In this PAGBioDay 2021 presentation, Michelle Vierra of PacBio provides the official welcome to PAGBio Day including a presentation on the latest in HiFi sequencing. Michelle is be followed by…
N6-methyladenosine (m6A) is a widespread RNA modification that influences nearly every aspect of the messenger RNA lifecycle. Our understanding of m6A has been facilitated by the development of global m6A mapping methods, which use antibodies to immunoprecipitate methylated RNA. However, these methods have several limitations, including high input RNA requirements and cross-reactivity to other RNA modifications. Here, we present DART-seq (deamination adjacent to RNA modification targets), an antibody-free method for detecting m6A sites. In DART-seq, the cytidine deaminase APOBEC1 is fused to the m6A-binding YTH domain. APOBEC1-YTH expression in cells induces C-to-U deamination at sites adjacent to m6A residues, which are detected using standard RNA-seq. DART-seq identifies thousands of m6A sites in cells from as little as 10?ng of total RNA and can detect m6A accumulation in cells over time. Additionally, we use long-read DART-seq to gain insights into m6A distribution along the length of individual transcripts.
The widespread occurrence of repetitive stretches of DNA in genomes of organisms across the tree of life imposes fundamental challenges for sequencing, genome assembly, and automated annotation of genes and proteins. This multi-level problem can lead to errors in genome and protein databases that are often not recognized or acknowledged. As a consequence, end users working with sequences with repetitive regions are faced with ‘ready-to-use’ deposited data whose trustworthiness is difficult to determine, let alone to quantify. Here, we provide a review of the problems associated with tandem repeat sequences that originate from different stages during the sequencing-assembly-annotation-deposition workflow, and that may proliferate in public database repositories affecting all downstream analyses. As a case study, we provide examples of the Atlantic cod genome, whose sequencing and assembly were hindered by a particularly high prevalence of tandem repeats. We complement this case study with examples from other species, where mis-annotations and sequencing errors have propagated into protein databases. With this review, we aim to raise the awareness level within the community of database users, and alert scientists working in the underlying workflow of database creation that the data they omit or improperly assemble may well contain important biological information valuable to others. © The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.
The genomes of classical inbred mouse strains include genes derived from all three major subspecies of the house mouse, Mus musculus. We recently posited that genetic diversity in the immunoglobulin heavy chain (IGH) gene loci of C57BL/6 and BALB/c mice reflect differences in subspecies origin. To investigate this hypothesis, we conducted high-throughput sequencing of IGH gene rearrangements to document IGH variable (IGHV), joining (IGHJ), and diversity (IGHD) genes in four inbred wild-derived mouse strains (CAST/EiJ, LEWES/EiJ, MSM/MsJ, and PWD/PhJ), and a single disease model strain (NOD/ShiLtJ), collectively representing genetic backgrounds of several major mouse subspecies. A total of 341 germline IGHV sequences were inferred in the wild-derived strains, including 247 not curated in the International Immunogenetics Information System. In contrast, 83/84 inferred NOD IGHV genes had previously been observed in C57BL/6 mice. Variability among the strains examined was observed for only a single IGHJ gene, involving a description of a novel allele. In contrast, unexpected variation was found in the IGHD gene loci, with four previously unreported IGHD gene sequences being documented. Very few IGHV sequences of C57BL/6 and BALB/c mice were shared with strains representing major subspecies, suggesting that their IGH loci may be complex mosaics of genes of disparate origins. This suggests a similar level of diversity is likely present in the IGH loci of other classical inbred strains. This must now be documented if we are to properly understand inter-strain variation in models of antibody-mediated disease. This article is protected by copyright. All rights reserved.This article is protected by copyright. All rights reserved.
