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September 22, 2019

Full-length transcriptome of Misgurnus anguillicaudatus provides insights into evolution of genus Misgurnus.

Reconstruction and annotation of transcripts, particularly for a species without reference genome, plays a critical role in gene discovery, investigation of genomic signatures, and genome annotation in the pre-genomic era. This study generated 33,330 full-length transcripts of diploid M. anguillicaudatus using PacBio SMRT Sequencing. A total of 6,918 gene families were identified with two or more isoforms, and 26,683 complete ORFs with an average length of 1,497?bp were detected. Totally, 1,208 high-confidence lncRNAs were identified, and most of these appeared to be precursor transcripts of miRNAs or snoRNAs. Phylogenetic tree of the Misgurnus species was inferred based on the 1,905 single copy orthologous genes. The tetraploid and diploid M. anguillicaudatus grouped into a clade, and M. bipartitus showed a closer relationship with the M. anguillicaudatus. The overall evolutionary rates of tetraploid M. anguillicaudatus were significantly higher than those of other Misgurnus species. Meanwhile, 28 positively selected genes were identified in M. anguillicaudatus clade. These positively selected genes may play critical roles in the adaptation to various habitat environments for M. anguillicaudatus. This study could facilitate further exploration of the genomic signatures of M. anguillicaudatus and provide potential insights into unveiling the evolutionary history of tetraploid loach.

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September 22, 2019

Analyses of intestinal microbiota: culture versus sequencing.

Analyzing human as well as animal microbiota composition has gained growing interest because structural components and metabolites of microorganisms fundamentally influence all aspects of host physiology. Originally dominated by culture-dependent methods for exploring these ecosystems, the development of molecular techniques such as high throughput sequencing has dramatically increased our knowledge. Because many studies of the microbiota are based on the bacterial 16S ribosomal RNA (rRNA) gene targets, they can, at least in principle, be compared to determine the role of the microbiome composition for developmental processes, host metabolism, and physiology as well as different diseases. In our review, we will summarize differences and pitfalls in current experimental protocols, including all steps from nucleic acid extraction to bioinformatical analysis which may produce variation that outweighs subtle biological differences. Future developments, such as integration of metabolomic, transcriptomic, and metagenomic data sets and standardization of the procedures, will be discussed. © The Author 2015. Published by Oxford University Press on behalf of the Institute for Laboratory Animal Research. All rights reserved. For permissions, please email: journals.permissions@oup.com.

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September 22, 2019

Molecular mechanisms of acclimatization to phosphorus starvation and recovery underlying full-length transcriptome profiling in barley (Hordeum vulgare L.).

A lack of phosphorus (P) in plants can severely constrain growth and development. Barley, one of the earliest domesticated crops, is extensively planted in poor soil around the world. To date, the molecular mechanisms of enduring low phosphorus, at the transcriptional level, in barley are still unclear. In the present study, two different barley genotypes (GN121 and GN42)-with contrasting phosphorus efficiency-were used to reveal adaptations to low phosphorus stress, at three time points, at the morphological, physiological, biochemical, and transcriptome level. GN121 growth was less affected by phosphorus starvation and recovery than that of GN42. The biomass and inorganic phosphorus concentration of GN121 and GN42 declined under the low phosphorus-induced stress and increased after recovery with normal phosphorus. However, the range of these parameters was higher in GN42 than in GN121. Subsequently, a more complete genome annotation was obtained by correcting with the data sequenced on Illumina HiSeq X 10 and PacBio RSII SMRT platform. A total of 6,182 and 5,270 differentially expressed genes (DEGs) were identified in GN121 and GN42, respectively. The majority of these DEGs were involved in phosphorus metabolism such as phospholipid degradation, hydrolysis of phosphoric enzymes, sucrose synthesis, phosphorylation/dephosphorylation and post-transcriptional regulation; expression of these genes was significantly different between GN121 and GN42. Specifically, six and seven DEGs were annotated as phosphorus transporters in roots and leaves, respectively. Furthermore, a putative model was constructed relying on key metabolic pathways related to phosphorus to illustrate the higher phosphorus efficiency of GN121 compared to GN42 under low phosphorus conditions. Results from this study provide a multi-transcriptome database and candidate genes for further study on phosphorus use efficiency (PUE).

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September 22, 2019

HIV-1 interacts with human endogenous retrovirus K (HML-2) envelopes derived from human primary lymphocytes.

