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September 22, 2019  |  

Metagenomic SMRT sequencing-based exploration of novel lignocellulose-degrading capability in wood detritus from Torreya nucifera in Bija forest on Jeju Island.

Lignocellulose, mostly composed of cellulose, hemicellulose and lignin generated through secondary growth of woody plant, is considered as promising resources for bio-fuel. In order to use lignocellulose as a biofuel, the biodegradation besides high-cost chemical treatments were applied, but its knowledge on decomposition of lignocellulose occurring in a natural environment were insufficient. We analyzed 16S rRNA gene and metagenome to understand how the lignocellulose are decomposed naturally in decayed Torreya nucifera (L) of Bija forest (Bijarim) in Gotjawal, an ecologically distinct environment. A total of 464,360 reads were obtained from 16S rRNA gene sequencing, representing diverse phyla; Proteobacteria (51%), Bacteroidetes (11%) and Actinobacteria (10%). The metagenome analysis using Single Molecules Real-Time Sequencing revealed that the assembled contigs determined by originated from Proteobacteria (58%) and Actinobacteria (10.3%). Carbohydrate Active enZYmes (CAZy) and Protein families (Pfam) based analysis showed that Proteobacteria was involved in degrading whole lignocellulose and Actinobacteria played a role only in a part of hemicellulose degradation. Combining these results, it suggested that Proteobacteria and Actinobacteria had selective biodegradation potential for different lignocellulose substrate. Thus, it is considered that understanding of the systemic microbial degradation pathways may be a useful strategy for recycle of lignocellulosic biomass and the microbial enzymes in Bija forest can be useful natural resources in industrial processes.


September 22, 2019  |  

The industrial melanism mutation in British peppered moths is a transposable element.

Discovering the mutational events that fuel adaptation to environmental change remains an important challenge for evolutionary biology. The classroom example of a visible evolutionary response is industrial melanism in the peppered moth (Biston betularia): the replacement, during the Industrial Revolution, of the common pale typica form by a previously unknown black (carbonaria) form, driven by the interaction between bird predation and coal pollution. The carbonaria locus has been coarsely localized to a 200-kilobase region, but the specific identity and nature of the sequence difference controlling the carbonaria-typica polymorphism, and the gene it influences, are unknown. Here we show that the mutation event giving rise to industrial melanism in Britain was the insertion of a large, tandemly repeated, transposable element into the first intron of the gene cortex. Statistical inference based on the distribution of recombined carbonaria haplotypes indicates that this transposition event occurred around 1819, consistent with the historical record. We have begun to dissect the mode of action of the carbonaria transposable element by showing that it increases the abundance of a cortex transcript, the protein product of which plays an important role in cell-cycle regulation, during early wing disc development. Our findings fill a substantial knowledge gap in the iconic example of microevolutionary change, adding a further layer of insight into the mechanism of adaptation in response to natural selection. The discovery that the mutation itself is a transposable element will stimulate further debate about the importance of ‘jumping genes’ as a source of major phenotypic novelty.


September 22, 2019  |  

Complex rearrangements and oncogene amplifications revealed by long-read DNA and RNA sequencing of a breast cancer cell line.

The SK-BR-3 cell line is one of the most important models for HER2+ breast cancers, which affect one in five breast cancer patients. SK-BR-3 is known to be highly rearranged, although much of the variation is in complex and repetitive regions that may be underreported. Addressing this, we sequenced SK-BR-3 using long-read single molecule sequencing from Pacific Biosciences and develop one of the most detailed maps of structural variations (SVs) in a cancer genome available, with nearly 20,000 variants present, most of which were missed by short-read sequencing. Surrounding the important ERBB2 oncogene (also known as HER2), we discover a complex sequence of nested duplications and translocations, suggesting a punctuated progression. Full-length transcriptome sequencing further revealed several novel gene fusions within the nested genomic variants. Combining long-read genome and transcriptome sequencing enables an in-depth analysis of how SVs disrupt the genome and sheds new light on the complex mechanisms involved in cancer genome evolution.© 2018 Nattestad et al.; Published by Cold Spring Harbor Laboratory Press.