Inherited skin disorders have been reported recently to have sporadic normal-looking areas, where a portion of the keratinocytes have recovered from causative gene mutations (revertant mosaicism). We observed a case of recessive dystrophic epidermolysis bullosa treated with cultured epidermal autografts (CEAs), whose CEA-grafted site remained epithelized for 16 years. We proved that the CEA product and the grafted area included cells with revertant mosaicism. Based on these findings, we conducted an investigator-initiated clinical trial of CEAs from clinically revertant skin for recessive dystrophic epidermolysis bullosa. The donor sites were analyzed by genetic analysis, immunofluorescence, electron microscopy, and quantification of the reverted mRNA with deep sequencing. The primary endpoint was the ulcer epithelization rate per patient at 4 weeks after the last CEA application. Three patients with recessive dystrophic epidermolysis bullosa with 8 ulcers were enrolled, and the epithelization rate for each patient at the primary endpoint was 87.7%, 100%, and 57.0%, respectively. The clinical effects were found to persist for at least 76 weeks after CEA transplantation. One of the three patients had apparent revertant mosaicism in the donor skin and in the post-transplanted area. CEAs from clinically normal skin are a potentially well-tolerated treatment for recessive dystrophic epidermolysis bullosa.Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.
We present high quality, phased genome assemblies representative of taurine and indicine cattle, subspecies that differ markedly in productivity-related traits and environmental adaptation. We report a new haplotype-aware scaffolding and polishing pipeline using contigs generated by the trio binning method to produce haplotype-resolved, chromosome-level genome assemblies of Angus (taurine) and Brahman (indicine) cattle breeds. These assemblies were used to identify structural and copy number variants that differentiate the subspecies and we found variant detection was sensitive to the specific reference genome chosen. Six gene families with immune related functions are expanded in the indicine lineage. Assembly of the genomes of both subspecies from a single individual enabled transcripts to be phased to detect allele-specific expression, and to study genome-wide selective sweeps. An indicus-specific extra copy of fatty acid desaturase is under positive selection and may contribute to indicine adaptation to heat and drought.
We report reference-quality genome assemblies and annotations for two accessions of soybean (Glycine max) and one of Glycine soja, the closest wild relative of G. max. The G. max assemblies are for widely used U.S. cultivars: the northern line ‘Williams 82’ (Wm82); and the southern line ‘Lee’. The Wm82 assembly improves the prior published assembly, and the Lee and G. soja assemblies are new for these accessions. Comparisons among the three accessions show generally high structural conservation, but nucleotide difference of 1.7 SNPs/kb between Wm82 and Lee, and 4.7 SNPs/kb between these lines and G. soja. SNP distributions and comparisons with genotypes of the Lee and Wm82 parents highlight patterns of introgressions and haplotype structure. Comparisons against the U.S. germplasm collection shows placement of the sequenced accessions relative to global soybean diversity. Analysis of a pan-gene collection shows generally high conservation, with variation occurring primarily in genomically clustered gene families. We found ~40-42 inversions per chromosome between either Lee or Wm82v4 and G. soja, and ~32 inversions per chromosome between Wm82 and Lee. We also investigated five domestication loci. For each locus, we found two different alleles with functional differences between G. soja and the two domesticated accessions. The genome assemblies for multiple cultivated accessions and for the closest wild ancestor of soybean provides a valuable set of resources for identifying causal variants that underlie traits for soybean’s domestication and improvement, serving as a basis for future research and crop improvement efforts for this important crop species. This article is protected by copyright. All rights reserved.This article is protected by copyright. All rights reserved.
China is the origin and evolutionary centre of Oriental pears. Pyrus betuleafolia is a wild species native to China and distributed in the northern region, and it is widely used as rootstock. Here, we report the de novo assembly of the genome of P. betuleafolia-Shanxi Duli using an integrated strategy that combines PacBio sequencing, BioNano mapping and chromosome conformation capture (Hi-C) sequencing. The genome assembly size was 532.7 Mb, with a contig N50 of 1.57 Mb. A total of 59 552 protein-coding genes and 247.4 Mb of repetitive sequences were annotated for this genome. The expansion genes in P. betuleafolia were significantly enriched in secondary metabolism, which may account for the organism’s considerable environmental adaptability. An alignment analysis of orthologous genes showed that fruit size, sugar metabolism and transport, and photosynthetic efficiency were positively selected in Oriental pear during domestication. A total of 573 nucleotide-binding site (NBS)-type resistance gene analogues (RGAs) were identified in the P. betuleafolia genome, 150 of which are TIR-NBS-LRR (TNL)-type genes, which represented the greatest number of TNL-type genes among the published Rosaceae genomes and explained the strong disease resistance of this wild species. The study of flavour metabolism-related genes showed that the anthocyanidin reductase (ANR) metabolic pathway affected the astringency of pear fruit and that sorbitol transporter (SOT) transmembrane transport may be the main factor affecting the accumulation of soluble organic matter. This high-quality P. betuleafolia genome provides a valuable resource for the utilization of wild pear in fundamental pear studies and breeding. © 2019 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.
