A FAST AND ACCURATE ALGORITHM FOR DIPLOID INDIVIDUAL HAPLOTYPE RECONSTRUCTION

Haplotypes can provide significant information in many research fields, including molecular biology and medical therapy. However, haplotyping is much more difficult than genotyping by using only biological techniques. With the development of sequencing technologies, it becomes possible to obtain haplotypes by combining sequence fragments. The haplotype reconstruction problem of diploid individual has received considerable attention in recent years. It assembles the two haplotypes for a chromosome given the collection of fragments coming from the two haplotypes. Fragment errors significantly increase the difficulty of the problem, and which has been shown to be NP-hard. In this paper, a fast and accurate algorithm, named FAHR, is proposed for haplotyping a single diploid individual. Algorithm FAHR reconstructs the SNP sites of a pair of haplotypes one after another. The SNP fragments that cover some SNP site are partitioned into two groups according to the alleles of the corresponding SNP site, and the SNP values of the pair of haplotypes are ascertained by using the fragments in the group that contains more SNP fragments. The experimental comparisons were conducted among the FAHR, the Fast Hare and the DGS algorithms by using the haplotypes on chromosome 1 of 60 individuals in CEPH samples, which were released by the International HapMap Project. Experimental results under different parameter settings indicate that the reconstruction rate of the FAHR algorithm is higher than those of the Fast Hare and the DGS algorithms, and the running time of the FAHR algorithm is shorter than those of the Fast Hare and the DGS algorithms. Moreover, the FAHR algorithm has high efficiency even for the reconstruction of long haplotypes and is very practical for realistic applications.

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CRISPR/Cas9-mediated knock-in of alligator cathelicidin gene in a non-coding region of channel catfish genome

Scientific Reports ◽

10.1038/s41598-020-79409-5 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Rhoda Mae C. Simora ◽

De Xing ◽

Max R. Bangs ◽

Wenwen Wang ◽

Xiaoli Ma ◽

...

Keyword(s):

Ictalurus Punctatus ◽

Channel Catfish ◽

High Efficiency ◽

Gene Knockout ◽

Chromosome 1 ◽

Model Organisms ◽

Coding Region ◽

Exogenous Dna ◽

Efficient System ◽

Gene Integration

AbstractCRISPR/Cas9-based gene knockout in animal cells, particularly in teleosts, has proven to be very efficient with regards to mutation rates, but the precise insertion of exogenous DNA or gene knock-in via the homology-directed repair (HDR) pathway has seldom been achieved outside of the model organisms. Here, we succeeded in integrating with high efficiency an exogenous alligator cathelicidin gene into a targeted non-coding region of channel catfish (Ictalurus punctatus) chromosome 1 using two different donor templates (synthesized linear dsDNA and cloned plasmid DNA constructs). We also tested two different promoters for driving the gene, zebrafish ubiquitin promoter and common carp β-actin promoter, harboring a 250-bp homologous region flanking both sides of the genomic target locus. Integration rates were found higher in dead fry than in live fingerlings, indicating either off-target effects or pleiotropic effects. Furthermore, low levels of mosaicism were detected in the tissues of P1 individuals harboring the transgene, and high transgene expression was observed in the blood of some P1 fish. This can be an indication of the localization of cathelicidin in neutrophils and macrophage granules as also observed in most antimicrobial peptides. This study marks the first use of CRISPR/Cas9 HDR for gene integration in channel catfish and may contribute to the generation of a more efficient system for precise gene integration in catfish and other aquaculture species, and the development of gene-edited, disease-resistant fish.

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Recent Advances in RNA Therapy and Its Carriers to Treat the Single-Gene Neurological Disorders

Biomedicines ◽

10.3390/biomedicines10010158 ◽

2022 ◽

Vol 10 (1) ◽

pp. 158

Author(s):

Ming-Jen Lee ◽

Inyoul Lee ◽

Kai Wang

Keyword(s):

Neurological Disorders ◽

High Efficiency ◽

Single Gene ◽

Treatment Strategies ◽

Specific Protein ◽

Rna Molecules ◽

Sequencing Technologies ◽

Short Rnas ◽

Single Gene Disorders ◽

New Treatment

The development of new sequencing technologies in the post-genomic era has accelerated the identification of causative mutations of several single gene disorders. Advances in cell and animal models provide insights into the underlining pathogenesis, which facilitates the development and maturation of new treatment strategies. The progress in biochemistry and molecular biology has established a new class of therapeutics—the short RNAs and expressible long RNAs. The sequences of therapeutic RNAs can be optimized to enhance their stability and translatability with reduced immunogenicity. The chemically-modified RNAs can also increase their stability during intracellular trafficking. In addition, the development of safe and high efficiency carriers that preserves the integrity of therapeutic RNA molecules also accelerates the transition of RNA therapeutics into the clinic. For example, for diseases that are caused by genetic defects in a specific protein, an effective approach termed “protein replacement therapy” can provide treatment through the delivery of modified translatable mRNAs. Short interference RNAs can also be used to treat diseases caused by gain of function mutations or restore the splicing aberration defects. Here we review the applications of newly developed RNA-based therapeutics and its delivery and discuss the clinical evidence supporting the potential of RNA-based therapy in single-gene neurological disorders.

