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October 23, 2019  |  

An online bioinformatics tool predicts zinc finger and TALE nuclease off-target cleavage.

Although engineered nucleases can efficiently cleave intracellular DNA at desired target sites, major concerns remain on potential ‘off-target’ cleavage that may occur throughout the genome. We developed an online tool: predicted report of genome-wide nuclease off-target sites (PROGNOS) that effectively identifies off-target sites. The initial bioinformatics algorithms in PROGNOS were validated by predicting 44 of 65 previously confirmed off-target sites, and by uncovering a new off-target site for the extensively studied zinc finger nucleases (ZFNs) targeting C-C chemokine receptor type 5. Using PROGNOS, we rapidly interrogated 128 potential off-target sites for newly designed transcription activator-like effector nucleases containing either Asn-Asn (NN) or Asn-Lys (NK) repeat variable di-residues (RVDs) and 3- and 4-finger ZFNs, and validated 13 bona fide off-target sites for these nucleases by DNA sequencing. The PROGNOS algorithms were further refined by incorporating additional features of nuclease-DNA interactions and the newly confirmed off-target sites into the training set, which increased the percentage of bona fide off-target sites found within the top PROGNOS rankings. By identifying potential off-target sites in silico, PROGNOS allows the selection of more specific target sites and aids the identification of bona fide off-target sites, significantly facilitating the design of engineered nucleases for genome editing applications.

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October 23, 2019  |  

Improved production of propionic acid using genome shuffling.

Traditionally derived from fossil fuels, biological production of propionic acid has recently gained interest. Propionibacterium species produce propionic acid as their main fermentation product. Production of other organic acids reduces propionic acid yield and productivity, pointing to by-products gene-knockout strategies as a logical solution to increase yield. However, removing by-product formation has seen limited success due to our inability to genetically engineer the best producing strains (i.e. Propionibacterium acidipropionici). To overcome this limitation, random mutagenesis continues to be the best path towards improving strains for biological propionic acid production. Recent advances in next generation sequencing opened new avenues to understand improved strains. In this work, we use genome shuffling on two wild type strains to generate a better propionic acid producing strain. Using next generation sequencing, we mapped the genomic changes leading to the improved phenotype. The best strain produced 25% more propionic acid than the wild type strain. Sequencing of the strains showed that genomic changes were restricted to single point mutations and gene duplications in well-conserved regions in the genomes. Such results confirm the involvement of gene conversion in genome shuffling as opposed to long genomic insertions. © 2016 The Authors. Biotechnology Journal published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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October 23, 2019  |  

Endogenous sequence patterns predispose the repair modes of CRISPR/Cas9-induced DNA double-stranded breaks in Arabidopsis thaliana.

The possibility to predict the outcome of targeted DNA double-stranded break (DSB) repair would be desirable for genome editing. Furthermore the consequences of mis-repair of potentially cell-lethal DSBs and the underlying pathways are not yet fully understood. Here we study the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-induced mutation spectra at three selected endogenous loci in Arabidopsis thaliana by deep sequencing of long amplicon libraries. Notably, we found sequence-dependent genomic features that affected the DNA repair outcome. Deletions of 1-bp to <1000-bp size and/or very short insertions, deletions >1 kbp (all due to NHEJ) and deletions combined with insertions between 5-bp to >100 bp [caused by a synthesis-dependent strand annealing (SDSA)-like mechanism] occurred most frequently at all three loci. The appearance of single-stranded annealing events depends on the presence and distance between repeats flanking the DSB. The frequency and size of insertions is increased if a sequence with high similarity to the target site was available in cis. Most deletions were linked to pre-existing microhomology. Deletion and/or insertion mutations were blunt-end ligated or via de novo generated microhomology. While most mutation types and, to some degree, their predictability are comparable with animal systems, the broad range of deletion mutations seems to be a peculiar feature of the plant A. thaliana.© 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

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October 23, 2019  |  

Bioengineered AAV capsids with combined high human liver transduction in vivo and unique humoral seroreactivity.

Existing recombinant adeno-associated virus (rAAV) serotypes for delivering in vivo gene therapy treatments for human liver diseases have not yielded combined high-level human hepatocyte transduction and favorable humoral neutralization properties in diverse patient groups. Yet, these combined properties are important for therapeutic efficacy. To bioengineer capsids that exhibit both unique seroreactivity profiles and functionally transduce human hepatocytes at therapeutically relevant levels, we performed multiplexed sequential directed evolution screens using diverse capsid libraries in both primary human hepatocytes in vivo and with pooled human sera from thousands of patients. AAV libraries were subjected to five rounds of in vivo selection in xenografted mice with human livers to isolate an enriched human-hepatotropic library that was then used as input for a sequential on-bead screen against pooled human immunoglobulins. Evolved variants were vectorized and validated against existing hepatotropic serotypes. Two of the evolved AAV serotypes, NP40 and NP59, exhibited dramatically improved functional human hepatocyte transduction in vivo in xenografted mice with human livers, along with favorable human seroreactivity profiles, compared with existing serotypes. These novel capsids represent enhanced vector delivery systems for future human liver gene therapy applications. Copyright © 2017. Published by Elsevier Inc.

