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July 19, 2019

Biomonitoring for traditional herbal medicinal products using DNA metabarcoding and single molecule, real-time sequencing.

Global concerns have been paid to the potential hazard of traditional herbal medicinal products (THMPs). Substandard and counterfeit THMPs, including traditional Chinese patent medicine, health foods, dietary supplements, etc. are potential threats to public health. Recent marketplace studies using DNA barcoding have determined that the current quality control methods are not sufficient for ensuring the presence of authentic herbal ingredients and detection of contaminants/adulterants. An efficient biomonitoring method for THMPs is of great needed. Herein, metabarcoding and single-molecule, real-time (SMRT) sequencing were used to detect the multiple ingredients in Jiuwei Qianghuo Wan (JWQHW), a classical herbal prescription widely used in China for the last 800 years. Reference experimental mixtures and commercial JWQHW products from the marketplace were used to confirm the method. Successful SMRT sequencing results recovered 5416 and 4342 circular-consensus sequencing (CCS) reads belonging to the ITS2 and psbA-trnH regions. The results suggest that with the combination of metabarcoding and SMRT sequencing, it is repeatable, reliable, and sensitive enough to detect species in the THMPs, and the error in SMRT sequencing did not affect the ability to identify multiple prescribed species and several adulterants/contaminants. It has the potential for becoming a valuable tool for the biomonitoring of multi-ingredient THMPs.

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July 19, 2019

Piercing the dark matter: bioinformatics of long-range sequencing and mapping.

Several new genomics technologies have become available that offer long-read sequencing or long-range mapping with higher throughput and higher resolution analysis than ever before. These long-range technologies are rapidly advancing the field with improved reference genomes, more comprehensive variant identification and more complete views of transcriptomes and epigenomes. However, they also require new bioinformatics approaches to take full advantage of their unique characteristics while overcoming their complex errors and modalities. Here, we discuss several of the most important applications of the new technologies, focusing on both the currently available bioinformatics tools and opportunities for future research.

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July 19, 2019

The genome of Schmidtea mediterranea and the evolution of core cellular mechanisms.

The planarian Schmidtea mediterranea is an important model for stem cell research and regeneration, but adequate genome resources for this species have been lacking. Here we report a highly contiguous genome assembly of S. mediterranea, using long-read sequencing and a de novo assembler (MARVEL) enhanced for low-complexity reads. The S. mediterranea genome is highly polymorphic and repetitive, and harbours a novel class of giant retroelements. Furthermore, the genome assembly lacks a number of highly conserved genes, including critical components of the mitotic spindle assembly checkpoint, but planarians maintain checkpoint function. Our genome assembly provides a key model system resource that will be useful for studying regeneration and the evolutionary plasticity of core cell biological mechanisms.

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July 19, 2019

Genomic repeats, misassembly and reannotation: a case study with long-read resequencing of Porphyromonas gingivalis reference strains.

Without knowledge of their genomic sequences, it is impossible to make functional models of the bacteria that make up human and animal microbiota. Unfortunately, the vast majority of publicly available genomes are only working drafts, an incompleteness that causes numerous problems and constitutes a major obstacle to genotypic and phenotypic interpretation. In this work, we began with an example from the class Bacteroidia in the phylum Bacteroidetes, which is preponderant among human orodigestive microbiota. We successfully identify the genetic loci responsible for assembly breaks and misassemblies and demonstrate the importance and usefulness of long-read sequencing and curated reannotation.We showed that the fragmentation in Bacteroidia draft genomes assembled from massively parallel sequencing linearly correlates with genomic repeats of the same or greater size than the reads. We also demonstrated that some of these repeats, especially the long ones, correspond to misassembled loci in three reference Porphyromonas gingivalis genomes marked as circularized (thus complete or finished). We prove that even at modest coverage (30X), long-read resequencing together with PCR contiguity verification (rrn operons and an integrative and conjugative element or ICE) can be used to identify and correct the wrongly combined or assembled regions. Finally, although time-consuming and labor-intensive, consistent manual biocuration of three P. gingivalis strains allowed us to compare and correct the existing genomic annotations, resulting in a more accurate interpretation of the genomic differences among these strains.In this study, we demonstrate the usefulness and importance of long-read sequencing in verifying published genomes (even when complete) and generating assemblies for new bacterial strains/species with high genomic plasticity. We also show that when combined with biological validation processes and diligent biocurated annotation, this strategy helps reduce the propagation of errors in shared databases, thus limiting false conclusions based on incomplete or misleading information.

