June 1, 2021  |  

From RNA to full-length transcripts: The PacBio Iso-Seq method for transcriptome analysis and genome annotation

A single gene may encode a surprising number of proteins, each with a distinct biological function. This is especially true in complex eukaryotes. Short- read RNA sequencing (RNA-seq) works by physically shearing transcript isoforms into smaller pieces and bioinformatically reassembling them, leaving opportunity for misassembly or incomplete capture of the full diversity of isoforms from genes of interest. The PacBio Isoform Sequencing (Iso-Seq™) method employs long reads to sequence transcript isoforms from the 5’ end to their poly-A tails, eliminating the need for transcript reconstruction and inference. These long reads result in complete, unambiguous information about alternatively spliced exons, transcriptional start sites, and poly- adenylation sites. This allows for the characterization of the full complement of isoforms within targeted genes, or across an entire transcriptome. Here we present improved genome annotations for two avian models of vocal learning, Anna’s hummingbird (Calypte anna) and zebra finch (Taeniopygia guttata), using the Iso-Seq method. We present graphical user interface and command line analysis workflows for the data sets. From brain total RNA, we characterize more than 15,000 isoforms in each species, 9% and 5% of which were previously unannotated in hummingbird and zebra finch, respectively. We highlight one example where capturing full-length transcripts identifies additional exons and UTRs.


June 1, 2021  |  

Full-length transcript profiling with the Iso-Seq method for improved genome annotations

Incomplete annotation of genomes represents a major impediment to understanding biological processes, functional differences between species, and evolutionary mechanisms. Often, genes that are large, embedded within duplicated genomic regions, or associated with repeats are difficult to study by short-read expression profiling and assembly. In addition, most genes in eukaryotic organisms produce alternatively spliced isoforms, broadening the diversity of proteins encoded by the genome, which are difficult to resolve with short-read methods. Short-read RNA sequencing (RNA-seq) works by physically shearing transcript isoforms into smaller pieces and bioinformatically reassembling them, leaving opportunity for misassembly or incomplete capture of the full diversity of isoforms from genes of interest. In contrast, Single Molecule, Real-Time (SMRT) Sequencing directly sequences full-length transcripts without the need for assembly and imputation. Here we apply the Iso-Seq method (long-read RNA sequencing) to detect full-length isoforms and the new IsoPhase algorithm to retrieve allele-specific isoform information for two avian models of vocal learning, Anna’s hummingbird (Calypte anna) and zebra finch (Taeniopygia guttata).


April 21, 2020  |  

Tandem repeats lead to sequence assembly errors and impose multi-level challenges for genome and protein databases.

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.


April 21, 2020  |  

The bracteatus pineapple genome and domestication of clonally propagated crops.

Domestication of clonally propagated crops such as pineapple from South America was hypothesized to be a ‘one-step operation’. We sequenced the genome of Ananas comosus var. bracteatus CB5 and assembled 513?Mb into 25 chromosomes with 29,412 genes. Comparison of the genomes of CB5, F153 and MD2 elucidated the genomic basis of fiber production, color formation, sugar accumulation and fruit maturation. We also resequenced 89 Ananas genomes. Cultivars ‘Smooth Cayenne’ and ‘Queen’ exhibited ancient and recent admixture, while ‘Singapore Spanish’ supported a one-step operation of domestication. We identified 25 selective sweeps, including a strong sweep containing a pair of tandemly duplicated bromelain inhibitors. Four candidate genes for self-incompatibility were linked in F153, but were not functional in self-compatible CB5. Our findings support the coexistence of sexual recombination and a one-step operation in the domestication of clonally propagated crops. This work guides the exploration of sexual and asexual domestication trajectories in other clonally propagated crops.


April 21, 2020  |  

Genome assembly provides insights into the genome evolution and flowering regulation of orchardgrass.

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.