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Generation of knock-in lampreys by CRISPR-Cas9-mediated genome engineering

Scientific Reports ◽

10.1038/s41598-021-99338-1 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Daichi G. Suzuki ◽

Hiroshi Wada ◽

Shin-ichi Higashijima

Keyword(s):

Genome Engineering ◽

High Efficiency ◽

Evolutionary Developmental Biology ◽

Model Organisms ◽

Donor Plasmid ◽

Guide Rna ◽

Research Fields ◽

Design Flexibility ◽

Genetic Techniques ◽

Mrna Targeting

AbstractThe lamprey represents the oldest group of living vertebrates and has been a key organism in various research fields such as evolutionary developmental biology and neuroscience. However, no knock-in technique for this animal has been established yet, preventing application of advanced genetic techniques. Here, we report efficient generation of F0 knock-in lampreys by CRISPR-Cas9-mediated genome editing. A donor plasmid containing a heat-shock promoter was co-injected with a short guide RNA (sgRNA) for genome digestion, a sgRNA for donor plasmid digestion, and Cas9 mRNA. Targeting different genetic loci, we succeeded in generating knock-in lampreys expressing photoconvertible protein Dendra2 as well as those expressing EGFP. With its simplicity, design flexibility, and high efficiency, we propose that the present method has great versatility for various experimental uses in lamprey research and that it can also be applied to other “non-model” organisms.

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Comparative Analysis of PacBio and Oxford Nanopore Sequencing Technologies for Transcriptomic Landscape Identification of Penaeus monodon

Life ◽

10.3390/life11080862 ◽

2021 ◽

Vol 11 (8) ◽

pp. 862

Author(s):

Zulema Udaondo ◽

Kanchana Sittikankaew ◽

Tanaporn Uengwetwanit ◽

Thidathip Wongsurawat ◽

Chutima Sonthirod ◽

...

Keyword(s):

Penaeus Monodon ◽

Influential Factors ◽

Read Length ◽

Sequencing Platform ◽

Sequencing Technologies ◽

Research Fields ◽

Oxford Nanopore ◽

Long Read ◽

Assembly Features ◽

Sequencing Platforms

With the advantages that long-read sequencing platforms such as Pacific Biosciences (Menlo Park, CA, USA) (PacBio) and Oxford Nanopore Technologies (Oxford, UK) (ONT) can offer, various research fields such as genomics and transcriptomics can exploit their benefits. Selecting an appropriate sequencing platform is undoubtedly crucial for the success of the research outcome, thus there is a need to compare these long-read sequencing platforms and evaluate them for specific research questions. This study aims to compare the performance of PacBio and ONT platforms for transcriptomic analysis by utilizing transcriptome data from three different tissues (hepatopancreas, intestine, and gonads) of the juvenile black tiger shrimp, Penaeus monodon. We compared three important features: (i) main characteristics of the sequencing libraries and their alignment with the reference genome, (ii) transcript assembly features and isoform identification, and (iii) correlation of the quantification of gene expression levels for both platforms. Our analyses suggest that read-length bias and differences in sequencing throughput are highly influential factors when using long reads in transcriptome studies. These comparisons can provide a guideline when designing a transcriptome study utilizing these two long-read sequencing technologies.

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Whole-Genome Characteristics and Polymorphic Analysis of Vietnamese Rice Landraces as a Comprehensive Information Resource for Marker-Assisted Selection

International Journal of Genomics ◽

10.1155/2017/9272363 ◽

2017 ◽

Vol 2017 ◽

pp. 1-11 ◽

Cited By ~ 2

Author(s):

Hien Trinh ◽

Khoa Truong Nguyen ◽

Lam Van Nguyen ◽

Huy Quang Pham ◽

Can Thu Huong ◽

...

Keyword(s):

Marker Assisted Selection ◽

Blast Resistance ◽

Nucleotide Polymorphisms ◽

Significant Information ◽

Single Nucleotide ◽

Sequencing Technologies ◽

Multiple Transcripts ◽

Rice Landraces ◽

Whole Plant ◽

Comprehensive Information

Next generation sequencing technologies have provided numerous opportunities for application in the study of whole plant genomes. In this study, we present the sequencing and bioinformatic analyses of five typical rice landraces including threeindicaand twojaponicawith potential blast resistance. A total of 688.4 million 100 bp paired-end reads have yielded approximately 30-fold coverage to compare with the Nipponbare reference genome. Among them, a small number of reads were mapped to both chromosomes and organellar genomes. Over two million and eight hundred thousand single nucleotide polymorphisms (SNPs) and insertions and deletions (InDels) inindicaandjaponicalines have been determined, which potentially have significant impacts on multiple transcripts of genes. SNP deserts, contiguous SNP-low regions, were found on chromosomes 1, 4, and 5 of all genomes of rice examined. Based on the distribution of SNPs per 100 kilobase pairs, the phylogenetic relationships among the landraces have been constructed. This is the first step towards revealing several salient features of rice genomes in Vietnam and providing significant information resources to further marker-assisted selection (MAS) in rice breeding programs.