September 22, 2019  |  

Next-generation sequencing for pathogen detection and identification

Over the past decade, the field of genomics has seen such drastic improvements in sequencing chemistries that high-throughput sequencing, or next-generation sequencing (NGS), is being applied to generate data across many disciplines. NGS instruments are becoming less expensive, faster, and smaller, and therefore are being adopted in an increasing number of laboratories, including clinical laboratories. Thus far, clinical use of NGS has been mostly focused on the human genome, for purposes such as characterizing the molecular basis of cancer or for diagnosing and understanding the basis of rare genetic disorders. There are, however, an increasing number of examples whereby NGS is employed to discover novel pathogens, and these cases provide precedent for the use of NGS in microbial diagnostics. NGS has many advantages over traditional microbial diagnostic methods, such as unbiased rather than pathogen-specific protocols, ability to detect fastidious or non-culturable organisms, and ability to detect co-infections. One of the most impressive advantages of NGS is that it requires little or no prior knowledge of the pathogen, unlike many other diagnostic assays; therefore for pathogen discovery, NGS is very valuable. However, despite these advantages, there are challenges involved in implementing NGS for routine clinical microbiological diagnosis. We discuss these advantages and challenges in the context of recently described research studies.


September 22, 2019  |  

Single molecule real-time (SMRT) sequencing comes of age: applications and utilities for medical diagnostics.

Short read massive parallel sequencing has emerged as a standard diagnostic tool in the medical setting. However, short read technologies have inherent limitations such as GC bias, difficulties mapping to repetitive elements, trouble discriminating paralogous sequences, and difficulties in phasing alleles. Long read single molecule sequencers resolve these obstacles. Moreover, they offer higher consensus accuracies and can detect epigenetic modifications from native DNA. The first commercially available long read single molecule platform was the RS system based on PacBio’s single molecule real-time (SMRT) sequencing technology, which has since evolved into their RSII and Sequel systems. Here we capsulize how SMRT sequencing is revolutionizing constitutional, reproductive, cancer, microbial and viral genetic testing.© The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research.


September 22, 2019  |  

Metataxonomic and metagenomic approaches vs. culture-based techniques for clinical pathology.

Diagnoses that are both timely and accurate are critically important for patients with life-threatening or drug resistant infections. Technological improvements in High-Throughput Sequencing (HTS) have led to its use in pathogen detection and its application in clinical diagnoses of infectious diseases. The present study compares two HTS methods, 16S rRNA marker gene sequencing (metataxonomics) and whole metagenomic shotgun sequencing (metagenomics), in their respective abilities to match the same diagnosis as traditional culture methods (culture inference) for patients with ventilator associated pneumonia (VAP). The metagenomic analysis was able to produce the same diagnosis as culture methods at the species-level for five of the six samples, while the metataxonomic analysis was only able to produce results with the same species-level identification as culture for two of the six samples. These results indicate that metagenomic analyses have the accuracy needed for a clinical diagnostic tool, but full integration in diagnostic protocols is contingent on technological improvements to decrease turnaround time and lower costs.


September 22, 2019  |  

Computational analysis of alternative splicing in plant genomes.

Computational analyses play crucial roles in characterizing splicing isoforms in plant genomes. In this review, we provide a survey of computational tools used in recently published, genome-scale splicing analyses in plants. We summarize the commonly used software and pipelines for read mapping, isoform reconstruction, isoform quantification, and differential expression analysis. We also discuss methods for analyzing long reads and the strategies to combine long and short reads in identifying splicing isoforms. We review several tools for characterizing local splicing events, splicing graphs, coding potential, and visualizing splicing isoforms. We further discuss the procedures for identifying conserved splicing isoforms across plant species. Finally, we discuss the outlook of integrating other genomic data with splicing analyses to identify regulatory mechanisms of AS on genome-wide scale. Copyright © 2018 Elsevier B.V. All rights reserved.


September 22, 2019  |  

Genome and evolution of the shade-requiring medicinal herb Panax ginseng.