April 21, 2020  |  

Rapid evolution of a-gliadin gene family revealed by analyzing Gli-2 locus regions of wild emmer wheat.

a-Gliadins are a major group of gluten proteins in wheat flour that contribute to the end-use properties for food processing and contain major immunogenic epitopes that can cause serious health-related issues including celiac disease (CD). a-Gliadins are also the youngest group of gluten proteins and are encoded by a large gene family. The majority of the gene family members evolved independently in the A, B, and D genomes of different wheat species after their separation from a common ancestral species. To gain insights into the origin and evolution of these complex genes, the genomic regions of the Gli-2 loci encoding a-gliadins were characterized from the tetraploid wild emmer, a progenitor of hexaploid bread wheat that contributed the AABB genomes. Genomic sequences of Gli-2 locus regions for the wild emmer A and B genomes were first reconstructed using the genome sequence scaffolds along with optical genome maps. A total of 24 and 16 a-gliadin genes were identified for the A and B genome regions, respectively. a-Gliadin pseudogene frequencies of 86% for the A genome and 69% for the B genome were primarily caused by C to T substitutions in the highly abundant glutamine codons, resulting in the generation of premature stop codons. Comparison with the homologous regions from the hexaploid wheat cv. Chinese Spring indicated considerable sequence divergence of the two A genomes at the genomic level. In comparison, conserved regions between the two B genomes were identified that included a-gliadin pseudogenes containing shared nested TE insertions. Analyses of the genomic organization and phylogenetic tree reconstruction indicate that although orthologous gene pairs derived from speciation were present, large portions of a-gliadin genes were likely derived from differential gene duplications or deletions after the separation of the homologous wheat genomes ~?0.5 MYA. The higher number of full-length intact a-gliadin genes in hexaploid wheat than that in wild emmer suggests that human selection through domestication might have an impact on a-gliadin evolution. Our study provides insights into the rapid and dynamic evolution of genomic regions harboring the a-gliadin genes in wheat.


April 21, 2020  |  

Chromosome-level hybrid de novo genome assemblies as an attainable option for non-model organisms

The emergence of third generation sequencing (3GS; long-reads) is making closer the goal of chromosome-size fragments in de novo genome assemblies. This allows the exploration of new and broader questions on genome evolution for a number of non-model organisms. However, long-read technologies result in higher sequencing error rates and therefore impose an elevated cost of sufficient coverage to achieve high enough quality. In this context, hybrid assemblies, combining short-reads and long-reads provide an alternative efficient and cost-effective approach to generate de novo, chromosome-level genome assemblies. The array of available software programs for hybrid genome assembly, sequence correction and manipulation is constantly being expanded and improved. This makes it difficult for non-experts to find efficient, fast and tractable computational solutions for genome assembly, especially in the case of non-model organisms lacking a reference genome or one from a closely related species. In this study, we review and test the most recent pipelines for hybrid assemblies, comparing the model organism Drosophila melanogaster to a non-model cactophilic Drosophila, D. mojavensis. We show that it is possible to achieve excellent contiguity on this non-model organism using the DBG2OLC pipeline.


April 21, 2020  |  

The Chinese chestnut genome: a reference for species restoration

Forest tree species are increasingly subject to severe mortalities from exotic pests, diseases, and invasive organisms, accelerated by climate change. Forest health issues are threatening multiple species and ecosystem sustainability globally. While sources of resistance may be available in related species, or among surviving trees, introgression of resistance genes into threatened tree species in reasonable time frames requires genome-wide breeding tools. Asian species of chestnut (Castanea spp.) are being employed as donors of disease resistance genes to restore native chestnut species in North America and Europe. To aid in the restoration of threatened chestnut species, we present the assembly of a reference genome with chromosome-scale sequences for Chinese chestnut (C. mollissima), the disease-resistance donor for American chestnut restoration. We also demonstrate the value of the genome as a platform for research and species restoration, including new insights into the evolution of blight resistance in Asian chestnut species, the locations in the genome of ecologically important signatures of selection differentiating American chestnut from Chinese chestnut, the identification of candidate genes for disease resistance, and preliminary comparisons of genome organization with related species.


