The best practice for microbiome analysis using R

With the gradual maturity of sequencing technology, many microbiome studies have published, driving the emergence and advance of related analysis tools. R language is the widely used platform for microbiome data analysis for powerful functions. However, tens of thousands of R packages and numerous similar analysis tools have brought major challenges for many researchers to explore microbiome data. How to choose suitable, efficient, convenient, and easy-to-learn tools from the numerous R packages has become a problem for many microbiome researchers. We have organized 324 common R packages for microbiome analysis and classified them according to application categories (diversity, difference, biomarker, correlation and network, functional prediction, and others), which could help researchers quickly find relevant R packages for microbiome analysis. Furthermore, we systematically sorted the integrated R packages (phyloseq, microbiome, MicrobiomeAnalystR, Animalcules, microeco, and amplicon) for microbiome analysis, and summarized the advantages and limitations, which will help researchers choose the appropriate tools. Finally, we thoroughly reviewed the R packages for microbiome analysis, summarized most of the common analysis content in the microbiome, and formed the most suitable pipeline for microbiome analysis. This paper is accompanied by hundreds of examples with 10,000 lines codes in GitHub, which can help beginners to learn, also help analysts compare and test different tools. This paper systematically sorts the application of R in microbiome, providing an important theoretical basis and practical reference for the development of better microbiome tools in the future. All the code is available at GitHub github.com/taowenmicro/EasyMicrobiomeR.

Comparison of ocular microbiomes between patients with and without dry eye / 中华实验眼科杂志

Objective:To explore the difference in ocular surface microbiota between patients with and without dry eye.Methods:Forty-two patients (42 eyes) diagnosed with dry eye were enrolled as dry eye group in the First Affiliated Hospital of Xi'an Jiaotong University from June to November 2020, and 37 controls without dry eye (37 eyes) were enrolled as control group in the same period.One eye was selected as the study eye, and the right eye was included when both eyes met the inclusion criteria.Swab samples from the conjunctival sac were obtained and sequenced.Sequencing of the V3-V4 region of the bacterial 16S rRNA gene was performed with Miseq PE301+ 8+ 301 platform.Operational taxonomic species (OTUs) clustering of microflora, comparison of alpha and beta diversity analysis of microflora between the two groups, annotation analysis of species and analysis of microbial markers were performed.The study protocol was approved by the Ethics Committee of The First Affiliated Hospital of Xi'an Jiaotong University (No.XJTU1AFCRC2018SJ-014). Written informed consent was obtained from each subject before any medical examination.Results:A total of 18 586 OTUs were obtained, and 3 674 OTUs were shared between the two groups.Alpha diversity analysis showed that there was no significant difference in observed species index, Chao index, Ace index, Shannon index and Simpson index between the two groups (all at P>0.05), suggesting there was no difference in microbiota richness between them.The PCoA analysis showed that the microbial compositions of the two groups were significantly different ( R2=0.039, F=3.100, P=0.022). The dominant flora of the two groups was similar, with Proteobacteria, Actinobacteria, Firmicutes, Bacteroidetes and Cyanobacteria as the top 5 abundant bacterial phyla, with Pelomonas, Corynebacterium, Propionibacterium, Pseudomonas and Herbaspirillum as the top 5 bacterial genera.LEfSe analysis identified Tissierellaceae, Enhydrobater and Finegoldia as dominant bacterial genera in dry eye group, and Caulobacter and Curvibacter in control group. Conclusions:The composition of ocular surface microbiomes is different between dry eye patients and controls.

Application of nanopore sequencing in environmental microbiology research / 生物工程学报

The development of high-throughput sequencing techniques enabled a deeper and more comprehensive understanding of environmental microbiology. Specifically, the third-generation sequencing techniques represented by nanopore sequencing have greatly promoted the development of environmental microbiology research due to its advantages such as long sequencing reads, fast sequencing speed, real-time monitoring of sequencing data, and convenient machine carrying, as well as no GC bias and no PCR amplification requirement. This review briefly summarized the technical principle and characteristics of nanopore sequencing, followed by discussing the application of nanopore sequencing techniques in the amplicon sequencing, metagenome sequencing and whole genome sequencing of environmental microorganisms. The advantages and challenges of nanopore sequencing in the application of environmental microbiology research were also analyzed.

