:Foundation Medicine, Inc.: Employment; F. Hoffman La Roche, Ltd.: Equity Ownership. Alexander:Foundation Medicine, Inc.: Employment; F. Hoffman La Roche, Ltd.: Equity Ownership. Ross:F. Hoffman La Roche, Ltd.: Equity Ownership; Foundation Medicine, Inc.: Employment. Brown:Foundation Medicine, Inc.: Employment; F. Hoffman La Roche, Ltd.: Equity Ownership. Ramkissoon:F. Hoffman La Roche, Ltd.: Equity Ownership; Foundation Medicine, Inc.: Employment. Severson:F. Hoffman La Roche, Ltd.: Equity Ownership; Foundation Medicine, Inc.: Employment.

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