April 21, 2020  |  

Draft genome sequence resource of switchgrass rust pathogen, Puccinia novopanici isolate Ard-01.

Puccinia novopanici is an important biotrophic fungal pathogen that causes rust disease in switchgrass. Lack of genomic resources for P. novopanici has hampered the progress towards developing effective disease resistance against this pathogen. Therefore, we have sequenced the whole genome of P. novopanici and generated a framework to understand pathogenicity mechanisms, identify effectors, repeat element invasion, genome evolution, and comparative genomics among Puccinia species in the future. Long and short read sequences were generated from P. novopanici genomic DNA by PacBio and Illumina technologies, respectively, and assembled a 99.9 megabase (Mb) genome. Transcripts of P. novopanici were predicted from assembled genome using MAKER and were further validated by RNAseq data. The genome sequence information of P. novopanici will be a valuable resource for researchers working on monocot rusts and plant disease resistance in general.


April 21, 2020  |  

Insect genomes: progress and challenges.

In the wake of constant improvements in sequencing technologies, numerous insect genomes have been sequenced. Currently, 1219 insect genome-sequencing projects have been registered with the National Center for Biotechnology Information, including 401 that have genome assemblies and 155 with an official gene set of annotated protein-coding genes. Comparative genomics analysis showed that the expansion or contraction of gene families was associated with well-studied physiological traits such as immune system, metabolic detoxification, parasitism and polyphagy in insects. Here, we summarize the progress of insect genome sequencing, with an emphasis on how this impacts research on pest control. We begin with a brief introduction to the basic concepts of genome assembly, annotation and metrics for evaluating the quality of draft assemblies. We then provide an overview of genome information for numerous insect species, highlighting examples from prominent model organisms, agricultural pests and disease vectors. We also introduce the major insect genome databases. The increasing availability of insect genomic resources is beneficial for developing alternative pest control methods. However, many opportunities remain for developing data-mining tools that make maximal use of the available insect genome resources. Although rapid progress has been achieved, many challenges remain in the field of insect genomics. © 2019 The Royal Entomological Society.


April 21, 2020  |  

Variation in genome content and predatory phenotypes between Bdellovibrio sp. NC01 isolated from soil and B. bacteriovorus type strain HD100

The range of naturally occurring variation in the ability of Bdellovibrio strains to attack and kill Gram-negative bacteria is not well understood. Defining phenotypic and associated genotypic variation among Bdellovibrio may further our understanding of how this genus impacts microbial communities. In addition, comparisons of the predatory phenotypes of divergent strains may inform the development of Bdellovibrio as biocontrol agents to combat bacterial infections. We isolated Bdellovibrio sp. NC01 from soil and compared its genome and predatory phenotypes to B. bacteriovorus type strain HD100. Based on analysis of 16S rRNA gene sequences and average amino acid identity, NC01 belongs to a different species than HD100. Genome-wide comparisons and individual gene analyses indicated that eight NC01 genome regions were likely acquired by horizontal gene transfer (HGT), further supporting an important role for HGT in Bdellovibrio genome evolution. Within these regions, multiple protein-coding sequences were assigned predicted functions related to transcriptional regulation and transport; however, most were annotated as hypothetical proteins. Compared to HD100, NC01 has a limited prey range and kills E. coli ML35 less efficiently. Whereas HD100 drastically reduces the ML35 population and then maintains low prey population density, NC01 causes a smaller reduction in ML35, after which the prey population recovers, accompanied by a decrease in NC01. In addition, NC01 forms turbid plaques on lawns of E. coli ML35, in contrast to clear plaques formed by HD100. Characterizing variation in interactions between Bdellovibrio and Gram-negative bacteria, such as observed with NC01 and HD100, is valuable for understanding the ecological significance of predatory bacteria and evaluating their effectiveness in clinical applications.