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October 23, 2019  |  

Cas9-mediated allelic exchange repairs compound heterozygous recessive mutations in mice.

We report a genome-editing strategy to correct compound heterozygous mutations, a common genotype in patients with recessive genetic disorders. Adeno-associated viral vector delivery of Cas9 and guide RNA induces allelic exchange and rescues the disease phenotype in mouse models of hereditary tyrosinemia type I and mucopolysaccharidosis type I. This approach recombines non-mutated genetic information present in two heterozygous alleles into one functional allele without using donor DNA templates.

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October 23, 2019  |  

Chromosomal-level assembly of yellow catfish genome using third-generation DNA sequencing and Hi-C analysis.

The yellow catfish, Pelteobagrus fulvidraco, belonging to the Siluriformes order, is an economically important freshwater aquaculture fish species in Asia, especially in Southern China. The aquaculture industry has recently been facing tremendous challenges in germplasm degeneration and poor disease resistance. As the yellow catfish exhibits notable sex dimorphism in growth, with adult males about two- to three-fold bigger than females, the way in which the aquaculture industry takes advantage of such sex dimorphism is another challenge. To address these issues, a high-quality reference genome of the yellow catfish would be a very useful resource.To construct a high-quality reference genome for the yellow catfish, we generated 51.2 Gb short reads and 38.9 Gb long reads using Illumina and Pacific Biosciences (PacBio) sequencing platforms, respectively. The sequencing data were assembled into a 732.8 Mb genome assembly with a contig N50 length of 1.1 Mb. Additionally, we applied Hi-C technology to identify contacts among contigs, which were then used to assemble contigs into scaffolds, resulting in a genome assembly with 26 chromosomes and a scaffold N50 length of 25.8 Mb. Using 24,552 protein-coding genes annotated in the yellow catfish genome, the phylogenetic relationships of the yellow catfish with other teleosts showed that yellow catfish separated from the common ancestor of channel catfish ~81.9 million years ago. We identified 1,717 gene families to be expanded in the yellow catfish, and those gene families are mainly enriched in the immune system, signal transduction, glycosphingolipid biosynthesis, and fatty acid biosynthesis.Taking advantage of Illumina, PacBio, and Hi-C technologies, we constructed the first high-quality chromosome-level genome assembly for the yellow catfish P. fulvidraco. The genomic resources generated in this work not only offer a valuable reference genome for functional genomics studies of yellow catfish to decipher the economic traits and sex determination but also provide important chromosome information for genome comparisons in the wider evolutionary research community.

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September 22, 2019  |  

A chromosome conformation capture ordered sequence of the barley genome.

Cereal grasses of the Triticeae tribe have been the major food source in temperate regions since the dawn of agriculture. Their large genomes are characterized by a high content of repetitive elements and large pericentromeric regions that are virtually devoid of meiotic recombination. Here we present a high-quality reference genome assembly for barley (Hordeum vulgare L.). We use chromosome conformation capture mapping to derive the linear order of sequences across the pericentromeric space and to investigate the spatial organization of chromatin in the nucleus at megabase resolution. The composition of genes and repetitive elements differs between distal and proximal regions. Gene family analyses reveal lineage-specific duplications of genes involved in the transport of nutrients to developing seeds and the mobilization of carbohydrates in grains. We demonstrate the importance of the barley reference sequence for breeding by inspecting the genomic partitioning of sequence variation in modern elite germplasm, highlighting regions vulnerable to genetic erosion.

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September 22, 2019  |  

Searching for convergent pathways in autism spectrum disorders: insights from human brain transcriptome studies.

Autism spectrum disorder (ASD) is one of the most heritable neuropsychiatric conditions. The complex genetic landscape of the disorder includes both common and rare variants at hundreds of genetic loci. This marked heterogeneity has thus far hampered efforts to develop genetic diagnostic panels and targeted pharmacological therapies. Here, we give an overview of the current literature on the genetic basis of ASD, and review recent human brain transcriptome studies and their role in identifying convergent pathways downstream of the heterogeneous genetic variants. We also discuss emerging evidence on the involvement of non-coding genomic regions and non-coding RNAs in ASD.