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July 19, 2019

Extreme sensitivity to ultraviolet light in the fungal pathogen causing white-nose syndrome of bats.

Bat white-nose syndrome (WNS), caused by the fungal pathogen Pseudogymnoascus destructans, has decimated North American hibernating bats since its emergence in 2006. Here, we utilize comparative genomics to examine the evolutionary history of this pathogen in comparison to six closely related nonpathogenic species. P. destructans displays a large reduction in carbohydrate-utilizing enzymes (CAZymes) and in the predicted secretome (~50%), and an increase in lineage-specific genes. The pathogen has lost a key enzyme, UVE1, in the alternate excision repair (AER) pathway, which is known to contribute to repair of DNA lesions induced by ultraviolet (UV) light. Consistent with a nonfunctional AER pathway, P. destructans is extremely sensitive to UV light, as well as the DNA alkylating agent methyl methanesulfonate (MMS). The differential susceptibility of P. destructans to UV light in comparison to other hibernacula-inhabiting fungi represents a potential “Achilles’ heel” of P. destructans that might be exploited for treatment of bats with WNS.

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July 19, 2019

Long-read sequence assembly of the firefly Pyrocoelia pectoralis genome.

Fireflies are a family of insects within the beetle order Coleoptera, or winged beetles, and they are one of the most well-known and loved insect species because of their bioluminescence. However, the firefly is in danger of extinction because of the massive destruction of its living environment. In order to improve the understanding of fireflies and protect them effectively, we sequenced the whole genome of the terrestrial firefly Pyrocoelia pectoralis.Here, we developed a highly reliable genome resource for the terrestrial firefly Pyrocoelia pectoralis (E. Oliv., 1883; Coleoptera: Lampyridae) using single molecule real time (SMRT) sequencing on the PacBio Sequel platform. In total, 57.8 Gb of long reads were generated and assembled into a 760.4-Mb genome, which is close to the estimated genome size and covered 98.7% complete and 0.7% partial insect Benchmarking Universal Single-Copy Orthologs. The k-mer analysis showed that this genome is highly heterozygous. However, our long-read assembly demonstrates continuousness with a contig N50 length of 3.04 Mb and the longest contig length of 13.69 Mb. Furthermore, 135 589 SSRs and 341 Mb of repeat sequences were detected. A total of 23 092 genes were predicted; 88.44% of genes were annotated with one or more related functions.We assembled a high-quality firefly genome, which will not only provide insights into the conservation and biodiversity of fireflies, but also provide a wealth of information to study the mechanisms of their sexual communication, bio-luminescence, and evolution.© The Authors 2017. Published by Oxford University Press.

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July 19, 2019

Single-molecule sequencing and optical mapping yields an improved genome of woodland strawberry (Fragaria vesca) with chromosome-scale contiguity.

Although draft genomes are available for most agronomically important plant species, the majority are incomplete, highly fragmented, and often riddled with assembly and scaffolding errors. These assembly issues hinder advances in tool development for functional genomics and systems biology.Here we utilized a robust, cost-effective approach to produce high-quality reference genomes. We report a near-complete genome of diploid woodland strawberry (Fragaria vesca) using single-molecule real-time sequencing from Pacific Biosciences (PacBio). This assembly has a contig N50 length of ~7.9 million base pairs (Mb), representing a ~300-fold improvement of the previous version. The vast majority (>99.8%) of the assembly was anchored to 7 pseudomolecules using 2 sets of optical maps from Bionano Genomics. We obtained ~24.96 Mb of sequence not present in the previous version of the F. vesca genome and produced an improved annotation that includes 1496 new genes. Comparative syntenic analyses uncovered numerous, large-scale scaffolding errors present in each chromosome in the previously published version of the F. vesca genome.Our results highlight the need to improve existing short-read based reference genomes. Furthermore, we demonstrate how genome quality impacts commonly used analyses for addressing both fundamental and applied biological questions.© The Authors 2017. Published by Oxford University Press.