RNA-Seq de novo assembly is an important method to generate transcriptomes for non-model organisms before any downstream analysis. Given many great de novo assembly methods developed by now, one critical issue is that there is no consensus on the evaluation of de novo assembly methods yet. Therefore, to set up a benchmark for evaluating the quality of de novo assemblies is very critical. Addressing this challenge will help us deepen the insights on the properties of different de novo assemblers and their evaluation methods, and provide hints on choosing the best assembly sets as transcriptomes of non-model organisms for the further functional analysis. In this article, we generate a textquotedblleftreal timetextquotedblright transcriptome using PacBio long reads as a benchmark for evaluating five de novo assemblers and two model-based de novo assembly evaluation methods. By comparing the de novo assmblies generated by RNA-Seq short reads with the textquotedblleftreal timetextquotedblright transcriptome from the same biological sample, we find that Trinity is best at the completeness by generating more assemblies than the alternative assemblers, but less continuous and having more misassemblies; Oases is best at the continuity and specificity, but less complete; The performance of SOAPdenovo-Trans, Trans-AByss and IDBA-Tran are in between of five assemblers. For evaluation methods, DETONATE leverages multiple aspects of the assembly set and ranks the assembly set with an average performance as the best, meanwhile the contig score can serve as a good metric to select assemblies with high completeness, specificity, continuity but not sensitive to misassemblies; TransRate contig score is useful for removing misassemblies, yet often the assemblies in the optimal set is too few to be used as a transcriptome.
The domestic pig (Sus scrofa) is important both as a food source and as a biomedical model with high anatomical and immunological similarity to humans. The draft reference genome (Sscrofa10.2) represented a purebred female pig from a commercial pork production breed (Duroc), and was established using older clone-based sequencing methods. The Sscrofa10.2 assembly was incomplete and unresolved redundancies, short range order and orientation errors and associated misassembled genes limited its utility. We present two highly contiguous chromosome-level genome assemblies created with more recent long read technologies and a whole genome shotgun strategy, one for the same Duroc female (Sscrofa11.1) and one for an outbred, composite breed male animal commonly used for commercial pork production (USMARCv1.0). Both assemblies are of substantially higher (>90-fold) continuity and accuracy compared to the earlier reference, and the availability of two independent assemblies provided an opportunity to identify large-scale variants and to error-check the accuracy of representation of the genome. We propose that the improved Duroc breed assembly (Sscrofa11.1) become the reference genome for genomic research in pigs.
Neisseria gonorrhoeae, the sole causative agent of gonorrhea, constitutively undergoes diversification of the Type IV pilus. Gene conversion occurs between one of the several donor silent copies located in distinct loci and the recipient pilE gene, encoding the major pilin subunit of the pilus. A guanine quadruplex (G4) DNA structure and a cis-acting sRNA (G4-sRNA) are located upstream of the pilE gene and both are required for pilin antigenic variation (Av). We show that the reduced sRNA transcription lowers pilin Av frequencies. Extended transcriptional elongation is not required for Av, since limiting the transcript to 32 nt allows for normal Av frequencies. Using chromatin immunoprecipitation (ChIP) assays, we show that cellular G4s are less abundant when sRNA transcription is lower. In addition, using ChIP, we demonstrate that the G4-sRNA forms a stable RNA:DNA hybrid (R-loop) with its template strand. However, modulating R-loop levels by controlling RNase HI expression does not alter G4 abundance quantified through ChIP. Since pilin Av frequencies were not altered when modulating R-loop levels by controlling RNase HI expression, we conclude that transcription of the sRNA is necessary, but stable R-loops are not required to promote pilin Av. © 2019 John Wiley & Sons Ltd.