Human endogenous retroviruses (HERVs) are viruses that have colonized the germ line and spread through vertical passage. Only the more recently acquired HERVs, such as the HERV-K (HML-2) group, maintain coding open reading frames. Expression of HERV-Ks has been linked to different pathological conditions, including HIV infection, but our knowledge on which specific HERV-Ks are expressed in primary lymphocytes currently is very limited. To identify the most expressed HERV-Ks in an unbiased manner, we analyzed their expression patterns in peripheral blood lymphocytes using Pacific Biosciences (PacBio) single-molecule real-time (SMRT) sequencing. We observe that three HERV-Ks (KII, K102, and K18) constitute over 90% of the total HERV-K expression in primary human lymphocytes of five different donors. We also show experimentally that two of these HERV-K env sequences (K18 and K102) retain their ability to produce full-length and posttranslationally processed envelope proteins in cell culture. We show that HERV-K18 Env can be incorporated into HIV-1 but not simian immunodeficiency virus (SIV) particles. Moreover, HERV-K18 Env incorporation into HIV-1 virions is dependent on HIV-1 matrix. Taken together, we generated high-resolution HERV-K expression profiles specific for activated human lymphocytes. We found that one of the most abundantly expressed HERV-K envelopes not only makes a full-length protein but also specifically interacts with HIV-1. Our findings raise the possibility that these endogenous retroviral Env proteins could directly influence HIV-1 replication.Here, we report the HERV-K expression profile of primary lymphocytes from 5 different healthy donors. We used a novel deep-sequencing technology (PacBio SMRT) that produces the long reads necessary to discriminate the complexity of HERV-K expression. We find that primary lymphocytes express up to 32 different HERV-K envelopes, and that at least two of the most expressed Env proteins retain their ability to make a protein. Importantly, one of them, the envelope glycoprotein of HERV-K18, is incorporated into HIV-1 in an HIV matrix-specific fashion. The ramifications of such interactions are discussed, as the possibility of HIV-1 target tissue broadening and immune evasion are considered.

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September 22, 2019

Clonal distribution of BCR-ABL1 mutations and splice isoforms by single-molecule long-read RNA sequencing.

The evolution of mutations in the BCR-ABL1 fusion gene transcript renders CML patients resistant to tyrosine kinase inhibitor (TKI) based therapy. Thus screening for BCR-ABL1 mutations is recommended particularly in patients experiencing poor response to treatment. Herein we describe a novel approach for the detection and surveillance of BCR-ABL1 mutations in CML patients.To detect mutations in the BCR-ABL1 transcript we developed an assay based on the Pacific Biosciences (PacBio) sequencing technology, which allows for single-molecule long-read sequencing of BCR-ABL1 fusion transcript molecules. Samples from six patients with poor response to therapy were analyzed both at diagnosis and follow-up. cDNA was generated from total RNA and a 1,6 kb fragment encompassing the BCR-ABL1 transcript was amplified using long range PCR. To estimate the sensitivity of the assay, a serial dilution experiment was performed.Over 10,000 full-length BCR-ABL1 sequences were obtained for all samples studied. Through the serial dilution analysis, mutations in CML patient samples could be detected down to a level of at least 1%. Notably, the assay was determined to be sufficiently sensitive even in patients harboring a low abundance of BCR-ABL1 levels. The PacBio sequencing successfully identified all mutations seen by standard methods. Importantly, we identified several mutations that escaped detection by the clinical routine analysis. Resistance mutations were found in all but one of the patients. Due to the long reads afforded by PacBio sequencing, compound mutations present in the same molecule were readily distinguished from independent alterations arising in different molecules. Moreover, several transcript isoforms of the BCR-ABL1 transcript were identified in two of the CML patients. Finally, our assay allowed for a quick turn around time allowing samples to be reported upon within 2 days.In summary the PacBio sequencing assay can be applied to detect BCR-ABL1 resistance mutations in both diagnostic and follow-up CML patient samples using a simple protocol applicable to routine diagnosis. The method besides its sensitivity, gives a complete view of the clonal distribution of mutations, which is of importance when making therapy decisions.

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September 22, 2019

Major histocompatibility complex haplotyping and long-amplicon allele discovery in cynomolgus macaques from Chinese breeding facilities.