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September 22, 2019  |  

TACO produces robust multisample transcriptome assemblies from RNA-seq.

Accurate transcript structure and abundance inference from RNA sequencing (RNA-seq) data is foundational for molecular discovery. Here we present TACO, a computational method to reconstruct a consensus transcriptome from multiple RNA-seq data sets. TACO employs novel change-point detection to demarcate transcript start and end sites, leading to improved reconstruction accuracy compared with other tools in its class. The tool is available at http://tacorna.github.io and can be readily incorporated into RNA-seq analysis workflows.

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September 22, 2019  |  

The transcriptome of human pluripotent stem cells.

Human Embryonic Stem Cells (hESCs) are in vitro derivatives of the inner cell mass of the blastocyst and are characterized by an undifferentiated and pluripotent state that can be perpetuated in time, indefinitely. hESCs provide a unique opportunity to both dissect the molecular mechanisms that are predisposed to the maintenance of pluripotency and model the ability to initiate differentiation and cell commitment within the developing embryo. To fully understand these mechanisms, it is necessary to accurately identify the specific transcriptome of hESCs. Many distinct gene annotation methods, such as cDNA and EST sequencing and RNA-Seq, have been used to identify the transcriptome of hESCs. Lately, we developed a new tool (IDP) to integrate the hybrid sequencing data to characterize a more reliable and comprehensive hESC transcriptome with discoveries of many novel transcripts. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.

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September 22, 2019  |  

PacBio metabarcoding of Fungi and other eukaryotes: errors, biases and perspectives.

Second-generation, high-throughput sequencing methods have greatly improved our understanding of the ecology of soil microorganisms, yet the short barcodes (< 500 bp) provide limited taxonomic and phylogenetic information for species discrimination and taxonomic assignment. Here, we utilized the third-generation Pacific Biosciences (PacBio) RSII and Sequel instruments to evaluate the suitability of full-length internal transcribed spacer (ITS) barcodes and longer rRNA gene amplicons for metabarcoding Fungi, Oomycetes and other eukaryotes in soil samples. Metabarcoding revealed multiple errors and biases: Taq polymerase substitution errors and mis-incorporating indels in sequencing homopolymers constitute major errors; sequence length biases occur during PCR, library preparation, loading to the sequencing instrument and quality filtering; primer-template mismatches bias the taxonomic profile when using regular and highly degenerate primers. The RSII and Sequel platforms enable the sequencing of amplicons up to 3000 bp, but the sequence quality remains slightly inferior to Illumina sequencing especially in longer amplicons. The full ITS barcode and flanking rRNA small subunit gene greatly improve taxonomic identification at the species and phylum levels, respectively. We conclude that PacBio sequencing provides a viable alternative for metabarcoding of organisms that are of relatively low diversity, require > 500-bp barcode for reliable identification or when phylogenetic approaches are intended.© 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

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September 22, 2019  |  

Transcriptional adaptations during long-term persistence of Staphylococcus aureus in the airways of a cystic fibrosis patient.

The lungs of Cystic fibrosis (CF) patients are often colonized and/or infected by Staphylococcus aureus for years, mostly by one predominant clone. For long-term survival in this environment, S. aureus needs to adapt during its interactions with host factors, antibiotics, and other pathogens. Here, we study long-term transcriptional as well as genomic adaptations of an isogenic pair of S. aureus isolates from a single patient using RNA sequencing (RNA-Seq) and whole genome sequencing (WGS). Mimicking in vivo conditions, we cultivated the S. aureus isolates using artificial sputum medium before harvesting RNA for subsequent analysis. We confirmed our RNA-Seq data using quantitative real-time (qRT)-PCR and additionally investigated intermediate isolates from the same patient representing in total 13.2 years of persistence in the CF airways. Comparative RNA-Seq analysis of the first and the last (“late”) isolate revealed significant differences in the late isolate after 13.2 years of persistence. Of the 2545 genes expressed in both isolates that were cultivated aerobically, 256 genes were up- and 161 were down-regulated with a minimum 2-fold change (2f). Focusing on 25 highly (=8f) up- (n=9) or down- (n=16) regulated genes, we identified several genes encoding for virulence factors involved in immune evasion, bacterial spread or secretion (e.g. spa, sak, and esxA). Moreover, these genes displayed similar expression trends under aerobic, microaerophilic and anaerobic conditions. Further qRT-PCR-experiments of highly up- or down-regulated genes within intermediate S. aureus isolates resulted in different gene expression patterns over the years. Using sequencing analysis of the differently expressed genes and their upstream regions in the late S. aureus isolate resulted in only few genomic alterations. Comparative transcriptomic analysis revealed adaptive changes affecting mainly genes involved in host-pathogen interaction. Although the underlying mechanisms were not known, our results suggest adaptive processes beyond genomic mutations triggered by local factors rather than by activation of global regulators. Copyright © 2014 The Authors. Published by Elsevier GmbH.. All rights reserved.

