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April 21, 2020  |  

Progression of the canonical reference malaria parasite genome from 2002-2019.

Here we describe the ways in which the sequence and annotation of the Plasmodium falciparum reference genome has changed since its publication in 2002. As the malaria species responsible for the most deaths worldwide, the richness of annotation and accuracy of the sequence are important resources for the P. falciparum research community as well as the basis for interpreting the genomes of subsequently sequenced species. At the time of publication in 2002 over 60% of predicted genes had unknown functions. As of March 2019, this number has been significantly decreased to 33%. The reduction is due to the inclusion of genes that were subsequently characterised experimentally and genes with significant similarity to others with known functions. In addition, the structural annotation of genes has been significantly refined; 27% of gene structures have been changed since 2002, comprising changes in exon-intron boundaries, addition or deletion of exons and the addition or deletion of genes. The sequence has also undergone significant improvements. In addition to the correction of a large number of single-base and insertion or deletion errors, a major miss-assembly between the subtelomeres of chromosome 7 and 8 has been corrected. As the number of sequenced isolates continues to grow rapidly, a single reference genome will not be an adequate basis for interpretating intra-species sequence diversity. We therefore describe in this publication a population reference genome of P. falciparum, called Pfref1. This reference will enable the community to map to regions that are not present in the current assembly. P. falciparum 3D7 will be continued to be maintained with ongoing curation ensuring continual improvements in annotation quality.

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October 23, 2019  |  

Simultaneous non-contiguous deletions using large synthetic DNA and site-specific recombinases.

Toward achieving rapid and large scale genome modification directly in a target organism, we have developed a new genome engineering strategy that uses a combination of bioinformatics aided design, large synthetic DNA and site-specific recombinases. Using Cre recombinase we swapped a target 126-kb segment of the Escherichia coli genome with a 72-kb synthetic DNA cassette, thereby effectively eliminating over 54 kb of genomic DNA from three non-contiguous regions in a single recombination event. We observed complete replacement of the native sequence with the modified synthetic sequence through the action of the Cre recombinase and no competition from homologous recombination. Because of the versatility and high-efficiency of the Cre-lox system, this method can be used in any organism where this system is functional as well as adapted to use with other highly precise genome engineering systems. Compared to present-day iterative approaches in genome engineering, we anticipate this method will greatly speed up the creation of reduced, modularized and optimized genomes through the integration of deletion analyses data, transcriptomics, synthetic biology and site-specific recombination. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

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October 23, 2019  |  

Transmission, evolution, and endogenization: Lessons learned from recent retroviral invasions.

Viruses of the subfamily Orthoretrovirinaeare defined by the ability to reverse transcribe an RNA genome into DNA that integrates into the host cell genome during the intracellular virus life cycle. Exogenous retroviruses (XRVs) are horizontally transmitted between host individuals, with disease outcome depending on interactions between the retrovirus and the host organism. When retroviruses infect germ line cells of the host, they may become endogenous retroviruses (ERVs), which are permanent elements in the host germ line that are subject to vertical transmission. These ERVs sometimes remain infectious and can themselves give rise to XRVs. This review integrates recent developments in the phylogenetic classification of retroviruses and the identification of retroviral receptors to elucidate the origins and evolution of XRVs and ERVs. We consider whether ERVs may recurrently pressure XRVs to shift receptor usage to sidestep ERV interference. We discuss how related retroviruses undergo alternative fates in different host lineages after endogenization, with koala retrovirus (KoRV) receiving notable interest as a recent invader of its host germ line. KoRV is heritable but also infectious, which provides insights into the early stages of germ line invasions as well as XRV generation from ERVs. The relationship of KoRV to primate and other retroviruses is placed in the context of host biogeography and the potential role of bats and rodents as vectors for interspecies viral transmission. Combining studies of extant XRVs and “fossil” endogenous retroviruses in koalas and other Australasian species has broadened our understanding of the evolution of retroviruses and host-retrovirus interactions. Copyright © 2017 American Society for Microbiology.