Screening Novel Drugresistant Mutations in Mycobacterium tuberculosis Strains by Using the Amplicon Sequencing Method / 中国生物化学与分子生物学报

Tuberculosis is one of the seriously public problems. The increasing drug-resistant tuberculosis is the key problem for controlling tuberculosis. Rapid and accurate diagnosis is important for further treatment. In this study‚ a next-generation sequencing method based on amplicon sequencing was constructed to screen the mutations in 17 drug-resistant genes of five first-line anti-tuberculosis drugs. A total of 65 mutations were identified in 26 clinic drug-resistant tuberculosis strains‚ including 33 hotspot mutations‚ 9 rare mutations‚ and 23 novel mutations. The pathogenesis‚ conservation‚ and partial structures caused by 18 novel missense mutations were predicted. The results showed that 14 novel mutations showed high conservation in nine species. All these 14 mutations could change the partial structure of protein. According to the detection and analysis results of this study‚ it is speculated that these newly discovered mutations may be potential drug-resistant mutations. It is a rapid‚ accurate and comprehensive method for the detection of drug-resistant mutations in first-line drug-resistant Mycobacterium tuberculosis‚ which could identify hotspot and rare mutations together with novel mutations. The detection method may be used for clinical diagnosis and basic research.

Investigation and research on fungal diversity of Platycladi Semen on Chinese market / 中国中药杂志

In the process of harvesting, production and processing, storage, and transportation, the traditional Chinese medicine Platycladi Semen is prone to mildew due to its own and environmental factors, which can nourish the production of toxic or pathogenic fungi, and even produce mycotoxins, which affects the safety of clinical medication. The 2020 edition of Chinese Pharmacopoeia limits the highest standard of aflatoxin content in Platycladi Semen. However, there are few studies on the fungal contamination of Platycladi Semen, and it is difficult to prevent and control it in a targeted manner. Therefore, based on the Illumina NovaSeq6000 platform, this article uses ITS sequence amplicon technology to analyze the distribution and diversity of fungi in 27 batches of commercially available Platycladi Semen in the Chinese market. A total of 10 phyla, 35 classes, 93 orders, 193 families, 336 genera, and 372 species of fungi were identified in China. Among them, Aspergillus, Alternaria spp. were dominant, 20 batches of samples were detected for A. flavus, 10 batches of samples were detected for A. nidulans, and all samples were detected for potential pathogenic fungi such as A. fumigatus and A. niger. According to diversity analysis, the diversity of the fungal communities in the samples from Gansu province was high, the samples in Shandong province contain the largest number of fungal species, and the samples in Guangxi province had the lo-west diversity and the least number of species. In most samples, pathogenic fungi such as A. fumigatus, A. niger, A. flavus, A. parasiticus were detected in varying degrees. This study systematically investigated the fungal contamination of Platycladi Semen from the markets in the last link of the its industrial chain, and clarified the distribution of Platycladi Semen fungi, especially toxin-producing fungi, and provided theoretical basis for the targeted prevention and control of fungal contamination in Platycladi Semen.

Indigenous bacterial community of heavy metal tolerance in the rhizosphere soils of Mimosa pudica naturally growing on an ex-tin mining area

Aims@#The purpose of this research was to explore the composition and genomic functions of bacterial community inhabiting the rhizosphere of Mimosa pudica, which were naturally growing on tailing and non-tailing soils of an ex-tin mining area.@*Methodology and results@#DNA were extracted from rhizosphere soils and PCR targeting the hypervariable region V3-V4 was carried out by Illumina 16S metagenomic library. Libraries were sequenced using Illumina MiSeq. The Operational Taxonomic Units (OTUs) were assigned to 23 bacterial phyla, 72 classes, 165 orders, 248 families and 357 genera. The most represented and dominant phylum was Proteobacteria, with an average abundance value of 41.2%. The most represented genera included Paraburkholderia, Bradyrhizobium, Bacillus, Candidatus, Acidothermus, Acidibacter and Nitrospira. Non-tailing soils had more number and richness of species while the tailings had more diversity of species. The metagenomes accommodate suspected genes for heavy metal tolerance of microbes (As, Cr, Co, Zn, Ni, Cu, Cd, Fe and Hg) and microbial plant-growth-promoting traits for hyperaccumulator plants (synthesis of indole acetic acid (IAA), siderophore and 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase; solubilization of phosphate and potassium and nitrogen fixation). @*Conclusion, significance and impact of study@#Bacteria and predicted genes discovered could be part of major factors influencing growth of M. pudica in heavy metal-contaminated soils. The study provides the first report and a basis into the bacterial community associated with M. pudica in ex-tin mining soils from the studied geographical location. The findings also provide fundamental knowledge on phytoremediation potential of heavy metal contaminated soils involving indigenous beneficial microbial populations.

