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September 22, 2019

Long read reference genome-free reconstruction of a full-length transcriptome from Astragalus membranaceus reveals transcript variants involved in bioactive compound biosynthesis.

Astragalus membranaceus, also known as Huangqi in China, is one of the most widely used medicinal herbs in Traditional Chinese Medicine. Traditional Chinese Medicine formulations from Astragalus membranaceus have been used to treat a wide range of illnesses, such as cardiovascular disease, type 2 diabetes, nephritis and cancers. Pharmacological studies have shown that immunomodulating, anti-hyperglycemic, anti-inflammatory, antioxidant and antiviral activities exist in the extract of Astragalus membranaceus. Therefore, characterising the biosynthesis of bioactive compounds in Astragalus membranaceus, such as Astragalosides, Calycosin and Calycosin-7-O-ß-d-glucoside, is of particular importance for further genetic studies of Astragalus membranaceus. In this study, we reconstructed the Astragalus membranaceus full-length transcriptomes from leaf and root tissues using PacBio Iso-Seq long reads. We identified 27 975 and 22 343 full-length unique transcript models in each tissue respectively. Compared with previous studies that used short read sequencing, our reconstructed transcripts are longer, and are more likely to be full-length and include numerous transcript variants. Moreover, we also re-characterised and identified potential transcript variants of genes involved in Astragalosides, Calycosin and Calycosin-7-O-ß-d-glucoside biosynthesis. In conclusion, our study provides a practical pipeline to characterise the full-length transcriptome for species without a reference genome and a useful genomic resource for exploring the biosynthesis of active compounds in Astragalus membranaceus.

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September 22, 2019

Functional mitochondria in health and disease.

The ability to rapidly adapt cellular bioenergetic capabilities to meet rapidly changing environmental conditions is mandatory for normal cellular function and for cancer progression. Any loss of this adaptive response has the potential to compromise cellular function and render the cell more susceptible to external stressors such as oxidative stress, radiation, chemotherapeutic drugs, and hypoxia. Mitochondria play a vital role in bioenergetic and biosynthetic pathways and can rapidly adjust to meet the metabolic needs of the cell. Increased demand is met by mitochondrial biogenesis and fusion of individual mitochondria into dynamic networks, whereas a decrease in demand results in the removal of superfluous mitochondria through fission and mitophagy. Effective communication between nucleus and mitochondria (mito-nuclear cross talk), involving the generation of different mitochondrial stress signals as well as the nuclear stress response pathways to deal with these stressors, maintains bioenergetic homeostasis under most conditions. However, when mitochondrial DNA (mtDNA) mutations accumulate and mito-nuclear cross talk falters, mitochondria fail to deliver critical functional outputs. Mutations in mtDNA have been implicated in neuromuscular and neurodegenerative mitochondriopathies and complex diseases such as diabetes, cardiovascular diseases, gastrointestinal disorders, skin disorders, aging, and cancer. In some cases, drastic measures such as acquisition of new mitochondria from donor cells occurs to ensure cell survival. This review starts with a brief discussion of the evolutionary origin of mitochondria and summarizes how mutations in mtDNA lead to mitochondriopathies and other degenerative diseases. Mito-nuclear cross talk, including various stress signals generated by mitochondria and corresponding stress response pathways activated by the nucleus are summarized. We also introduce and discuss a small family of recently discovered hormone-like mitopeptides that modulate body metabolism. Under conditions of severe mitochondrial stress, mitochondria have been shown to traffic between cells, replacing mitochondria in cells with damaged and malfunctional mtDNA. Understanding the processes involved in cellular bioenergetics and metabolic adaptation has the potential to generate new knowledge that will lead to improved treatment of many of the metabolic, degenerative, and age-related inflammatory diseases that characterize modern societies.

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September 22, 2019

Cell-type-specific splicing of Piezo2 regulates mechanotransduction.

Piezo2 is a mechanically activated ion channel required for touch discrimination, vibration detection, and proprioception. Here, we discovered that Piezo2 is extensively spliced, producing different Piezo2 isoforms with distinct properties. Sensory neurons from both mice and humans express a large repertoire of Piezo2 variants, whereas non-neuronal tissues express predominantly a single isoform. Notably, even within sensory ganglia, we demonstrate the splicing of Piezo2 to be cell type specific. Biophysical characterization revealed substantial differences in ion permeability, sensitivity to calcium modulation, and inactivation kinetics among Piezo2 splice variants. Together, our results describe, at the molecular level, a potential mechanism by which transduction is tuned, permitting the detection of a variety of mechanosensory stimuli. Published by Elsevier Inc.

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September 22, 2019

Isoform evolution in primates through independent combination of alternative RNA processing events.