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October 23, 2019  |  

Development of a Novel Reference Transcriptome for Scleractinian Coral Porites lutea Using Single-Molecule Long-Read Isoform Sequencing (Iso-Seq)

Elevation in seawater temperature associated with global climate change has caused coral bleaching problems and posed a significant threat to coral health and survival worldwide. Several studies have explored the effects of thermal stress on changes in gene expression levels of both coral hosts and their algal endosymbionts and provided evidences suggesting that corals could acclimatize to environmental stressors through differential regulation of their gene expression (Desalvo et al., 2008, 2010; Császár et al., 2009; Rodriguez-Lanetty et al., 2009; Polato et al., 2010; Meyer et al., 2011; Kenkel et al., 2013). Such information is crucial for understanding the adaptive capacity of the coral holobionts (Hughes et al., 2003). The availability of transcriptome data from a number of coral species and their associated Symbiodinium allows us to probe the molecular stress response of the organisms to heat stress (Traylor-Knowles et al., 2011; Moya et al., 2012; Kenkel et al., 2013; Shinzato et al., 2014; Kitchen et al., 2015; Anderson et al., 2016; Davies et al., 2016). Here, we report the first reference transcriptome for a scleractinian coral Porites lutea, one of the dominant reef-builders in the Indo-West Pacific (Yeemin et al., 2009). We applied both short-read Ion S5 RNA sequencing and long-read Pacific Biosciences (PacBio) isoform sequencing (Iso-seq) to generate transcriptome sequences of P. lutea under normal and heat stress conditions. The key advantage of PacBio’s Iso-seq technology lies within its ability to capture full-length mRNA sequences. These full-length transcripts enable the identification of novel genes/isoforms and the detection of alternative splice variants, which have been shown to be overrepresented in stress responses (Iida et al., 2004; Reddy et al., 2013; Liu and Guo, 2017). We envision that this reference transcriptome will provide a coral research community a valuable resource for investigating changes in gene expression under various biotic/abiotic stress conditions.

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September 22, 2019  |  

Comparison of the mitochondrial genomes and steady state transcriptomes of two strains of the trypanosomatid parasite, Leishmania tarentolae.

U-insertion/deletion RNA editing is a post-transcriptional mitochondrial RNA modification phenomenon required for viability of trypanosomatid parasites. Small guide RNAs encoded mainly by the thousands of catenated minicircles contain the information for this editing. We analyzed by NGS technology the mitochondrial genomes and transcriptomes of two strains, the old lab UC strain and the recently isolated LEM125 strain. PacBio sequencing provided complete minicircle sequences which avoided the assembly problem of short reads caused by the conserved regions. Minicircles were identified by a characteristic size, the presence of three short conserved sequences, a region of inherently bent DNA and the presence of single gRNA genes at a fairly defined location. The LEM125 strain contained over 114 minicircles encoding different gRNAs and the UC strain only ~24 minicircles. Some LEM125 minicircles contained no identifiable gRNAs. Approximate copy numbers of the different minicircle classes in the network were determined by the number of PacBio CCS reads that assembled to each class. Mitochondrial RNA libraries from both strains were mapped against the minicircle and maxicircle sequences. Small RNA reads mapped to the putative gRNA genes but also to multiple regions outside the genes on both strands and large RNA reads mapped in many cases over almost the entire minicircle on both strands. These data suggest that minicircle transcription is complete and bidirectional, with 3′ processing yielding the mature gRNAs. Steady state RNAs in varying abundances are derived from all maxicircle genes, including portions of the repetitive divergent region. The relative extents of editing in both strains correlated with the presence of a cascade of cognate gRNAs. These data should provide the foundation for a deeper understanding of this dynamic genetic system as well as the evolutionary variation of editing in different strains.

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September 22, 2019  |  

PacBio full-length transcriptome profiling of insect mitochondrial gene expression.

