June 1, 2021  |  

Single Molecule, Real-Time Sequencing for base modification detection in eukaryotic organisms: Coprinopsis cinerea.

Single Molecule Real-Time (SMRT) DNA sequencing provides a wealth of kinetic information beyond the extraction of the primary DNA sequence, and this kinetic information can provide for the direct detection of modified bases present in genomic DNA. This method has been demonstrated for base modification detection in prokaryotes at base and strand resolutions. In eukaryotes, the common base modifications known to exist are the cytosine variants including methyl, hydroxymethyl, formyl and carboxyl forms. Each of these modifications exhibits different signatures in SMRT kinetic data, allowing for unprecedented possibilities to differentiate between them in direct sequencing data. We present early results of directly sequencing different base modifications in eukaryotic genomic DNA using this method.


June 1, 2021  |  

Isoform sequencing: Unveiling the complex landscape of the eukaryotic transcriptome on the PacBio RS II.

Alternative splicing of RNA is an important mechanism that increases protein diversity and is pervasive in the most complex biological functions. While advances in RNA sequencing methods have accelerated our understanding of the transcriptome, isoform discovery remains computationally challenging due to short read lengths. Here, we describe the Isoform Sequencing (Iso-Seq) method using long reads generated by the PacBio RS II. We sequenced rat heart and lung RNA using the Clontech® SMARTer® cDNA preparation kit followed by size selection using agarose gel. Additionally, we tested the BluePippin™ device from Sage Science for efficiently extracting longer transcripts = 3 kb. Post-sequencing, we developed a novel isoform-level clustering algorithm to generate high-quality transcript consensus sequences. We show that our method recovered alternative splice forms as well as alternative stop sites, antisense transcription, and retained introns. To conclude, the Iso-Seq method provides a new opportunity for researchers to study the complex eukaryotic transcriptome even in the absence of reference genomes or annotated transcripts.


June 1, 2021  |  

Integrative biology of a fungus: Using PacBio SMRT Sequencing to interrogate the genome, epigenome, and transcriptome of Neurospora crassa.

PacBio SMRT Sequencing has the unique ability to directly detect base modifications in addition to the nucleotide sequence of DNA. Because eukaryotes use base modifications to regulate gene expression, the absence or presence of epigenetic events relative to the location of genes is critical to elucidate the function of the modification. Therefore an integrated approach that combines multiple omic-scale assays is necessary to study complex organisms. Here, we present an integrated analysis of three sequencing experiments: 1) DNA sequencing, 2) base-modification detection, and 3) Iso-seq analysis, in Neurospora crassa, a filamentous fungus that has been used to make many landmark discoveries in biochemistry and genetics. We show that de novo assembly of a new strain yields complete assemblies of entire chromosomes, and additionally contains entire centromeric sequences. Base-modification analyses reveal candidate sites of increased interpulse duration (IPD) ratio, that may signify regions of 5mC, 5hmC, or 6mA base modifications. Iso-seq method provides full-length transcript evidence for comprehensive gene annotation, as well as context to the base-modifications in the newly assembled genome. Projects that integrate multiple genome-wide assays could become common practice for identifying genomic elements and understanding their function in new strains and organisms.


June 1, 2021  |  

Isoform sequencing: Unveiling the complex landscape in eukaryotic transcriptome on the PacBio RS II.

Advances in RNA sequencing have accelerated our understanding of the transcriptome, however isoform discovery remains challenging due to short read lengths. The Iso-Seq Application provides a new alternative to sequence full-length cDNA libraries using long reads from the PacBio RS II. Identification of long and often rare isoforms is demonstrated with rat heart and lung RNA prepared using the Clontech® SMARTer® cDNA preparation kit, followed by agarose-gel size selection in fractions of 1-2 kb, 2-3 kb and 3-6 kb. For each tissue, 1.8 and 1.2 million reads were obtained from 32 and 26 SMRT Cells, respectively. Filtering for reads with both adapters and polyA tail signals yielded >50% putative full-length transcripts. To improve consensus accuracy, we developed an isoform-level clustering algorithm ICE (Iterative Clustering for Error Correction), and polished full-length consensus sequences from ICE using Quiver. This method generated full-length transcripts up to 4.5 kb with = 99% post-correction accuracy. Compared with known rat genes, the Iso-Seq method not only recovered the majority of currently annotated isoforms, but also several unannotated novel isoforms with identified homologs in the RefSeq database. Additionally, alternative stop sites, extended UTRs, and retained introns were detected.