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July 19, 2019

Editing out five Serpina1 paralogs to create a mouse model of genetic emphysema.

Chronic obstructive pulmonary disease affects 10% of the worldwide population, and the leading genetic cause is a-1 antitrypsin (AAT) deficiency. Due to the complexity of the murine locus, which includes up to six Serpina1 paralogs, no genetic animal model of the disease has been successfully generated until now. Here we create a quintuple Serpina1a-e knockout using CRISPR/Cas9-mediated genome editing. The phenotype recapitulates the human disease phenotype, i.e., absence of hepatic and circulating AAT translates functionally to a reduced capacity to inhibit neutrophil elastase. With age, Serpina1 null mice develop emphysema spontaneously, which can be induced in younger mice by a lipopolysaccharide challenge. This mouse models not only AAT deficiency but also emphysema and is a relevant genetic model and not one based on developmental impairment of alveolarization or elastase administration. We anticipate that this unique model will be highly relevant not only to the preclinical development of therapeutics for AAT deficiency, but also to emphysema and smoking research. Copyright © 2018 the Author(s). Published by PNAS.

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July 19, 2019

Expanding an expanded genome: long-read sequencing of Trypanosoma cruzi.

Although the genome of Trypanosoma cruzi, the causative agent of Chagas disease, was first made available in 2005, with additional strains reported later, the intrinsic genome complexity of this parasite (the abundance of repetitive sequences and genes organized in tandem) has traditionally hindered high-quality genome assembly and annotation. This also limits diverse types of analyses that require high degrees of precision. Long reads generated by third-generation sequencing technologies are particularly suitable to address the challenges associated with T. cruzi’s genome since they permit direct determination of the full sequence of large clusters of repetitive sequences without collapsing them. This, in turn, not only allows accurate estimation of gene copy numbers but also circumvents assembly fragmentation. Here, we present the analysis of the genome sequences of two T. cruzi clones: the hybrid TCC (TcVI) and the non-hybrid Dm28c (TcI), determined by PacBio Single Molecular Real-Time (SMRT) technology. The improved assemblies herein obtained permitted us to accurately estimate gene copy numbers, abundance and distribution of repetitive sequences (including satellites and retroelements). We found that the genome of T. cruzi is composed of a ‘core compartment’ and a ‘disruptive compartment’ which exhibit opposite GC content and gene composition. Novel tandem and dispersed repetitive sequences were identified, including some located inside coding sequences. Additionally, homologous chromosomes were separately assembled, allowing us to retrieve haplotypes as separate contigs instead of a unique mosaic sequence. Finally, manual annotation of surface multigene families, mucins and trans-sialidases allows now a better overview of these complex groups of genes.

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July 19, 2019

Ultradeep single-molecule real-time sequencing of HIV envelope reveals complete compartmentalization of highly macrophage-tropic R5 proviral variants in brain and CXCR4-using variants in immune and peripheral tissues.

Despite combined antiretroviral therapy (cART), HIV+ patients still develop neurological disorders, which may be due to persistent HIV infection and selective evolution in brain tissues. Single-molecule real-time (SMRT) sequencing technology offers an improved opportunity to study the relationship among HIV isolates in the brain and lymphoid tissues because it is capable of generating thousands of long sequence reads in a single run. Here, we used SMRT sequencing to generate ~?50,000 high-quality full-length HIV envelope sequences (>?2200 bp) from seven autopsy tissues from an HIV+/cART+ subject, including three brain and four non-brain sites. Sanger sequencing was used for comparison with SMRT data and to clone functional pseudoviruses for in vitro tropism assays. Phylogenetic analysis demonstrated that brain-derived HIV was compartmentalized from HIV outside the brain and that the variants from each of the three brain tissues grouped independently. Variants from all peripheral tissues were intermixed on the tree but independent of the brain clades. Due to the large number of sequences, a clustering analysis at three similarity thresholds (99, 99.5, and 99.9%) was also performed. All brain sequences clustered exclusive of any non-brain sequences at all thresholds; however, frontal lobe sequences clustered independently of occipital and parietal lobes. Translated sequences revealed potentially functional differences between brain and non-brain sequences in the location of putative N-linked glycosylation sites (N-sites), V1 length, V3 charge, and the number of V4 N-sites. All brain sequences were predicted to use the CCR5 co-receptor, while most non-brain sequences were predicted to use CXCR4 co-receptor. Tropism results were confirmed by in vitro infection assays. The study is the first to use a SMRT sequencing approach to study HIV compartmentalization in tissues and supports other reports of limited trafficking between brain and non-brain sequences during cART. Due to the long sequence length, we could observe changes along the entire envelope gene, likely caused by differential selective pressure in the brain that may contribute to neurological disease.