In this study, we sequenced the first full-length insect transcriptome using the Erthesina fullo Thunberg based on the PacBio platform. We constructed the first quantitative transcription map of animal mitochondrial genomes and built a straightforward and concise methodology to investigate mitochondrial gene transcription, RNA processing, mRNA maturation and several other related topics. Most of the results were consistent with the previous studies, while to the best of our knowledge some findings were reported for the first time in this study. The new findings included the high levels of mitochondrial gene expression, the 3′ polyadenylation and possible 5′ m(7)G caps of rRNAs, the isoform diversity of 12S rRNA, the polycistronic transcripts and natural antisense transcripts of mitochondrial genes et al. These findings could challenge and enrich fundamental concepts of mitochondrial gene transcription and RNA processing, particularly of the rRNA primary (sequence) structure. The methodology constructed in this study can also be used to study gene expression or RNA processing of nuclear genomes.

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September 22, 2019  |  

Single-molecule long-read transcriptome profiling of Platysternon megacephalum mitochondrial genome with gene rearrangement and control region duplication.

Platysternon megacephalum is the sole living representative of the poorly studied turtle lineage Platysternidae. Their mitochondrial genome has been subject to gene rearrangement and control region duplication, resulting in a unique mitochondrial gene order in vertebrates. In this study, we sequenced the first full-length turtle (P. megacephalum) liver transcriptome using single-molecule real-time sequencing to study the transcriptional mechanisms of its mitochondrial genome. ND5 and ND6 anti-sense (ND6AS) forms a single transcript with the same expression in the human mitochondrial genome, but here we demonstrated differential expression of the rearranged ND5 and ND6AS genes in P. megacephalum. And some polycistronic transcripts were also reported in this study. Notably, we detected some novel long non-coding RNAs with alternative polyadenylation from the duplicated control region, and a novel ND6AS transcript composed of a long non-coding sequence, ND6AS, and tRNA-GluAS. These results provide the first description of a mtDNA transcriptome with gene rearrangement and control region duplication. These findings further our understanding of the fundamental concepts of mitochondrial gene transcription and RNA processing, and provide a new insight into the mechanism of transcription regulation of the mitochondrial genome.

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September 22, 2019  |  

Gene activity in primary T cells infected with HIV89.6: intron retention and induction of genomic repeats.

HIV infection has been reported to alter cellular gene activity, but published studies have commonly assayed transformed cell lines and lab-adapted HIV strains, yielding inconsistent results. Here we carried out a deep RNA-Seq analysis of primary human T cells infected with the low passage HIV isolate HIV89.6.Seventeen percent of cellular genes showed altered activity 48 h after infection. In a meta-analysis including four other studies, our data differed from studies of HIV infection in cell lines but showed more parallels with infections of primary cells. We found a global trend toward retention of introns after infection, suggestive of a novel cellular response to infection. HIV89.6 infection was also associated with activation of several human endogenous retroviruses (HERVs) and retrotransposons, of interest as possible novel antigens that could serve as vaccine targets. The most highly activated group of HERVs was a subset of the ERV-9. Analysis showed that activation was associated with a particular variant of ERV-9 long terminal repeats that contains an indel near the U3-R border. These data also allowed quantification of >70 splice forms of the HIV89.6 RNA and specified the main types of chimeric HIV89.6-host RNAs. Comparison to over 100,000 integration site sequences from the same infected cell populations allowed quantification of authentic versus artifactual chimeric reads, showing that 5′ read-in, splicing out of HIV89.6 from the D4 donor and 3′ read-through were the most common HIV89.6-host cell chimeric RNA forms.Analysis of RNA abundance after infection of primary T cells with the low passage HIV89.6 isolate disclosed multiple novel features of HIV-host interactions, notably intron retention and induction of transcription of retrotransposons and endogenous retroviruses.

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September 22, 2019  |  

A high-quality annotated transcriptome of swine peripheral blood.