Preface for microbiome sequencing and analysis / 生物工程学报

Microbes are the most important commensal organisms in humans, animals and plants, and are the major habitants in soil, sediment, water, air and other habitats. The analysis of microbiome in these habitats has become a basic research technique. As a fast developing technology in recent years, microbiome sequencing and analysis have been widely used in human health, environmental pollution control, food industry, agriculture and animal husbandry and other fields. In order to sort out and summarize the current status, development and application prospects of microbiome sequencing and analysis technologies, this special issue has prepared a collection of 16 papers in this field, that comprise sample preservation and processing, single microbe genome sequencing and analysis, and microbiome feature analysis in special habitats, microbiome related databases and algorithms, and microbiome sequencing and analysis expert consensus. It also introduced in detail the development trend of the microbiome sequencing and analysis, in order to promote the rapid development of the microbiome sequencing and analysis industry and scientific research in China, and provide necessary reference for the healthy development of related industries.

A new quantitative 16S rRNA amplicon sequencing method / 生物工程学报

In recent years, 16S rRNA amplicon sequencing technology has been widely used to study human gut microbiota and to detect unknown pathogens in clinical samples. However, its resolution to bacterial population can only reach the relative abundance of genus level, and different factors affect the final bacterial profile, such as sample concentrations, PCR cycle numbers and amplification primers. In order to solve these problems, we developed a quantitative 16S rRNA amplicon sequencing method by combining random tag and internal marker method. The new methods improved the accuracy of human gut microbiota, reduced the impact of experimental operation on the results, and improved the comparability between sequencing and other molecular biological methods.

Repeated mild traumatic brain injury affects microbial diversity in rat jejunum

Traumatic brain injuries (TBI) manifest into post-traumatic stress disorders such as anxiety comorbid with gut ailments. Theperturbations in gut microbial communities are often linked to intestinal and neuropsychological disorders. We havepreviously reported anxiety and abnormalities in gut function in mild TBI (MTBI)-exposed rats. The current studydemonstrates the changes in gut microbiome of MTBI-exposed animals and discusses its implications in intestinal healthand behaviours. The rats were subjected to repeated MTBI (rMTBI) and microbial composition in jejunum was examinedafter 6 h, 48 h and 30 days of rMTBI. Significant reduction in bacterial diversity was observed in the rMTBI-exposedanimals at all the time points. Principal coordinate analysis based on weighted UniFrac distances indicated substantialdifferences in gut microbial diversity and abundances in rMTBI-exposed animals as compared to that in healthy controls.The abundance of Proteobacteria increased dramatically with reciprocal decrease in Firmicutes after rMTBI. At the genuslevel, Helicobacter, Lactobacillus, Campylobacter, and Streptococcus were found to be differentially abundant in thejejunum of rMTBI-exposed rats as compared to sham controls indicating profound dysbiosis from the healthy state.Furthermore, substantial depletion in butyrate-producing bacterial communities was observed in rMTBI-exposed animals.These results suggest that the traumatic stress alters the gut microbiome with possible implications in gut health andneuropsychopathology.

HLA-B*27 subtypes and its clinical implications characterization by studying sequence-specific alleles

Ankylosing spondylitis (AS) is a form of spondylitis in which spine is affected primarily along with otherjoints. The vertebrae fuse together and form a rigid structure. Individuals with AS are also positive for humanleukocyte antigen (HLA) B-27. One hundred and eighty-three symptomatic patients of AS were used in thestudy. Sequence-specific priming (SSP) PCR technique was used to detect the HLA B-27 specific allele. Thisstudy showed (out of 183 suspected cases, 45 cases were detected positive with HLA B-27 allele, while theremaining 138 were negative) that the positivity rate for AS with HLA B-27 allele is less. In 183 cases, 63were females, whereas 7 cases were positive and 56 negative, whereas 38 cases were positive for male and 82cases were negative. When sample was analyzed in term of age groups, it was found that out of 45 positivesamples, the positivity rate was maximum, i.e., 57.14% in the patient above 37 years of age. Furthermore, itwas observed that the males above 30 years are more prone to develop AS with HLA B27 specific allele. Forthe diagnosis of AS, conventional PCR technology is a promising diagnostic method and can be considered asan important tool along with other diagnostics parameters.