April 21, 2020  |  

Precise temporal regulation of Dux is important for embryo development.

Zygotic genome activation (ZGA) following fertilization is accomplished through a process termed the maternal-to-zygotic transition, during which the maternal RNAs and proteins are degraded and zygotic genome is transcriptionally activated.1 In mice, minor ZGA occurs from S phase of the zygote to G1 phase of the two-cell (2C) embryo, while major ZGA takes place during the middle-to-late 2C stage with a burst of transcription of totipotent cleavage stage-specific genes and retrotransposons.2Dux has been recently identified and considered as a master inducer that regulates the ZGA process.3–5Dux can directly bind and robustly activate 2C stage-specific ZGA transcripts and convert mouse embryonic stem cells (mESCs) to a 2C-like state with unique features that resembles the 2C embryos.4Intriguingly, ~20% embryos with zygotic depletion of Dux unexpectedly reached morula or blastocyst stage even though defective ZGA program was detected.


April 21, 2020  |  

A comparison of immunoglobulin IGHV, IGHD and IGHJ genes in wild-derived and classical inbred mouse strains.

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.


April 21, 2020  |  

Large Fragment Deletions Induced by Cas9 Cleavage While Not in BEs System in Rabbit

CRISPR-Cas9 and BEs system are poised to become the gene editing tool of choice in clinical contexts, however large fragment deletion was found in Cas9-mediated mutation cells without animal level validation. By analyzing 16 gene-edited rabbit lines (including 112 rabbits) generated using SpCas9, BEs, xCas9 and xCas9-BEs with long-range PCR genotyping and long-read sequencing by PacBio platform, we show that extending thousands of bases fragment deletions in single-guide RNA/Cas9 and xCas9 system mutation rabbit, but few large deletions were found in BEs-induced mutation rabbits. We firstly validated that no large fragment deletion induced by BEs system at animal level, suggesting that BE systems can be beneficial tools for the further development of highly accurate and secure gene therapy for the clinical treatment of human genetic disorders


April 21, 2020  |  

Disruption of the kringle 1 domain of prothrombin leads to late onset mortality in zebrafish

The ability to prevent blood loss in response to injury is a critical, evolutionarily conserved function of all vertebrates. Prothrombin (F2) contributes to both primary and secondary hemostasis through the activation of platelets and the conversion of soluble fibrinogen to insoluble fibrin, respectively. Complete prothrombin deficiency has never been observed in humans and is incompatible with life in mice, limiting the ability to understand the entirety of prothrombin’s in vivo functions. We have previously demonstrated the ability of zebrafish to tolerate loss of both pro- and anticoagulant factors that are embryonic lethal in mammals, making them an ideal model for the study of prothrombin deficiency. Using genome editing with TALENs, we have generated a null allele in zebrafish f2. Homozygous mutant embryos develop normally into early adulthood, but demonstrate eventual complete mortality with the majority of fish succumbing to internal hemorrhage by 2 months of age. We show that despite the extended survival, the mutants are unable to form occlusive thrombi in both the venous and arterial systems as early as 3-5 days of life, and we were able to phenocopy this early hemostatic defect using direct oral anticoagulants. When the equivalent mutation was engineered into the homologous residues of human prothrombin, there were severe reductions in secretion and activation, suggesting a possible role for kringle 1 in thrombin maturation, and the possibility that the F1.2 fragment has a functional role in exerting the procoagulant effects of thrombin. Together, our data demonstrate the conserved function of thrombin in zebrafish, as well as the requirement for kringle 1 for biosynthesis and activation by prothrombinase. Understanding how zebrafish are able to develop normally and survive into early adulthood without prothrombin will provide important insight into its pleiotropic functions as well as the management of patients with bleeding disorders.


April 21, 2020  |  

A critical comparison of technologies for a plant genome sequencing project.

A high-quality genome sequence of any model organism is an essential starting point for genetic and other studies. Older clone-based methods are slow and expensive, whereas faster, cheaper short-read-only assemblies can be incomplete and highly fragmented, which minimizes their usefulness. The last few years have seen the introduction of many new technologies for genome assembly. These new technologies and associated new algorithms are typically benchmarked on microbial genomes or, if they scale appropriately, on larger (e.g., human) genomes. However, plant genomes can be much more repetitive and larger than the human genome, and plant biochemistry often makes obtaining high-quality DNA that is free from contaminants difficult. Reflecting their challenging nature, we observe that plant genome assembly statistics are typically poorer than for vertebrates.Here, we compare Illumina short read, Pacific Biosciences long read, 10x Genomics linked reads, Dovetail Hi-C, and BioNano Genomics optical maps, singly and combined, in producing high-quality long-range genome assemblies of the potato species Solanum verrucosum. We benchmark the assemblies for completeness and accuracy, as well as DNA compute requirements and sequencing costs.The field of genome sequencing and assembly is reaching maturity, and the differences we observe between assemblies are surprisingly small. We expect that our results will be helpful to other genome projects, and that these datasets will be used in benchmarking by assembly algorithm developers. © The Author(s) 2019. Published by Oxford University Press.