High throughput gene expression profiling assays of peripheral blood are widely used in biomedicine, as well as in animal genetics and physiology research. Accurate, comprehensive, and precise interpretation of such high throughput assays relies on well-characterized reference genomes and/or transcriptomes. However, neither the reference genome nor the peripheral blood transcriptome of the pig have been sufficiently assembled and annotated to support such profiling assays in this emerging biomedical model organism. We aimed to assemble published and novel RNA-seq data to provide a comprehensive, well-annotated blood transcriptome for pigs by integrating a de novo assembly with a genome-guided assembly.A de novo and a genome-guided transcriptome of porcine whole peripheral blood was assembled with ~162 million pairs of paired-end and ~183 million single-end, trimmed and normalized Illumina RNA-seq reads (~6 billion initial reads from 146 RNA-seq libraries) from five independent studies by using the Trinity and Cufflinks software, respectively. We then removed putative transcripts (PTs) of low confidence from both assemblies and merged the remaining PTs into an integrated transcriptome consisting of 132,928 PTs, with 126,225 (~95%) PTs from the de novo assembly and more than 91% of PTs spliced. In the integrated transcriptome, ~90% and 63% of PTs had significant sequence similarity to sequences in the NCBI NT and NR databases, respectively; 68,754 (~52%) PTs were annotated with 15,965 unique gene ontology (GO) terms; and 7618 PTs annotated with Enzyme Commission codes were assigned to 134 pathways curated by the Kyoto Encyclopedia of Genes and Genomes (KEGG). Full exon-intron junctions of 17,528 PTs were validated by PacBio IsoSeq full-length cDNA reads from 3 other porcine tissues, NCBI pig RefSeq mRNAs and transcripts from Ensembl Sscrofa10.2 annotation. Completeness of the 5′ termini of 37,569 PTs was validated by public cap analysis of gene expression (CAGE) data. By comparison to the Ensembl transcripts, we found that (1) the deduced precursors of 54,402 PTs shared at least one intron or exon with those of 18,437 Ensembl transcripts; (2) 12,262 PTs had both longer 5′ and 3′ termini than their maximally overlapping Ensembl transcripts; and (3) 41,838 spliced PTs were totally missing from the Sscrofa10.2 annotation. Similar results were obtained when the PTs were compared to the pig NCBI RefSeq mRNA collection.We built, validated and annotated a comprehensive porcine blood transcriptome with significant improvement over the annotation of Ensembl Sscrofa10.2 and the pig NCBI RefSeq mRNAs, and laid a foundation for blood-based high throughput transcriptomic assays in pigs and for advancing annotation of the pig genome.

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September 22, 2019  |  

Sixteen diverse laboratory mouse reference genomes define strain-specific haplotypes and novel functional loci.

We report full-length draft de novo genome assemblies for 16 widely used inbred mouse strains and find extensive strain-specific haplotype variation. We identify and characterize 2,567 regions on the current mouse reference genome exhibiting the greatest sequence diversity. These regions are enriched for genes involved in pathogen defence and immunity and exhibit enrichment of transposable elements and signatures of recent retrotransposition events. Combinations of alleles and genes unique to an individual strain are commonly observed at these loci, reflecting distinct strain phenotypes. We used these genomes to improve the mouse reference genome, resulting in the completion of 10 new gene structures. Also, 62 new coding loci were added to the reference genome annotation. These genomes identified a large, previously unannotated, gene (Efcab3-like) encoding 5,874 amino acids. Mutant Efcab3-like mice display anomalies in multiple brain regions, suggesting a possible role for this gene in the regulation of brain development.

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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.

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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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September 22, 2019  |  

A survey of the complex transcriptome from the highly polyploid sugarcane genome using full-length isoform sequencing and de novo assembly from short read sequencing.