Aseguramiento de Calidad en el Laboratorio de Biología Molecular: Control de Contaminación Ambiental / Quality Assurance in the Laboratory of Molecular Biology: Control of Environmental Contamination / Garantia de Qualidade no Laboratório de Biologia Molecular: Controle da Contaminação Ambiental

La reacción en cadena de la polimerasa (PCR) es la técnica más importante en Biología Molecular. Su principal deficiencia es la susceptibilidad a la contaminación de la muestra, ya sea con productos de amplificación generados en reacciones previas o con material genético proveniente de otras muestras. Se entiende por contaminación a la presencia indeseada de moléculas de ADN o ARN, que pueden actuar como templados en reacciones de amplificación. El objetivo del trabajo fue demostrar la importancia de la implementación de un control de contaminación ambiental periódico monitoreando la integridad del circuito de PCR. Para ello, se hisoparon puntos correspondientes a diferentes áreas de trabajo y se investigó la presencia de amplicones mediante PCR Real Time en Lighcycler 2.0, Roche® y Ampliprep Cobas s201, Roche®. En puntos críticos del circuito (flujo laminar o la cabina de seguridad del área de pre-PCR) no se detectaron amplicones, aunque sí se hallaron en las mesadas y heladeras. En el resto del circuito, la distribución de los amplicones fue variable. De esta forma se demuestra la importancia de la realización de un control de contaminación ambiental periódico en laboratorios que realizan la técnica de PCR, pudiendo detectar contaminación de forma precoz y tomar acciones correctivas a tiempo.

Polymerase chain reaction (PCR) is the most important technique in Molecular Biology. Its main deficiency is susceptibility to contamination of the sample, either with amplification products generated in previous reactions or with genetic material from other samples. Contamination is understood as the undesired presence of DNA or RNA molecules, which may act as annealing in amplification reactions. The objective of this work is to demonstrate the importance of implementing a periodic environmental pollution control by monitoring the integrity of the PCR. To do this, different areas of work were swabbed and the presence of amplicons were investigated by Real Time PCR in Lighcycler 2.0, Roche® and Ampliprep Cobas s201, Roche®. At critical points in the circuit (laminar flow or pre-PCR area booth) no amplicons were detected, although they were found in countertops and refrigerators. In the rest of the circuit, the distribution of the amplicons was variable. Thus, the importance of conducting a periodic environmental contamination control in laboratories that perform the PCR technique is demonstrated, enabling early detection contamination and corrective actions being taken on time.

A reação em cadeia da polimerase (PCR) é a técnica mais importante em Biologia Molecular. Sua principal deficiência é a suscetibilidade à contaminação da amostra, seja com produtos de amplificação gerados em reações prévias ou com material genético em reações prévias ou com material genético proveniente de outras amostras. Entende-se por contaminação a presença não desejada de moléculas de DNA ou ARN, que podem agir como temperados em reações de amplificação. O objetivo do trabalho foi demonstrar a importância da implementação de um controle de contaminação ambiental periódico monitorando a integridade do circuito de PCR. Para isso, se fez esfregaço de pontos correspondentes a diferentes áreas de trabalho e foi investigada a presença de amplicons mediante PCR Real Time em Lighcycler 2.0, Roche® e Ampliprep Cobas s201, Roche®. Em pontos críticos do circuito (fluxo laminar ou a cabine de segurança da área de pré-PCR), não foram detectados amplicons, apesar de serem encontrados nas bancadas e refrigeradores. No resto do circuito, a distribuição dos amplicons foi variável. Assim mostra a importância da realização de um controle de contaminação ambiental periódico em laboratórios que realizam a técnica de PCR, podendo detectar a contaminação de forma precoce e tomar ações corretivas a tempo.