Panax ginseng C. A. Meyer, reputed as the king of medicinal herbs, has slow growth, long generation time, low seed production and complicated genome structure that hamper its study. Here, we unveil the genomic architecture of tetraploid P. ginseng by de novo genome assembly, representing 2.98 Gbp with 59 352 annotated genes. Resequencing data indicated that diploid Panax species diverged in association with global warming in Southern Asia, and two North American species evolved via two intercontinental migrations. Two whole genome duplications (WGD) occurred in the family Araliaceae (including Panax) after divergence with the Apiaceae, the more recent one contributing to the ability of P. ginseng to overwinter, enabling it to spread broadly through the Northern Hemisphere. Functional and evolutionary analyses suggest that production of pharmacologically important dammarane-type ginsenosides originated in Panax and are produced largely in shoot tissues and transported to roots; that newly evolved P. ginseng fatty acid desaturases increase freezing tolerance; and that unprecedented retention of chlorophyll a/b binding protein genes enables efficient photosynthesis under low light. A genome-scale metabolic network provides a holistic view of Panax ginsenoside biosynthesis. This study provides valuable resources for improving medicinal values of ginseng either through genomics-assisted breeding or metabolic engineering.© 2018 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.


September 22, 2019  |  

Hybrid error correction and de novo assembly of single-molecule sequencing reads.

Single-molecule sequencing instruments can generate multikilobase sequences with the potential to greatly improve genome and transcriptome assembly. However, the error rates of single-molecule reads are high, which has limited their use thus far to resequencing bacteria. To address this limitation, we introduce a correction algorithm and assembly strategy that uses short, high-fidelity sequences to correct the error in single-molecule sequences. We demonstrate the utility of this approach on reads generated by a PacBio RS instrument from phage, prokaryotic and eukaryotic whole genomes, including the previously unsequenced genome of the parrot Melopsittacus undulatus, as well as for RNA-Seq reads of the corn (Zea mays) transcriptome. Our long-read correction achieves >99.9% base-call accuracy, leading to substantially better assemblies than current sequencing strategies: in the best example, the median contig size was quintupled relative to high-coverage, second-generation assemblies. Greater gains are predicted if read lengths continue to increase, including the prospect of single-contig bacterial chromosome assembly.


September 22, 2019  |  

Next generation multilocus sequence typing (NGMLST) and the analytical software program MLSTEZ enable efficient, cost-effective, high-throughput, multilocus sequencing typing.

Multilocus sequence typing (MLST) has become the preferred method for genotyping many biological species, and it is especially useful for analyzing haploid eukaryotes. MLST is rigorous, reproducible, and informative, and MLST genotyping has been shown to identify major phylogenetic clades, molecular groups, or subpopulations of a species, as well as individual strains or clones. MLST molecular types often correlate with important phenotypes. Conventional MLST involves the extraction of genomic DNA and the amplification by PCR of several conserved, unlinked gene sequences from a sample of isolates of the taxon under investigation. In some cases, as few as three loci are sufficient to yield definitive results. The amplicons are sequenced, aligned, and compared by phylogenetic methods to distinguish statistically significant differences among individuals and clades. Although MLST is simpler, faster, and less expensive than whole genome sequencing, it is more costly and time-consuming than less reliable genotyping methods (e.g. amplified fragment length polymorphisms). Here, we describe a new MLST method that uses next-generation sequencing, a multiplexing protocol, and appropriate analytical software to provide accurate, rapid, and economical MLST genotyping of 96 or more isolates in single assay. We demonstrate this methodology by genotyping isolates of the well-characterized, human pathogenic yeast Cryptococcus neoformans. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.


September 22, 2019  |  

High-resolution comparative analysis of great ape genomes.

Genetic studies of human evolution require high-quality contiguous ape genome assemblies that are not guided by the human reference. We coupled long-read sequence assembly and full-length complementary DNA sequencing with a multiplatform scaffolding approach to produce ab initio chimpanzee and orangutan genome assemblies. By comparing these with two long-read de novo human genome assemblies and a gorilla genome assembly, we characterized lineage-specific and shared great ape genetic variation ranging from single- to mega-base pair-sized variants. We identified ~17,000 fixed human-specific structural variants identifying genic and putative regulatory changes that have emerged in humans since divergence from nonhuman apes. Interestingly, these variants are enriched near genes that are down-regulated in human compared to chimpanzee cerebral organoids, particularly in cells analogous to radial glial neural progenitors. Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.