Recent RNA-seq technology revealed thousands of splicing events that are under rapid evolution in primates, whereas the reliability of these events, as well as their combination on the isoform level, have not been adequately addressed due to its limited sequencing length. Here, we performed comparative transcriptome analyses in human and rhesus macaque cerebellum using single molecule long-read sequencing (Iso-seq) and matched RNA-seq. Besides 359 million RNA-seq reads, 4,165,527 Iso-seq reads were generated with a mean length of 14,875?bp, covering 11,466 human genes, and 10,159 macaque genes. With Iso-seq data, we substantially expanded the repertoire of alternative RNA processing events in primates, and found that intron retention and alternative polyadenylation are surprisingly more prevalent in primates than previously estimated. We then investigated the combinatorial mode of these alternative events at the whole-transcript level, and found that the combination of these events is largely independent along the transcript, leading to thousands of novel isoforms missed by current annotations. Notably, these novel isoforms are selectively constrained in general, and 1,119 isoforms have even higher expression than the previously annotated major isoforms in human, indicating that the complexity of the human transcriptome is still significantly underestimated. Comparative transcriptome analysis further revealed 502 genes encoding selectively constrained, lineage-specific isoforms in human but not in rhesus macaque, linking them to some lineage-specific functions. Overall, we propose that the independent combination of alternative RNA processing events has contributed to complex isoform evolution in primates, which provides a new foundation for the study of phenotypic difference among primates.© The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

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September 22, 2019

Assessment of an organ-specific de novo transcriptome of the nematode trap-crop, Solanum sisymbriifolium

Solanum sisymbriifolium, also known as “Litchi Tomato” or “Sticky Nightshade,” is an undomesticated and poorly researched plant related to potato and tomato. Unlike the latter species, S. sisymbriifolium induces eggs of the cyst nematode, Globodera pallida, to hatch and migrate into its roots, but then arrests further nematode maturation. In order to provide researchers with a partial blueprint of its genetic make-up so that the mechanism of this response might be identified, we used single molecule real time (SMRT) sequencing to compile a high quality de novo transcriptome of 41,189 unigenes drawn from individually sequenced bud, root, stem, and leaf RNA populations. Functional annotation and BUSCO analysis showed that this transcriptome was surprisingly complete, even though it represented genes expressed at a single time point. By sequencing the 4 organ libraries separately, we found we could get a reliable snapshot of transcript distributions in each organ. A divergent site analysis of the merged transcriptome indicated that this species might have undergone a recent genome duplication and re-diploidization. Further analysis indicated that the plant then retained a disproportionate number of genes associated with photosynthesis and amino acid metabolism in comparison to genes with characteristics of R-proteins or involved in secondary metabolism. The former processes may have given S. sisymbriifolium a bigger competitive advantage than the latter did. Copyright © 2018 Wixom et al.

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September 22, 2019

Single molecule RNA sequencing uncovers trans-splicing and improves annotations in Anopheles stephensi.

Single molecule real-time (SMRT) sequencing has recently been used to obtain full-length cDNA sequences that improve genome annotation and reveal RNA isoforms. Here, we used one such method called isoform sequencing from Pacific Biosciences (PacBio) to sequence a cDNA library from the Asian malaria mosquito Anopheles stephensi. More than 600 000 full-length cDNAs, referred to as reads of insert, were identified. Owing to the inherently high error rate of PacBio sequencing, we tested different approaches for error correction. We found that error correction using Illumina RNA sequencing (RNA-seq) generated more data than using the default SMRT pipeline. The full-length error-corrected PacBio reads greatly improved the gene annotation of Anopheles stephensi: 4867 gene models were updated and 1785 alternatively spliced isoforms were added to the annotation. In addition, six trans-splicing events, where exons from different primary transcripts were joined together, were identified in An. stephensi. All six trans-splicing events appear to be conserved in Culicidae, as they are also found in Anopheles gambiae and Aedes aegypti. The proteins encoded by trans-splicing events are also highly conserved and the orthologues of these proteins are cis-spliced in outgroup species, indicating that trans-splicing may arise as a mechanism to rescue genes that broke up during evolution.© 2017 The Royal Entomological Society.

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September 22, 2019

A human-specific switch of alternatively spliced AFMID isoforms contributes to TP53 mutations and tumor recurrence in hepatocellular carcinoma.

Pre-mRNA splicing can contribute to the switch of cell identity that occurs in carcinogenesis. Here, we analyze a large collection of RNA-seq data sets and report that splicing changes in hepatocyte-specific enzymes, such as AFMID and KHK, are associated with HCC patients’ survival and relapse. The switch of AFMID isoforms is an early event in HCC development and is associated with driver mutations in TP53 and ARID1A The switch of AFMID isoforms is human-specific and not detectable in other species, including primates. Finally, we show that overexpression of the full-length AFMID isoform leads to a higher NAD+ level, lower DNA-damage response, and slower cell growth in HepG2 cells. The integrative analysis uncovered a mechanistic link between splicing switches, de novo NAD+ biosynthesis, driver mutations, and HCC recurrence.© 2018 Lin et al.; Published by Cold Spring Harbor Laboratory Press.