April 21, 2020  |  

Evolutionary superscaffolding and chromosome anchoring to improve Anopheles genome assemblies

Background New sequencing technologies have lowered financial barriers to whole genome sequencing, but resulting assemblies are often fragmented and far from textquoteleftfinishedtextquoteright. Updating multi-scaffold drafts to chromosome-level status can be achieved through experimental mapping or re-sequencing efforts. Avoiding the costs associated with such approaches, comparative genomic analysis of gene order conservation (synteny) to predict scaffold neighbours (adjacencies) offers a potentially useful complementary method for improving draft assemblies.Results We employed three gene synteny-based methods applied to 21 Anopheles mosquito assemblies to produce consensus sets of scaffold adjacencies. For subsets of the assemblies we integrated these with additional supporting data to confirm and complement the synteny-based adjacencies: six with physical mapping data that anchor scaffolds to chromosome locations, 13 with paired-end RNA sequencing (RNAseq) data, and three with new assemblies based on re-scaffolding or Pacific Biosciences long-read data. Our combined analyses produced 20 new superscaffolded assemblies with improved contiguities: seven for which assignments of non-anchored scaffolds to chromosome arms span more than 75% of the assemblies, and a further seven with chromosome anchoring including an 88% anchored Anopheles arabiensis assembly and, respectively, 73% and 84% anchored assemblies with comprehensively updated cytogenetic photomaps for Anopheles funestus and Anopheles stephensi.Conclusions Experimental data from probe mapping, RNAseq, or long-read technologies, where available, all contribute to successful upgrading of draft assemblies. Our comparisons show that gene synteny-based computational methods represent a valuable alternative or complementary approach. Our improved Anopheles reference assemblies highlight the utility of applying comparative genomics approaches to improve community genomic resources.ADADSEQAGOAGOUTI-basedAGOUTIannotated genome optimization using transcriptome information toolALNalignment-basedCAMSAcomparative analysis and merging of scaffold assemblies toolDPdynamic programmingFISHfluorescence in situ hybridizationGAGOS-ASMGOS-ASMGene order scaffold assemblerKbpkilobasepairsMbpmegabasepairsOSORTHOSTITCHPacBioPacific BiosciencesPBPacBio-basedPHYphysical-mapping-basedRNAseqRNA sequencingQTLquantitative trait lociSYNsynteny-based.


April 21, 2020  |  

Genome-wide selection footprints and deleterious variations in young Asian allotetraploid rapeseed.

Brassica napus (AACC, 2n = 38) is an important oilseed crop grown worldwide. However, little is known about the population evolution of this species, the genomic difference between its major genetic groups, such as European and Asian rapeseed, and the impacts of historical large-scale introgression events on this young tetraploid. In this study, we reported the de novo assembly of the genome sequences of an Asian rapeseed (B. napus), Ningyou 7, and its four progenitors and compared these genomes with other available genomic data from diverse European and Asian cultivars. Our results showed that Asian rapeseed originally derived from European rapeseed but subsequently significantly diverged, with rapid genome differentiation after hybridization and intensive local selective breeding. The first historical introgression of B. rapa dramatically broadened the allelic pool but decreased the deleterious variations of Asian rapeseed. The second historical introgression of the double-low traits of European rapeseed (canola) has reshaped Asian rapeseed into two groups (double-low and double-high), accompanied by an increase in genetic load in the double-low group. This study demonstrates distinctive genomic footprints and deleterious SNP (single nucleotide polymorphism) variants for local adaptation by recent intra- and interspecies introgression events and provides novel insights for understanding the rapid genome evolution of a young allopolyploid crop. © 2019 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.


Talk with an expert

If you have a question, need to check the status of an order, or are interested in purchasing an instrument, we're here to help.