Very little is currently known about the major histocompatibility complex (MHC) region of cynomolgus macaques (Macaca fascicularis; Mafa) from Chinese breeding centers. We performed comprehensive MHC class I haplotype analysis of 100 cynomolgus macaques from two different centers, with animals from different reported original geographic origins (Vietnamese, Cambodian, and Cambodian/Indonesian mixed-origin). Many of the samples were of known relation to each other (sire, dam, and progeny sets), making it possible to characterize lineage-level haplotypes in these animals. We identified 52 Mafa-A and 74 Mafa-B haplotypes in this cohort, many of which were restricted to specific sample origins. We also characterized full-length MHC class I transcripts using Pacific Biosciences (PacBio) RS II single-molecule real-time (SMRT) sequencing. This technology allows for complete read-through of unfragmented MHC class I transcripts (~1100 bp in length), so no assembly is required to unambiguously resolve novel full-length sequences. Overall, we identified 311 total full-length transcripts in a subset of 72 cynomolgus macaques from these Chinese breeding facilities; 130 of these sequences were novel and an additional 115 extended existing short database sequences to span the complete open reading frame. This significantly expands the number of Mafa-A, Mafa-B, and Mafa-I full-length alleles in the official cynomolgus macaque MHC class I database. The PacBio technique described here represents a general method for full-length allele discovery and genotyping that can be extended to other complex immune loci such as MHC class II, killer immunoglobulin-like receptors, and Fc gamma receptors.

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September 22, 2019

Melanization of mycorrhizal fungal necromass structures microbial decomposer communities

Mycorrhizal fungal necromass is increasingly recognized as an important contributor to soil organic carbon pools, particularly in forest ecosystems. While its decomposition rate is primarily determined by biochemical composition, how traits such as melanin content affect the structure of necromass decomposer communities remains poorly understood. To assess the role of biochemical traits on microbial decomposer community composition and functioning, we incubated melanized and non-melanized necromass of the mycorrhizal fungus Meliniomyces bicolor in Pinus- and Quercus-dominated forests in Minnesota, USA and then assessed the associated fungal and bacterial decomposer communities after 1, 2 and 3 months using high-throughput sequencing. Melanized necromass decomposed significantly slower than non-melanized necromass in both forests. The structure of the microbial decomposer communities depended significantly on necromass melanin content, although the effect was stronger for fungi than bacteria. On non-melanized necromass, fungal communities were dominated by r-selected ascomycete and mucoromycete microfungi early and then replaced by basidiomycete ectomycorrhizal fungi, while on melanized necromass these groups were co-dominant throughout the incubation. Bacterial communities were dominated by both specialist mycophageous and generalist taxa. Synthesis. Our results indicate that necromass biochemistry not only strongly affects rates of decomposition but also the structure of the associated decomposer communities. Furthermore, the observed colonization patterns suggest that fungi, and particularly ectomycorrhizal fungi, may play a more important role in necromass decomposition than previously recognized.

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September 22, 2019

Evaluation of bacterial contamination in raw milk, ultra-high temperature milk and infant formula using single molecule, real-time sequencing technology.

The Pacific Biosciences (Menlo Park, CA) single molecule, real-time sequencing technology (SMRT) was reported to have some advantages in analyzing the bacterial profile of environmental samples. In this study, the presence of bacterial contaminants in raw milk, UHT milk, and infant formula was determined by SMRT sequencing of the full length 16S rRNA gene. The bacterial profiles obtained at different taxonomic levels revealed clear differences in bacterial community structure across the 16 analyzed dairy samples. No indicative pathogenic bacteria were found in any of these tested samples. However, some of the detected bacterial species (e.g., Bacillus cereus, Enterococcus casseliflavus, and Enterococcus gallinarum) might potentially relate with product quality defects and bacterial antibiotic gene transfer. Although only a limited number of dairy samples were analyzed here, our data have demonstrated for the first time the feasibility of using the SMRT sequencing platform in detecting bacterial contamination. Our paper also provides interesting reference information for future development of new precautionary strategies for controlling the dairy safety in large-scale industrialized production lines. Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

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September 22, 2019

Caught in the middle with multiple displacement amplification: the myth of pooling for avoiding multiple displacement amplification bias in a metagenome.