Despite the economic importance of sugarcane in sugar and bioenergy production, there is not yet a reference genome available. Most of the sugarcane transcriptomic studies have been based on Saccharum officinarum gene indices (SoGI), expressed sequence tags (ESTs) and de novo assembled transcript contigs from short-reads; hence knowledge of the sugarcane transcriptome is limited in relation to transcript length and number of transcript isoforms.The sugarcane transcriptome was sequenced using PacBio isoform sequencing (Iso-Seq) of a pooled RNA sample derived from leaf, internode and root tissues, of different developmental stages, from 22 varieties, to explore the potential for capturing full-length transcript isoforms. A total of 107,598 unique transcript isoforms were obtained, representing about 71% of the total number of predicted sugarcane genes. The majority of this dataset (92%) matched the plant protein database, while just over 2% was novel transcripts, and over 2% was putative long non-coding RNAs. About 56% and 23% of total sequences were annotated against the gene ontology and KEGG pathway databases, respectively. Comparison with de novo contigs from Illumina RNA-Sequencing (RNA-Seq) of the internode samples from the same experiment and public databases showed that the Iso-Seq method recovered more full-length transcript isoforms, had a higher N50 and average length of largest 1,000 proteins; whereas a greater representation of the gene content and RNA diversity was captured in RNA-Seq. Only 62% of PacBio transcript isoforms matched 67% of de novo contigs, while the non-matched proportions were attributed to the inclusion of leaf/root tissues and the normalization in PacBio, and the representation of more gene content and RNA classes in the de novo assembly, respectively. About 69% of PacBio transcript isoforms and 41% of de novo contigs aligned with the sorghum genome, indicating the high conservation of orthologs in the genic regions of the two genomes.The transcriptome dataset should contribute to improved sugarcane gene models and sugarcane protein predictions; and will serve as a reference database for analysis of transcript expression in sugarcane.

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September 22, 2019  |  

GMAP and GSNAP for genomic sequence alignment: enhancements to speed, accuracy, and functionality.

The programs GMAP and GSNAP, for aligning RNA-Seq and DNA-Seq datasets to genomes, have evolved along with advances in biological methodology to handle longer reads, larger volumes of data, and new types of biological assays. The genomic representation has been improved to include linear genomes that can compare sequences using single-instruction multiple-data (SIMD) instructions, compressed genomic hash tables with fast access using SIMD instructions, handling of large genomes with more than four billion bp, and enhanced suffix arrays (ESAs) with novel data structures for fast access. Improvements to the algorithms have included a greedy match-and-extend algorithm using suffix arrays, segment chaining using genomic hash tables, diagonalization using segmental hash tables, and nucleotide-level dynamic programming procedures that use SIMD instructions and eliminate the need for F-loop calculations. Enhancements to the functionality of the programs include standardization of indel positions, handling of ambiguous splicing, clipping and merging of overlapping paired-end reads, and alignments to circular chromosomes and alternate scaffolds. The programs have been adapted for use in pipelines by integrating their usage into R/Bioconductor packages such as gmapR and HTSeqGenie, and these pipelines have facilitated the discovery of numerous biological phenomena.

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September 22, 2019  |  

How far can mitochondrial DNA drive the disease?

Mitochondria are one of the dominant drivers for producing cellular energy to meet a large number of biological functions, of which the mitochondrial DNA (mtDNA) is the control center of energetic driving force and the dominant driver of mitochondrial molecular diversification. mtDNA transcription generates the necessary RNAs to regulate the extent and nature of mtRNA post-transcriptional modifications and the activity of nucleus-encoded enzymes. With a special focus on mtDNA, the current volume aims to overview the biology and structures of mtDNA, regulatory roles of mtDNA in lung diseases, or involvement of mtDNA in metabolism. We explore the significance of mtDNA sequencing, methylation, stability, and mutation in the pathogenesis of the diseases. Molecular mechanisms by which mtDNA contribute to the regulation of mitochondrial homeostasis and drug resistance are also discussed. We also point out the importance of mitochondrial ribosome, single cell biology, and gene editing in the understanding of the development of mitochondrial dysfunction in lung disease.

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