Microbiota bacteriana asociada al papel moneda de circulación en Colombia / Bacterial microbiota on circulating Colombian banknotes

Diariamente los seres humanos están en interacción con objetos de uso continuo, como el papel moneda, sin el conocimiento de que estos almacenan microorganismos y de que nos exponemos al contacto con potenciales patógenos. La composición de la comunidad bacteriana en un billete colombiano fue determinada mediante el secuenciamiento profundo de librerías de amplicones 16S. Se encontraron 233 géneros bacterianos; 12 de estos géneros corresponden a especies con potencial patogénico. El género más abundante fue Propionibacterium, seguido de Streptococcus, Staphylococcus Pseudomonas . Este es el primer reporte de la diversidad bacteriana que puede ser alojada en este objeto de alta circulación en Colombia. Pocos estudios en el mundo han mostrado este nivel de detalle de la microbiota en billetes de circulación y ofrece un panorama mucho más amplio de la exposición diaria a microorganismos al utilizar papel moneda en las condiciones en las que se utiliza en Colombia.

Commonly used objects such as currency paper can be colonised by bacteria and can serve as carriers of microbes. This colonisation might expose us to unnoticed pathogenic bacteria. In this study, the researchers obtained a detailed panorama of the microbes that can be carried on currency notes in Colombia by using 454 next-generation deep sequencing of 16S amplicón libraries. A total of 233 bacterial genera were detected and classified, 12 of which are potential human pathogens. The most abundant genera were Propionibacterium, Streptococcus, Staphylococcus and Pseudomonas. To date, this is the first in-depth analysis of the microbiota carried by circulating banknotes in our continent and it offers insights into daily exposure to microbes when using banknotes in Colombia.

Amplicon restriction patterns associated with nitrogenase activity of root nodules for selection of superior Myrica seedlings.

Trees of Myrica sp. grow abundantly in the forests of Meghalaya, India. These trees are actinorhizal and harbour nitrogen-fixing Frankia in their root nodules and contribute positively towards the enhancement of nitrogen status of forest areas. They can be used in rejuvenation of mine spoils and nitrogen-depleted fallow lands generated due to slash and burn agriculture practiced in the area. We have studied the association of amplicon restriction patterns (ARPs) of Myrica ribosomal RNA gene and internal transcribed spacer (ITS) region and nitrogenase activity of its root nodules. We found that ARPs thus obtained could be used as markers for early screening of seedlings that could support strains of Frankia that fix atmospheric nitrogen more efficiently.

Induction of humoral responses to BHV-1 glycoprotein D expressed by HSV-1 amplicon vectors

Herpes simplex virus type-1 (HSV-1) amplicon vectors are versatile and useful tools for transferring genes into cells that are capable of stimulating a specific immune response to their expressed antigens. In this work, two HSV-1-derived amplicon vectors were generated. One of these expressed the full-length glycoprotein D (gD) of bovine herpesvirus 1 while the second expressed the truncated form of gD (gDtr) which lacked the trans-membrane region. After evaluating gD expression in the infected cells, the ability of both vectors to induce a specific gD immune response was tested in BALB/c mice that were intramuscularly immunized. Specific serum antibody responses were detected in mice inoculated with both vectors, and the response against truncated gD was higher than the response against full-length gD. These results reinforce previous findings that HSV-1 amplicon vectors can potentially deliver antigens to animals and highlight the prospective use of these vectors for treating infectious bovine rhinotracheitis disease.

SSR-Primer Generator: A Tool for Finding Simple Sequence Repeats and Designing SSR-Primers

Simple sequence repeats (SSRs) are ubiquitous short tandem duplications found within eukaryotic genomes. Their length variability and abundance throughout the genome has led them to be widely used as molecular markers for crop-breeding programs, facilitating the use of marker-assisted selection as well as estimation of genetic population structure. Here, we report a software application, "SSR-Primer Generator" for SSR discovery, SSR-primer design, and homology-based search of in silico amplicons from a DNA sequence dataset. On submission of multiple FASTA-format DNA sequences, those analyses are batch processed in a Java runtime environment (JRE) platform, in a pipeline, and the resulting data are visualized in HTML tabular format. This application will be a useful tool for reducing the time and costs associated with the development and application of SSR markers.