April 21, 2020  |  

Full-Length Transcriptome Sequencing and the Discovery of New Transcripts in the Unfertilized Eggs of Zebrafish (Danio rerio).

Understanding early gene expression in zebrafish embryos is a prerequisite for developmental biology research. In this study, 1,629,447 polymerase reads were obtained from the unfertilized eggs of zebrafish via full-length transcriptome sequencing using the PacBio RS II platform first. Then, 102,920 unique isoforms were obtained by correction, clustering and comparison with the zebrafish genome. 12,782 genes in the genome were captured, accounting for 39.71% of the all annotated genes. Approximately 62.27% of the 12,782 genes have been alternatively spliced. GO and KEGG annotations revealed that the unfertilized eggs primarily stored genes that participate in RNA processing and nuclear protein complex composition. According to this PacBio data that aligned with the genome, 3,970 fusion genes, 819 ncRNAs, and 84 new transcripts were predicted. Illumina RNA-seq and RT-qPCR detection found that the expression of two new transcripts, PB.5289.1 and PB.10209.1, were significantly up-regulated at the 2-cell stage and down-regulated rapidly thereafter, suggesting their involvement in minor ZGA during early embryonic development. This study indicated that the unfertilized eggs of zebrafish may have retained genes directly related to cell division and development to initiate the subsequent development in a limited space and time. On the other hand, NTRs or new transcriptome regions in the genome were discovered, which provided new clues regarding ZGA of MZT during early embryonic development in fish.Copyright © 2019 Mehjabin et al.


April 21, 2020  |  

Using Cre-recombinase-driven Polylox barcoding for in vivo fate mapping in mice.

Fate mapping is a powerful genetic tool for linking stem or progenitor cells with their progeny, and hence for defining cell lineages in vivo. The resolution of fate mapping depends on the numbers of distinct markers that are introduced in the beginning into stem or progenitor cells; ideally, numbers should be sufficiently large to allow the tracing of output from individual cells. Highly diverse genetic barcodes can serve this purpose. We recently developed an endogenous genetic barcoding system, termed Polylox. In Polylox, random DNA recombination can be induced by transient activity of Cre recombinase in a 2.1-kb-long artificial recombination substrate that has been introduced into a defined locus in mice (Rosa26Polylox reporter mice). Here, we provide a step-by-step protocol for the use of Polylox, including barcode induction and estimation of induction efficiency, barcode retrieval with single-molecule real-time (SMRT) DNA sequencing followed by computational barcode identification, and the calculation of barcode-generation probabilities, which is key for estimations of single-cell labeling for a given number of stem cells. Thus, Polylox barcoding enables high-resolution fate mapping in essentially all tissues in mice for which inducible Cre driver lines are available. Alternative methods include ex vivo cell barcoding, inducible transposon insertion and CRISPR-Cas9-based barcoding; Polylox currently allows combining non-invasive and cell-type-specific labeling with high label diversity. The execution time of this protocol is ~2-3 weeks for experimental data generation and typically <2 d for computational Polylox decoding and downstream analysis.


April 21, 2020  |  

Development and Genome Sequencing of a Laboratory-Inbred Miniature Pig Facilitates Study of Human Diabetic Disease.

Pig has been proved to be a valuable large animal model used for research on diabetic disease. However, their translational value is limited given their distinct anatomy and physiology. For the last 30 years, we have been developing a laboratory Asian miniature pig inbred line (Bama miniature pig [BM]) from the primitive Bama xiang pig via long-term selective inbreeding. Here, we assembled a BM reference genome at full chromosome-scale resolution with a total length of 2.49 Gb. Comparative and evolutionary genomic analyses identified numerous variations between the BM and commercial pig (Duroc), particularly those in the genetic loci associated with the features advantageous to diabetes studies. Resequencing analyses revealed many differentiated gene loci associated with inbreeding and other selective forces. These together with transcriptome analyses of diabetic pig models provide a comprehensive genetic basis for resistance to diabetogenic environment, especially related to energy metabolism.Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.


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