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July 19, 2019

Advances in Sequencing and Resequencing in Crop Plants.

DNA sequencing technologies have changed the face of biological research over the last 20 years. From reference genomes to population level resequencing studies, these technologies have made significant contributions to our understanding of plant biology and evolution. As the technologies have increased in power, the breadth and complexity of the questions that can be asked has increased. Along with this, the challenges of managing unprecedented quantities of sequence data are mounting. This chapter describes a few aspects of the journey so far and looks forward to what may lie ahead.

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July 19, 2019

A near-complete haplotype-phased genome of the dikaryotic wheat stripe rust fungus Puccinia striiformis f. sp. tritici reveals high interhaplotype diversity.

A long-standing biological question is how evolution has shaped the genomic architecture of dikaryotic fungi. To answer this, high-quality genomic resources that enable haplotype comparisons are essential. Short-read genome assemblies for dikaryotic fungi are highly fragmented and lack haplotype-specific information due to the high heterozygosity and repeat content of these genomes. Here, we present a diploid-aware assembly of the wheat stripe rust fungus Puccinia striiformis f. sp. tritici based on long reads using the FALCON-Unzip assembler. Transcriptome sequencing data sets were used to infer high-quality gene models and identify virulence genes involved in plant infection referred to as effectors. This represents the most complete Puccinia striiformis f. sp. tritici genome assembly to date (83 Mb, 156 contigs, N50 of 1.5 Mb) and provides phased haplotype information for over 92% of the genome. Comparisons of the phase blocks revealed high interhaplotype diversity of over 6%. More than 25% of all genes lack a clear allelic counterpart. When we investigated genome features that potentially promote the rapid evolution of virulence, we found that candidate effector genes are spatially associated with conserved genes commonly found in basidiomycetes. Yet, candidate effectors that lack an allelic counterpart are more distant from conserved genes than allelic candidate effectors and are less likely to be evolutionarily conserved within the P. striiformis species complex and Pucciniales In summary, this haplotype-phased assembly enabled us to discover novel genome features of a dikaryotic plant-pathogenic fungus previously hidden in collapsed and fragmented genome assemblies.IMPORTANCE Current representations of eukaryotic microbial genomes are haploid, hiding the genomic diversity intrinsic to diploid and polyploid life forms. This hidden diversity contributes to the organism’s evolutionary potential and ability to adapt to stress conditions. Yet, it is challenging to provide haplotype-specific information at a whole-genome level. Here, we take advantage of long-read DNA sequencing technology and a tailored-assembly algorithm to disentangle the two haploid genomes of a dikaryotic pathogenic wheat rust fungus. The two genomes display high levels of nucleotide and structural variations, which lead to allelic variation and the presence of genes lacking allelic counterparts. Nonallelic candidate effector genes, which likely encode important pathogenicity factors, display distinct genome localization patterns and are less likely to be evolutionary conserved than those which are present as allelic pairs. This genomic diversity may promote rapid host adaptation and/or be related to the age of the sequenced isolate since last meiosis. Copyright © 2018 Schwessinger et al.

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July 19, 2019

RNAi is a critical determinant of centromere evolution in closely related fungi.

