Jeremy Schmutz discusses the increased throughput and reduced project costs using HiFi reads from the PacBio Sequel II System in his work sequencing, assembling, and analyzing a variety of genomes…
Highly accurate long reads, known as HiFi reads, are a new tool in scientists’ sequencing toolbox. Hear PacBio users share how they are using HiFi reads to explore the genomes,…
Accurate sequencing data is key for University of Florida scientist Ana Conesa. She is using PacBio HiFi reads from the Sequel II System to identity alternative isoforms and determine the…
The utility of new highly accurate long reads, or HiFi reads, was first demonstrated for calling all variant types in human genomes. It has since been shown that HiFi reads…
In this ASHG 2020 CoLab presentation hear Principal Scientists, Aaron Wenger and Elizabeth Tseng share how highly accurate long reads (HiFi reads) provide comprehensive variant detection for both genomes and…
Long-read mRNA sequencing such as PacBio’s Iso-Seq method offer high-throughput transcriptome profiling that circumvents the transcript assembly problem by sequencing full-length cDNA. The Iso-Seq method has emerged as the most…
Most genes in eukaryotic organisms produce alternative isoforms, broadening the diversity of proteins and non-coding RNAs encoded by the genome. In contrast to other RNA sequencing platforms that rely on…
During the past decade, the search for pathogenic mutations in rare human genetic diseases has involved huge efforts to sequence coding regions, or the entire genome, using massively parallel short-read sequencers. However, the approximate current diagnostic rate is <50% using these approaches, and there remain many rare genetic diseases with unknown cause. There may be many reasons for this, but one plausible explanation is that the responsible mutations are in regions of the genome that are difficult to sequence using conventional technologies (e.g., tandem-repeat expansion or complex chromosomal structural aberrations). Despite the drawbacks of high cost and a shortage of standard analytical methods, several studies have analyzed pathogenic changes in the genome using long-read sequencers. The results of these studies provide hope that further application of long-read sequencers to identify the causative mutations in unsolved genetic diseases may expand our understanding of the human genome and diseases. Such approaches may also be applied to molecular diagnosis and therapeutic strategies for patients with genetic diseases in the future.
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.
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.
Chlorella vulgaris is a fast-growing fresh-water microalga cultivated at the industrial scale for applications ranging from food to biofuel production. To advance our understanding of its biology and to establish genetics tools for biotechnological manipulation, we sequenced the nuclear and organelle genomes of Chlorella vulgaris 211/11P by combining next generation sequencing and optical mapping of isolated DNA molecules. This hybrid approach allowed to assemble the nuclear genome in 14 pseudo-molecules with an N50 of 2.8 Mb and 98.9% of scaffolded genome. The integration of RNA-seq data obtained at two different irradiances of growth (high light-HL versus low light -LL) enabled to identify 10,724 nuclear genes, coding for 11,082 transcripts. Moreover 121 and 48 genes were respectively found in the chloroplast and mitochondrial genome. Functional annotation and expression analysis of nuclear, chloroplast and mitochondrial genome sequences revealed peculiar features of Chlorella vulgaris. Evidence of horizontal gene transfers from chloroplast to mitochondrial genome was observed. Furthermore, comparative transcriptomic analyses of LL vs HL provide insights into the molecular basis for metabolic rearrangement in HL vs. LL conditions leading to enhanced de novo fatty acid biosynthesis and triacylglycerol accumulation. The occurrence of a cytosolic fatty acid biosynthetic pathway can be predicted and its upregulation upon HL exposure is observed, consistent with increased lipid amount under HL. These data provide a rich genetic resource for future genome editing studies, and potential targets for biotechnological manipulation of Chlorella vulgaris or other microalgae species to improve biomass and lipid productivity.This article is protected by copyright. All rights reserved.
Background Assemblies of diploid genomes are generally unphased, pseudo-haploid representations that do not correctly reconstruct the two parental haplotypes present in the individual sequenced. Instead, the assembly alternates between parental haplotypes and may contain duplications in regions where the parental haplotypes are sufficiently different. Trio binning is an approach to genome assembly that uses short reads from both parents to classify long reads from the offspring according to maternal or paternal haplotype origin, and is thus helped rather than impeded by heterozygosity. Using this approach, it is possible to derive two assemblies from an individual, accurately representing both parental contributions in their entirety with higher continuity and accuracy than is possible with other methods.Results We used trio binning to assemble reference genomes for two species from a single individual using an interspecies cross of yak (Bos grunniens) and cattle (Bos taurus). The high heterozygosity inherent to interspecies hybrids allowed us to confidently assign >99% of long reads from the F1 offspring to parental bins using unique k-mers from parental short reads. Both the maternal (yak) and paternal (cattle) assemblies contain over one third of the acrocentric chromosomes, including the two largest chromosomes, in single haplotigs.Conclusions These haplotigs are the first vertebrate chromosome arms to be assembled gap-free and fully phased, and the first time assemblies for two species have been created from a single individual. Both assemblies are the most continuous currently available for non-model vertebrates.MbmegabaseskbkilobasesMYAmillions of years agoMHCmajor histocompatibility complexSMRTsingle molecule real time
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.
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.
Third-generation sequencing technologies benefit transcriptome analysis by generating longer sequencing reads. However, not all single-molecule long reads represent full transcripts due to incomplete cDNA synthesis and the sequencing length limit of the platform. This drives a need for long read transcript assembly. We quantify the benefit that can be achieved by using a transcript assembler on long reads. Adding long-read-specific algorithms, we evolved Scallop to make Scallop-LR, a long-read transcript assembler, to handle the computational challenges arising from long read lengths and high error rates. Analyzing 26 SRA PacBio datasets using Scallop-LR, Iso-Seq Analysis, and StringTie, we quantified the amount by which assembly improved Iso-Seq results. Through combined evaluation methods, we found that Scallop-LR identifies 2100–4000 more (for 18 human datasets) or 1100–2200 more (for eight mouse datasets) known transcripts than Iso-Seq Analysis, which does not do assembly. Further, Scallop-LR finds 2.4–4.4 times more potentially novel isoforms than Iso-Seq Analysis for the human and mouse datasets. StringTie also identifies more transcripts than Iso-Seq Analysis. Adding long-read-specific optimizations in Scallop-LR increases the numbers of predicted known transcripts and potentially novel isoforms for the human transcriptome compared to several recent short-read assemblers (e.g. StringTie). Our findings indicate that transcript assembly by Scallop-LR can reveal a more complete human transcriptome.
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