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September 22, 2019  |  

Novel syntrophic populations dominate an ammonia-tolerant methanogenic microbiome.

Biogas reactors operating with protein-rich substrates have high methane potential and industrial value; however, they are highly susceptible to process failure because of the accumulation of ammonia. High ammonia levels cause a decline in acetate-utilizing methanogens and instead promote the conversion of acetate via a two-step mechanism involving syntrophic acetate oxidation (SAO) to H2 and CO2, followed by hydrogenotrophic methanogenesis. Despite the key role of syntrophic acetate-oxidizing bacteria (SAOB), only a few culturable representatives have been characterized. Here we show that the microbiome of a commercial, ammonia-tolerant biogas reactor harbors a deeply branched, uncultured phylotype (unFirm_1) accounting for approximately 5% of the 16S rRNA gene inventory and sharing 88% 16S rRNA gene identity with its closest characterized relative. Reconstructed genome and quantitative metaproteomic analyses imply unFirm_1’s metabolic dominance and SAO capabilities, whereby the key enzymes required for acetate oxidation are among the most highly detected in the reactor microbiome. While culturable SAOB were identified in genomic analyses of the reactor, their limited proteomic representation suggests that unFirm_1 plays an important role in channeling acetate toward methane. Notably, unFirm_1-like populations were found in other high-ammonia biogas installations, conjecturing a broader importance for this novel clade of SAOB in anaerobic fermentations. IMPORTANCE The microbial production of methane or “biogas” is an attractive renewable energy technology that can recycle organic waste into biofuel. Biogas reactors operating with protein-rich substrates such as household municipal or agricultural wastes have significant industrial and societal value; however, they are highly unstable and frequently collapse due to the accumulation of ammonia. We report the discovery of a novel uncultured phylotype (unFirm_1) that is highly detectable in metaproteomic data generated from an ammonia-tolerant commercial reactor. Importantly, unFirm_1 is proposed to perform a key metabolic step in biogas microbiomes, whereby it syntrophically oxidizes acetate to hydrogen and carbon dioxide, which methanogens then covert to methane. Only very few culturable syntrophic acetate-oxidizing bacteria have been described, and all were detected at low in situ levels compared to unFirm_1. Broader comparisons produced the hypothesis that unFirm_1 is a key mediator toward the successful long-term stable operation of biogas production using protein-rich substrates.

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September 22, 2019  |  

The standard operating procedure of the DOE-JGI Microbial Genome Annotation Pipeline (MGAP v.4).

The DOE-JGI Microbial Genome Annotation Pipeline performs structural and functional annotation of microbial genomes that are further included into the Integrated Microbial Genome comparative analysis system. MGAP is applied to assembled nucleotide sequence datasets that are provided via the IMG submission site. Dataset submission for annotation first requires project and associated metadata description in GOLD. The MGAP sequence data processing consists of feature prediction including identification of protein-coding genes, non-coding RNAs and regulatory RNA features, as well as CRISPR elements. Structural annotation is followed by assignment of protein product names and functions.

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September 22, 2019  |  

Evaluating approaches to find exon chains based on long reads.

Transcript prediction can be modeled as a graph problem where exons are modeled as nodes and reads spanning two or more exons are modeled as exon chains. Pacific Biosciences third-generation sequencing technology produces significantly longer reads than earlier second-generation sequencing technologies, which gives valuable information about longer exon chains in a graph. However, with the high error rates of third-generation sequencing, aligning long reads correctly around the splice sites is a challenging task. Incorrect alignments lead to spurious nodes and arcs in the graph, which in turn lead to incorrect transcript predictions. We survey several approaches to find the exon chains corresponding to long reads in a splicing graph, and experimentally study the performance of these methods using simulated data to allow for sensitivity/precision analysis. Our experiments show that short reads from second-generation sequencing can be used to significantly improve exon chain correctness either by error-correcting the long reads before splicing graph creation, or by using them to create a splicing graph on which the long-read alignments are then projected. We also study the memory and time consumption of various modules, and show that accurate exon chains lead to significantly increased transcript prediction accuracy.The simulated data and in-house scripts used for this article are available at http://www.cs.helsinki.fi/group/gsa/exon-chains/exon-chains-bib.tar.bz2.

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