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September 22, 2019

Splicing of nascent RNA coincides with intron exit from RNA Polymerase II.

Protein-coding genes in eukaryotes are transcribed by RNA polymerase II (Pol II) and introns are removed from pre-mRNA by the spliceosome. Understanding the time lag between Pol II progression and splicing could provide mechanistic insights into the regulation of gene expression. Here, we present two single-molecule nascent RNA sequencing methods that directly determine the progress of splicing catalysis as a function of Pol II position. Endogenous genes were analyzed on a global scale in budding yeast. We show that splicing is 50% complete when Pol II is only 45 nt downstream of introns, with the first spliced products observed as introns emerge from Pol II. Perturbations that slow the rate of spliceosome assembly or speed up the rate of transcription caused splicing delays, showing that regulation of both processes determines in vivo splicing profiles. We propose that matched rates streamline the gene expression pathway, while allowing regulation through kinetic competition. Copyright © 2016 Elsevier Inc. All rights reserved.

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September 22, 2019

A statistical method for observing personal diploid methylomes and transcriptomes with Single-Molecule Real-Time sequencing.

We address the problem of observing personal diploid methylomes, CpG methylome pairs of homologous chromosomes that are distinguishable with respect to phased heterozygous variants (PHVs), which is challenging due to scarcity of PHVs in personal genomes. Single molecule real-time (SMRT) sequencing is promising as it outputs long reads with CpG methylation information, but a serious concern is whether reliable PHVs are available in erroneous SMRT reads with an error rate of ~15%. To overcome the issue, we propose a statistical model that reduces the error rate of phasing CpG site to 1%, thereby calling CpG hypomethylation in each haplotype with >90% precision and sensitivity. Using our statistical model, we examined GNAS complex locus known for a combination of maternally, paternally, or biallelically expressed isoforms, and observed allele-specific methylation pattern almost perfectly reflecting their respective allele-specific expression status, demonstrating the merit of elucidating comprehensive personal diploid methylomes and transcriptomes.

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September 22, 2019

First draft genome sequence of the rock bream in the family Oplegnathidae.

The rock bream (Oplegnathus fasciatus) is one of the most economically valuable marine fish in East Asia, and due to various environmental factors, there is substantial revenue loss in the production sector. Therefore, knowledge of its genome is required to uncover the genetic factors and the solutions to these problems. In this study, we constructed the first draft genome of O. fasciatus as a reference for the family Oplegnathidae. The genome size is estimated to be 749?Mb, and it was assembled into 766?Mb by combining Illumina and PacBio sequences. A total of 24,053 transcripts (23,338 genes) are predicted, and among those transcripts, 23,362 (97%), are annotated with functional terms. Finally, the completeness of the genome assembly was assessed by CEGMA, which resulted in the complete mapping of 220 (88.7%) core genes in the genome. To the best of our knowledge, this is the first draft genome for the family Oplegnathidae.

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September 22, 2019

The chromosome-level quality genome provides insights into the evolution of the biosynthesis genes for aroma compounds of Osmanthus fragrans.

Sweet osmanthus (Osmanthus fragrans) is a very popular ornamental tree species throughout Southeast Asia and USA particularly for its extremely fragrant aroma. We constructed a chromosome-level reference genome of O. fragrans to assist in studies of the evolution, genetic diversity, and molecular mechanism of aroma development. A total of over 118?Gb of polished reads was produced from HiSeq (45.1?Gb) and PacBio Sequel (73.35?Gb), giving 100× depth coverage for long reads. The combination of Illumina-short reads, PacBio-long reads, and Hi-C data produced the final chromosome quality genome of O. fragrans with a genome size of 727?Mb and a heterozygosity of 1.45 %. The genome was annotated using de novo and homology comparison and further refined with transcriptome data. The genome of O. fragrans was predicted to have?45,542 genes, of which 95.68 % were functionally annotated. Genome annotation found 49.35 % as the repetitive sequences, with long terminal repeats (LTR) being the richest (28.94 %). Genome evolution analysis indicated the evidence of whole-genome duplication 15 million years ago, which contributed to the current content of 45,242 genes. Metabolic analysis revealed that linalool, a monoterpene is the main aroma compound. Based on the genome and transcriptome, we further demonstrated the direct connection between terpene synthases (TPSs) and the rich aromatic molecules in O. fragrans. We identified three new flower-specific TPS genes, of which the expression coincided with the production of linalool. Our results suggest that the high number of TPS genes and the flower tissue- and stage-specific TPS genes expressions might drive the strong unique aroma production of O. fragrans.