Shotgun metagenomics has become an important tool for investigating the ecology of microorganisms. Underlying these investigations is the assumption that metagenome sequence data accurately estimates the census of microbial populations. Multiple displacement amplification (MDA) of microbial community DNA is often used in cases where it is difficult to obtain enough DNA for sequencing; however, MDA can result in amplification biases that may impact subsequent estimates of population census from metagenome data. Some have posited that pooling replicate MDA reactions negates these biases and restores the accuracy of population analyses. This assumption has not been empirically tested.Using mock viral communities, we examined the influence of pooling on population-scale analyses. In pooled and single reaction MDA treatments, sequence coverage of viral populations was highly variable and coverage patterns across viral genomes were nearly identical, indicating that initial priming biases were reproducible and that pooling did not alleviate biases. In contrast, control unamplified sequence libraries showed relatively even coverage across phage genomes.MDA should be avoided for metagenomic investigations that require quantitative estimates of microbial taxa and gene functional groups. While MDA is an indispensable technique in applications such as single-cell genomics, amplification biases cannot be overcome by combining replicate MDA reactions. Alternative library preparation techniques should be utilized for quantitative microbial ecology studies utilizing metagenomic sequencing approaches.

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September 22, 2019

PCR and omics based techniques to study the diversity, ecology and biology of anaerobic fungi: Insights, challenges andopportunities.

Anaerobic fungi (phylum Neocallimastigomycota) are common inhabitants of the digestive tract of mammalian herbivores, and in the rumen, can account for up to 20% of the microbial biomass. Anaerobic fungi play a primary role in the degradation of lignocellulosic plant material. They also have a syntrophic interaction with methanogenic archaea, which increases their fiber degradation activity. To date, nine anaerobic fungal genera have been described, with further novel taxonomic groupings known to exist based on culture-independent molecular surveys. However, the true extent of their diversity may be even more extensively underestimated as anaerobic fungi continue being discovered in yet unexplored gut and non-gut environments. Additionally many studies are now known to have used primers that provide incomplete coverage of the Neocallimastigomycota. For ecological studies the internal transcribed spacer 1 region (ITS1) has been the taxonomic marker of choice, but due to various limitations the large subunit rRNA (LSU) is now being increasingly used. How the continued expansion of our knowledge regarding anaerobic fungal diversity will impact on our understanding of their biology and ecological role remains unclear; particularly as it is becoming apparent that anaerobic fungi display niche differentiation. As a consequence, there is a need to move beyond the broad generalization of anaerobic fungi as fiber-degraders, and explore the fundamental differences that underpin their ability to exist in distinct ecological niches. Application of genomics, transcriptomics, proteomics and metabolomics to their study in pure/mixed cultures and environmental samples will be invaluable in this process. To date the genomes and transcriptomes of several characterized anaerobic fungal isolates have been successfully generated. In contrast, the application of proteomics and metabolomics to anaerobic fungal analysis is still in its infancy. A central problem for all analyses, however, is the limited functional annotation of anaerobic fungal sequence data. There is therefore an urgent need to expand information held within publicly available reference databases. Once this challenge is overcome, along with improved sample collection and extraction, the application of these techniques will be key in furthering our understanding of the ecological role and impact of anaerobic fungi in the wide range of environments they inhabit.

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September 22, 2019

A quantitative SMRT cell sequencing method for ribosomal amplicons.

Advances in sequencing technologies continue to provide unprecedented opportunities to characterize microbial communities. For example, the Pacific Biosciences Single Molecule Real-Time (SMRT) platform has emerged as a unique approach harnessing DNA polymerase activity to sequence template molecules, enabling long reads at low costs. With the aim to simultaneously classify and enumerate in situ microbial populations, we developed a quantitative SMRT (qSMRT) approach that involves the addition of exogenous standards to quantify ribosomal amplicons derived from environmental samples. The V7-9 regions of 18S SSU rDNA were targeted and quantified from protistan community samples collected in the Ross Sea during the Austral summer of 2011. We used three standards of different length and optimized conditions to obtain accurate quantitative retrieval across the range of expected amplicon sizes, a necessary criterion for analyzing taxonomically diverse 18S rDNA molecules from natural environments. The ability to concurrently identify and quantify microorganisms in their natural environment makes qSMRT a powerful, rapid and cost-effective approach for defining ecosystem diversity and function. Copyright © 2017 Elsevier B.V. All rights reserved.

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September 22, 2019

Current developments in molecular monitoring in chronic myeloid leukemia.