September 22, 2019  |  

Long-read based assembly and annotation of a Drosophila simulans genome

Long-read sequencing technologies enable high-quality, contiguous genome assemblies. Here we used SMRT sequencing to assemble the genome of a Drosophila simulans strain originating from Madagascar, the ancestral range of the species. We generated 8 Gb of raw data (~50x coverage) with a mean read length of 6,410 bp, a NR50 of 9,125 bp and the longest subread at 49 kb. We benchmarked six different assemblers and merged the best two assemblies from Canu and Falcon. Our final assembly was 127.41 Mb with a N50 of 5.38 Mb and 305 contigs. We anchored more than 4 Mb of novel sequence to the major chromosome arms, and significantly improved the assembly of peri-centromeric and telomeric regions. Finally, we performed full-length transcript sequencing and used this data in conjunction with short-read RNAseq data to annotate 13,422 genes in the genome, improving the annotation in regions with complex, nested gene structures.


September 22, 2019  |  

Genomic and metabolic diversity of Marine Group I Thaumarchaeota in the mesopelagic of two subtropical gyres.

Marine Group I (MGI) Thaumarchaeota are one of the most abundant and cosmopolitan chemoautotrophs within the global dark ocean. To date, no representatives of this archaeal group retrieved from the dark ocean have been successfully cultured. We used single cell genomics to investigate the genomic and metabolic diversity of thaumarchaea within the mesopelagic of the subtropical North Pacific and South Atlantic Ocean. Phylogenetic and metagenomic recruitment analysis revealed that MGI single amplified genomes (SAGs) are genetically and biogeographically distinct from existing thaumarchaea cultures obtained from surface waters. Confirming prior studies, we found genes encoding proteins for aerobic ammonia oxidation and the hydrolysis of urea, which may be used for energy production, as well as genes involved in 3-hydroxypropionate/4-hydroxybutyrate and oxidative tricarboxylic acid pathways. A large proportion of protein sequences identified in MGI SAGs were absent in the marine cultures Cenarchaeum symbiosum and Nitrosopumilus maritimus, thus expanding the predicted protein space for this archaeal group. Identifiable genes located on genomic islands with low metagenome recruitment capacity were enriched in cellular defense functions, likely in response to viral infections or grazing. We show that MGI Thaumarchaeota in the dark ocean may have more flexibility in potential energy sources and adaptations to biotic interactions than the existing, surface-ocean cultures.


September 22, 2019  |  

Next generation sequencing technology: Advances and applications.

Impressive progress has been made in the field of Next Generation Sequencing (NGS). Through advancements in the fields of molecular biology and technical engineering, parallelization of the sequencing reaction has profoundly increased the total number of produced sequence reads per run. Current sequencing platforms allow for a previously unprecedented view into complex mixtures of RNA and DNA samples. NGS is currently evolving into a molecular microscope finding its way into virtually every fields of biomedical research. In this chapter we review the technical background of the different commercially available NGS platforms with respect to template generation and the sequencing reaction and take a small step towards what the upcoming NGS technologies will bring. We close with an overview of different implementations of NGS into biomedical research. This article is part of a Special Issue entitled: From Genome to Function. Copyright © 2014 Elsevier B.V. All rights reserved.


September 22, 2019  |  

Defining cell identity with single cell omics.

Cells are a fundamental unit of life, and the ability to study the phenotypes and behaviors of individual cells is crucial to understanding the workings of complex biological systems. Cell phenotypes (epigenomic, transcriptomic, proteomic, and metabolomic) exhibit dramatic heterogeneity between and within the different cell types and states underlying cellular functional diversity. Cell genotypes can also display heterogeneity throughout an organism, in the form of somatic genetic variation-most notably in the emergence and evolution of tumors. Recent technical advances in single-cell isolation and the development of omics approaches sensitive enough to reveal these aspects of cell identity have enabled a revolution in the study of multicellular systems. In this review, we discuss the technologies available to resolve the genomes, epigenomes, transcriptomes, proteomes, and metabolomes of single cells from a wide variety of living systems.© 2018 The Authors. Proteomics Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.


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