Influence of amplicon size on the polymerase chain reaction of Parvovirus B19 genome in formalin-fixed specimens / Influência do tamanho do amplicon na reação em cadeia da polimerase na detecção do genoma do PB19 em amostras fixadas em formalina

The polymerase chain reaction (PCR) has provided diagnosis of archival material, but some fixation methods such as formalin damage DNA and, subsequently, affect PCR analysis, particularly paraffin-embedded tissues. PCR is known due to its high specificity and sensitivity, although some difficulties arise when formalinfixed and paraffin-embedded tissue is used. Not only does this occur due to protein cross-linking, which increases with longer fixation time, but it also happens due to the direct damage that formalin causes in the DNA itself. PCR was used to analyze placenta and fetal organs from 34 samples with suspected Parvovirus B19 infection. It was not possible to amplify Parvovirus B19 DNA using nested-PCR, probably due to the size of the amplicon generated with the first set of primers. We approached this problem by using only the second set of primers. Two out of 34 tissue samples (5,9 percent) were positive by PCR. However, PCR performed on corresponding fetal organs was negative in one of the two. We also observed a negative relation between the thickness of the tissue fragment and the positivity of the samples. In conclusion, although PCR is highly specific and sensitive in fresh or ideally fixed material, a careful standardization of PCR assays is necessary when using formalin fixed paraffin-embedded tissues by applying primers that require smaller DNA fragments for amplification.

A reação em cadeia da polimerase (PCR) tem fornecido diagnóstico de material de arquivo, mas alguns métodos de fixação, tais como formalina, provocam danos ao DNA e subsequentemente afetam sua análise, particularmente tecidos embebidos em parafina. A PCR é conhecida pela sua alta especificidade e sensibilidade, embora algumas dificuldades ocorram quando o material utilizado foi fixado em formalina e embebido em parafina. Isso não se deve somente pela formação de cross-linkings com proteínas, a qual aumenta com o maior tempo de fixação, mas também pelo dano direto que a formalina causa no DNA. PCR foi usada para analisar placenta e órgão fetais de 34 amostras com suspeita de infecção pelo Parvovírus B19 (PB19). Não foi possível amplificar o DNA do PB19 usando nested-PCR, provavelmente devido ao tamanho do amplicon gerado com o primeiro passo dos primers. Adequamos o problema utilizando somente o segundo par de primers e pudemos observar que das 34 amostras, duas eram positivas para PCR (5,9 por cento). Entretanto, a PCR dos órgãos fetais foi negativa em um de dois casos. Observamos também uma relação negativa entre a espessura do corte dos materiais com a positividade das amostras. Em conclusão, embora a PCR seja altamente específica e sensível em amostras a fresco ou idealmente fixadas, uma padronização cuidadosa para análise com a PCR é necessária quando se utiliza tecidos fixados em formalina e embebidos em parafina utilizando primers que requerem menor fragmento de DNA para a amplificação.

HSV AMPLICON-MEDIATED NEUROTROPHIN-3 TRANSDUCTION PREVENTS MOUSE SPIRAL GANGLION NEURONS FROM DAMAGE / 解剖学报

Objective To determine whether NT 3 overexpression could abrogate DDP toxicity in vitro . Methods We constructed a herpes simplex virus(HSV) amplicon vector to transduce a c myc tagged version of rat NT 3. In the HSVnt 3myc vector, the chimeric neurotrophin cDNA was placed under the transcriptional control of the CMVIE promoter. The resultant vector was packaged utilizing the recently developed helper virus free method. Results Transduction of NT 3myc in cultured mouse cochlear explants at a multiplicity of infection(MOI) of 0 25 resulted in production of NT 3 up to 3?g/L over a 48 hours period. The mouse cochlear explants were transduced with HSVnt 3myc or HSVmiap(control vector expressing the reporter gene, murine intestinal alkaline phosphatase) for 48 hours and the exposed to cisplatin for 48 hours.The cochlear explants transduced with HSVnt 3myc had a significantly greater number of SGN survival than the control group. NT 3 powerfully enhanced the process length and the density of SGNs.Conclusion These data demonstrate that the neurotrophic cDNA transduction via HSV amplicon helper free virus system can attenuate the ototoxic action of DDP on organotypic culture. The potency of NT 3 in protecting spiral ganglion neurons from degenerating suggests that neurotrophins might be useful for the prevention or treatment of hearing disorders. [