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September 21, 2019

Repair of double-strand breaks induced by CRISPR-Cas9 leads to large deletions and complex rearrangements.

CRISPR-Cas9 is poised to become the gene editing tool of choice in clinical contexts. Thus far, exploration of Cas9-induced genetic alterations has been limited to the immediate vicinity of the target site and distal off-target sequences, leading to the conclusion that CRISPR-Cas9 was reasonably specific. Here we report significant on-target mutagenesis, such as large deletions and more complex genomic rearrangements at the targeted sites in mouse embryonic stem cells, mouse hematopoietic progenitors and a human differentiated cell line. Using long-read sequencing and long-range PCR genotyping, we show that DNA breaks introduced by single-guide RNA/Cas9 frequently resolved into deletions extending over many kilobases. Furthermore, lesions distal to the cut site and crossover events were identified. The observed genomic damage in mitotically active cells caused by CRISPR-Cas9 editing may have pathogenic consequences.

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July 19, 2019

The genome of Chenopodium quinoa.

Chenopodium quinoa (quinoa) is a highly nutritious grain identified as an important crop to improve world food security. Unfortunately, few resources are available to facilitate its genetic improvement. Here we report the assembly of a high-quality, chromosome-scale reference genome sequence for quinoa, which was produced using single-molecule real-time sequencing in combination with optical, chromosome-contact and genetic maps. We also report the sequencing of two diploids from the ancestral gene pools of quinoa, which enables the identification of sub-genomes in quinoa, and reduced-coverage genome sequences for 22 other samples of the allotetraploid goosefoot complex. The genome sequence facilitated the identification of the transcription factor likely to control the production of anti-nutritional triterpenoid saponins found in quinoa seeds, including a mutation that appears to cause alternative splicing and a premature stop codon in sweet quinoa strains. These genomic resources are an important first step towards the genetic improvement of quinoa.

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July 19, 2019

De novo PacBio long-read and phased avian genome assemblies correct and add to reference genes generated with intermediate and short reads.

Reference-quality genomes are expected to provide a resource for studying gene structure, function, and evolution. However, often genes of interest are not completely or accurately assembled, leading to unknown errors in analyses or additional cloning efforts for the correct sequences. A promising solution is long-read sequencing. Here we tested PacBio-based long-read sequencing and diploid assembly for potential improvements to the Sanger-based intermediate-read zebra finch reference and Illumina-based short-read Anna’s hummingbird reference, 2 vocal learning avian species widely studied in neuroscience and genomics. With DNA of the same individuals used to generate the reference genomes, we generated diploid assemblies with the FALCON-Unzip assembler, resulting in contigs with no gaps in the megabase range, representing 150-fold and 200-fold improvements over the current zebra finch and hummingbird references, respectively. These long-read and phased assemblies corrected and resolved what we discovered to be numerous misassemblies in the references, including missing sequences in gaps, erroneous sequences flanking gaps, base call errors in difficult-to-sequence regions, complex repeat structure errors, and allelic differences between the 2 haplotypes. These improvements were validated by single long-genome and transcriptome reads and resulted for the first time in completely resolved protein-coding genes widely studied in neuroscience and specialized in vocal learning species. These findings demonstrate the impact of long reads, sequencing of previously difficult-to-sequence regions, and phasing of haplotypes on generating the high-quality assemblies necessary for understanding gene structure, function, and evolution.© The Authors 2017. Published by Oxford University Press.

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July 19, 2019

Dissecting the causal mechanism of X-linked Dystonia-Parkinsonism by integrating genome and transcriptome assembly.

X-linked Dystonia-Parkinsonism (XDP) is a Mendelian neurodegenerative disease that is endemic to the Philippines and is associated with a founder haplotype. We integrated multiple genome and transcriptome assembly technologies to narrow the causal mutation to the TAF1 locus, which included a SINE-VNTR-Alu (SVA) retrotransposition into intron 32 of the gene. Transcriptome analyses identified decreased expression of the canonical cTAF1 transcript among XDP probands, and de novo assembly across multiple pluripotent stem-cell-derived neuronal lineages discovered aberrant TAF1 transcription that involved alternative splicing and intron retention (IR) in proximity to the SVA that was anti-correlated with overall TAF1 expression. CRISPR/Cas9 excision of the SVA rescued this XDP-specific transcriptional signature and normalized TAF1 expression in probands. These data suggest an SVA-mediated aberrant transcriptional mechanism associated with XDP and may provide a roadmap for layered technologies and integrated assembly-based analyses for other unsolved Mendelian disorders. Copyright © 2018 Elsevier Inc. All rights reserved.

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