Molecular monitoring plays an essential role in the clinical management of chronic myeloid leukemia (CML) patients, and now guides clinical decision making. Quantitative reverse-transcriptase-polymerase-chain-reaction (qRT-PCR) assessment of BCR-ABL1 transcript levels has become the standard of care protocol in CML. However, further developments are required to assess leukemic burden more efficiently, monitor minimal residual disease (MRD), detect mutations that drive resistance to tyrosine kinase inhibitor (TKI) therapy and identify predictors of response to TKI therapy. Cartridge-based BCR-ABL1 quantitation, digital PCR and next generation sequencing are examples of technologies which are currently being explored, evaluated and translated into the clinic. Here we review the emerging molecular methods/technologies currently being developed to advance molecular monitoring in CML.

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September 22, 2019

A single-cell genome for Thiovulum sp.

We determined a significant fraction of the genome sequence of a representative of Thiovulum, the uncultivated genus of colorless sulfur Epsilonproteobacteria, by analyzing the genome sequences of four individual cells collected from phototrophic mats from Elkhorn Slough, California. These cells were isolated utilizing a microfluidic laser-tweezing system, and their genomes were amplified by multiple-displacement amplification prior to sequencing. Thiovulum is a gradient bacterium found at oxic-anoxic marine interfaces and noted for its distinctive morphology and rapid swimming motility. The genomic sequences of the four individual cells were assembled into a composite genome consisting of 221 contigs covering 2.083 Mb including 2,162 genes. This single-cell genome represents a genomic view of the physiological capabilities of isolated Thiovulum cells. Thiovulum is the second-fastest bacterium ever observed, swimming at 615 µm/s, and this genome shows that this rapid swimming motility is a result of a standard flagellar machinery that has been extensively characterized in other bacteria. This suggests that standard flagella are capable of propelling bacterial cells at speeds much faster than typically thought. Analysis of the genome suggests that naturally occurring Thiovulum populations are more diverse than previously recognized and that studies performed in the past probably address a wide range of unrecognized genotypic and phenotypic diversities of Thiovulum. The genome presented in this article provides a basis for future isolation-independent studies of Thiovulum, where single-cell and metagenomic tools can be used to differentiate between different Thiovulum genotypes.

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September 22, 2019

Great differences in performance and outcome of high-throughput sequencing data analysis platforms for fungal metabarcoding.

Along with recent developments in high-throughput sequencing (HTS) technologies and thus fast accumulation of HTS data, there has been a growing need and interest for developing tools for HTS data processing and communication. In particular, a number of bioinformatics tools have been designed for analysing metabarcoding data, each with specific features, assumptions and outputs. To evaluate the potential effect of the application of different bioinformatics workflow on the results, we compared the performance of different analysis platforms on two contrasting high-throughput sequencing data sets. Our analysis revealed that the computation time, quality of error filtering and hence output of specific bioinformatics process largely depends on the platform used. Our results show that none of the bioinformatics workflows appears to perfectly filter out the accumulated errors and generate Operational Taxonomic Units, although PipeCraft, LotuS and PIPITS perform better than QIIME2 and Galaxy for the tested fungal amplicon dataset. We conclude that the output of each platform requires manual validation of the OTUs by examining the taxonomy assignment values.

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September 22, 2019

High-resolution community profiling of arbuscular mycorrhizal fungi.

Community analyses of arbuscular mycorrhizal fungi (AMF) using ribosomal small subunit (SSU) or internal transcribed spacer (ITS) DNA sequences often suffer from low resolution or coverage. We developed a novel sequencing based approach for a highly resolving and specific profiling of AMF communities. We took advantage of previously established AMF-specific PCR primers that amplify a c. 1.5-kb long fragment covering parts of SSU, ITS and parts of the large ribosomal subunit (LSU), and we sequenced the resulting amplicons with single molecule real-time (SMRT) sequencing. The method was applicable to soil and root samples, detected all major AMF families and successfully discriminated closely related AMF species, which would not be discernible using SSU sequences. In inoculation tests we could trace the introduced AMF inoculum at the molecular level. One of the introduced strains almost replaced the local strain(s), revealing that AMF inoculation can have a profound impact on the native community. The methodology presented offers researchers a powerful new tool for AMF community analysis because it unifies improved specificity and enhanced resolution, whereas the drawback of medium sequencing throughput appears of lesser importance for low-diversity groups such as AMF.© 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

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