The centromere DNA locus on a eukaryotic chromosome facilitates faithful chromosome segregation. Despite performing such a conserved function, centromere DNA sequence as well as the organization of sequence elements is rapidly evolving in all forms of eukaryotes. The driving force that facilitates centromere evolution remains an enigma. Here, we studied the evolution of centromeres in closely related species in the fungal phylum of Basidiomycota. Using ChIP-seq analysis of conserved inner kinetochore proteins, we identified centromeres in three closely related Cryptococcus species: two of which are RNAi-proficient, while the other lost functional RNAi. We find that the centromeres in the RNAi-deficient species are significantly shorter than those of the two RNAi-proficient species. While centromeres are LTR retrotransposon-rich in all cases, the RNAi-deficient species lost all full-length retroelements from its centromeres. In addition, centromeres in RNAi-proficient species are associated with a significantly higher level of cytosine DNA modifications compared with those of RNAi-deficient species. Furthermore, when an RNAi-proficient Cryptococcus species and its RNAi-deficient mutants were passaged under similar conditions, the centromere length was found to be occasionally shortened in RNAi mutants. In silico analysis of predicted centromeres in a group of closely related Ustilago species, also belonging to the Basidiomycota, were found to have undergone a similar transition in the centromere length in an RNAi-dependent fashion. Based on the correlation found in two independent basidiomycetous species complexes, we present evidence suggesting that the loss of RNAi and cytosine DNA methylation triggered transposon attrition, which resulted in shortening of centromere length during evolution. Copyright © 2018 the Author(s). Published by PNAS.

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July 19, 2019

Utility of DNA, RNA, protein, and functional approaches to solve cryptic immunodeficiencies.

We report a female infant identified by newborn screening for severe combined immunodeficiencies (NBS SCID) with T cell lymphopenia (TCL). The patient had persistently elevated alpha-fetoprotein (AFP) with IgA deficiency, and elevated IgM. Gene sequencing for a SCID panel was uninformative. We sought to determine the cause of the immunodeficiency in this infant.We performed whole-exome sequencing (WES) on the patient and parents to identify a genetic diagnosis. Based on the WES result, we developed a novel flow cytometric panel for rapid assessment of DNA repair defects using blood samples. We also performed whole transcriptome sequencing (WTS) on fibroblast RNA from the patient and father for abnormal transcript analysis.WES revealed a pathogenic paternally inherited indel in ATM. We used the flow panel to assess several proteins in the DNA repair pathway in lymphocyte subsets. The patient had absent phosphorylation of ATM, resulting in absent or aberrant phosphorylation of downstream proteins, including ?H2AX. However, ataxia-telangiectasia (AT) is an autosomal recessive condition, and the abnormal functional data did not correspond with a single ATM variant. WTS revealed in-frame reciprocal fusion transcripts involving ATM and SLC35F2 indicating a chromosome 11 inversion within 11q22.3, of maternal origin. Inversion breakpoints were identified within ATM intron 16 and SLC35F2 intron 7.We identified a novel ATM-breaking chromosome 11 inversion in trans with a pathogenic indel (compound heterozygote) resulting in non-functional ATM protein, consistent with a diagnosis of AT. Utilization of several molecular and functional assays allowed successful resolution of this case.

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July 19, 2019

Genome sequence of the progenitor of wheat A subgenome Triticum urartu.

Triticum urartu (diploid, AA) is the progenitor of the A subgenome of tetraploid (Triticum turgidum, AABB) and hexaploid (Triticum aestivum, AABBDD) wheat1,2. Genomic studies of T. urartu have been useful for investigating the structure, function and evolution of polyploid wheat genomes. Here we report the generation of a high-quality genome sequence of T. urartu by combining bacterial artificial chromosome (BAC)-by-BAC sequencing, single molecule real-time whole-genome shotgun sequencing 3 , linked reads and optical mapping4,5. We assembled seven chromosome-scale pseudomolecules and identified protein-coding genes, and we suggest a model for the evolution of T. urartu chromosomes. Comparative analyses with genomes of other grasses showed gene loss and amplification in the numbers of transposable elements in the T. urartu genome. Population genomics analysis of 147 T. urartu accessions from across the Fertile Crescent showed clustering of three groups, with differences in altitude and biostress, such as powdery mildew disease. The T. urartu genome assembly provides a valuable resource for studying genetic variation in wheat and related grasses, and promises to facilitate the discovery of genes that could be useful for wheat improvement.

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