Orchardgrass (Dactylis glomerata L.) is an important forage grass for cultivating livestock worldwide. Here, we report an ~1.84-Gb chromosome-scale diploid genome assembly of orchardgrass, with a contig N50 of 0.93 Mb, a scaffold N50 of 6.08 Mb and a super-scaffold N50 of 252.52 Mb, which is the first chromosome-scale assembled genome of a cool-season forage grass. The genome includes 40 088 protein-coding genes, and 69% of the assembled sequences are transposable elements, with long terminal repeats (LTRs) being the most abundant. The LTRretrotransposons may have been activated and expanded in the grass genome in response to environmental changes during the Pleistocene between 0 and 1 million years ago. Phylogenetic analysis reveals that orchardgrass diverged after rice but before three Triticeae species, and evolutionarily conserved chromosomes were detected by analysing ancient chromosome rearrangements in these grass species. We also resequenced the whole genome of 76 orchardgrass accessions and found that germplasm from Northern Europe and East Asia clustered together, likely due to the exchange of plants along the ‘Silk Road’ or other ancient trade routes connecting the East and West. Last, a combined transcriptome, quantitative genetic and bulk segregant analysis provided insights into the genetic network regulating flowering time in orchardgrass and revealed four main candidate genes controlling this trait. This chromosome-scale genome and the online database of orchardgrass developed here will facilitate the discovery of genes controlling agronomically important traits, stimulate genetic improvement of and functional genetic research on orchardgrass and provide comparative genetic resources for other forage grasses. © 2019 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.
Bread wheat (Triticum aestivum L.) is an allohexaploid, and the transcriptional characteristics of the wheat embryo and endosperm during grain development remain unclear. To analyze the transcriptome, we performed isoform sequencing (Iso-Seq) for wheat grain and RNA sequencing (RNA-Seq) for the embryo and de-embryonated kernels. The differential regulation between the embryo and de-embryonated kernels was found to be greater than the difference between the two time points for each tissue. Exactly 2264 and 4790 tissue-specific genes were found at 14 days post-anthesis (DPA), while 5166 and 3784 genes were found at 25 DPA in the embryo and de-embryonated kernels, respectively. Genes expressed in the embryo were more likely to be related to nucleic acid and enzyme regulation. In de-embryonated kernels, genes were rich in substance metabolism and enzyme activity functions. Moreover, 4351, 4641, 4516, and 4453 genes with the A, B, and D homoeoloci were detected for each of the four tissues. Expression characteristics suggested that the D genome may be the largest contributor to the transcriptome in developing grain. Among these, 48, 66, and 38 silenced genes emerged in the A, B, and D genomes, respectively. Gene ontology analysis showed that silenced genes could be inclined to different functions in different genomes. Our study provided specific gene pools of the embryo and de-embryonated kernels and a homoeolog expression bias model on a large scale. This is helpful for providing new insights into the molecular physiology of wheat.
Over the past decade, RNA sequencing (RNA-seq) has become an indispensable tool for transcriptome-wide analysis of differential gene expression and differential splicing of mRNAs. However, as next-generation sequencing technologies have developed, so too has RNA-seq. Now, RNA-seq methods are available for studying many different aspects of RNA biology, including single-cell gene expression, translation (the translatome) and RNA structure (the structurome). Exciting new applications are being explored, such as spatial transcriptomics (spatialomics). Together with new long-read and direct RNA-seq technologies and better computational tools for data analysis, innovations in RNA-seq are contributing to a fuller understanding of RNA biology, from questions such as when and where transcription occurs to the folding and intermolecular interactions that govern RNA function.
Since the genome of herpes simplex virus 1 (HSV-1) was first sequenced more than 30 years ago, its predicted 80 genes have been intensively studied. Here, we unravel the complete viral transcriptome and translatome during lytic infection with base-pair resolution by computational integration of multi-omics data. We identified a total of 201 viral transcripts and 284 open reading frames (ORFs) including all known and 46 novel large ORFs. Multiple transcript isoforms expressed from individual gene loci explain translation of the vast majority of novel viral ORFs as well as N-terminal extensions (NTEs) and truncations thereof. We show that key viral regulators and structural proteins possess NTEs, which initiate from non-canonical start codons and govern subcellular protein localization and packaging. We validated a novel non-canonical large spliced ORF in the ICP0 locus and identified a 93 aa ORF overlapping ICP34.5 that is thus also deleted in the FDA-approved oncolytic virus Imlygic. Finally, we extend the current nomenclature to include all novel viral gene products. Taken together, this work provides a valuable resource for future functional studies, vaccine design and oncolytic therapies.
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