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Comparative Genomics across Three Ensifer Species Using a New Complete Genome Sequence of the Medicago Symbiont Sinorhizobium (Ensifer) meliloti WSM1022

Microorganisms ◽

10.3390/microorganisms9122428 ◽

2021 ◽

Vol 9 (12) ◽

pp. 2428

Author(s):

Laura Baxter ◽

Proyash Roy ◽

Emma Picot ◽

Jess Watts ◽

Alex Jones ◽

...

Keyword(s):

Genome Sequence ◽

Complete Genome Sequence ◽

Complete Genome ◽

High Efficiency ◽

Gc Content ◽

Protein Coding ◽

Sequencing Technologies ◽

Ensifer Meliloti ◽

Meliloti Strain ◽

Symbiotic Properties

Here, we report an improved and complete genome sequence of Sinorhizobium (Ensifer) meliloti strain WSM1022, a microsymbiont of Medicago species, revealing its tripartite structure. This improved genome sequence was generated combining Illumina and Oxford nanopore sequencing technologies to better understand the symbiotic properties of the bacterium. The 6.75 Mb WSM1022 genome consists of three scaffolds, corresponding to a chromosome (3.70 Mb) and the pSymA (1.38 Mb) and pSymB (1.66 Mb) megaplasmids. The assembly has an average GC content of 62.2% and a mean coverage of 77X. Genome annotation of WSM1022 predicted 6058 protein coding sequences (CDSs), 202 pseudogenes, 9 rRNAs (3 each of 5S, 16S, and 23S), 55 tRNAs, and 4 ncRNAs. We compared the genome of WSM1022 to two other rhizobial strains, closely related Sinorhizobium (Ensifer) meliloti Sm1021 and Sinorhizobium (Ensifer) medicae WSM419. Both WSM1022 and WSM419 species are high-efficiency rhizobial strains when in symbiosis with Medicago truncatula, whereas Sm1021 is ineffective. Our findings report significant genomic differences across the three strains with some similarities between the meliloti strains and some others between the high efficiency strains WSM1022 and WSM419. The addition of this high-quality rhizobial genome sequence in conjunction with comparative analyses will help to unravel the features that make a rhizobial symbiont highly efficient for nitrogen fixation.

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Computational Methods for Chromosome-Scale Haplotype Reconstruction

10.20944/preprints202101.0116.v1 ◽

2021 ◽

Author(s):

Shilpa Garg

Keyword(s):

Genetic Variation ◽

Whole Genome ◽

Haplotype Reconstruction ◽

High Quality ◽

Short Read ◽

Short Read Sequencing ◽

Sequencing Technologies ◽

Evolutionary Studies ◽

Long Read ◽

Haplotype Information

High-quality chromosome-scale haplotype sequences— of diploid genomes, polyploid genomes and metagenomes — provide important insights into genetic variation associated with disease and biodiversity. However, whole-genome short read sequencing does not yield haplotype information that spans whole chromosomes directly. Computational assembly of shorter haplotype fragments is required for haplotype reconstruction, which can be challenging owing to limited fragment lengths and high haplotype and repeat variability across genomes. Recent advancements in long-read and chromosome-scale sequencing technologies, alongside computational innovations, are improving the reconstruction of haplotypes at the level of whole chromosomes. Here, we review recent methodological progress in these areas and discuss perspectives that could enable routine high-quality haplotype reconstruction in clinical and evolutionary studies.

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Computational methods for chromosome-scale haplotype reconstruction

Genome Biology ◽

10.1186/s13059-021-02328-9 ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Shilpa Garg

Keyword(s):

Genetic Variation ◽

Computational Methods ◽

Whole Genome ◽

Haplotype Reconstruction ◽

High Quality ◽

Short Read ◽

Short Read Sequencing ◽

Sequencing Technologies ◽

Long Read ◽

Haplotype Information

AbstractHigh-quality chromosome-scale haplotype sequences of diploid genomes, polyploid genomes, and metagenomes provide important insights into genetic variation associated with disease and biodiversity. However, whole-genome short read sequencing does not yield haplotype information spanning whole chromosomes directly. Computational assembly of shorter haplotype fragments is required for haplotype reconstruction, which can be challenging owing to limited fragment lengths and high haplotype and repeat variability across genomes. Recent advancements in long-read and chromosome-scale sequencing technologies, alongside computational innovations, are improving the reconstruction of haplotypes at the level of whole chromosomes. Here, we review recent and discuss methodological progress and perspectives in these areas.

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High-Throughput Cell Trapping in the Dentate Spiral Microfluidic Channel

Micromachines ◽

10.3390/mi12030288 ◽

2021 ◽

Vol 12 (3) ◽

pp. 288

Author(s):

Jiawei Lu ◽

Bo Dai ◽

Kan Wang ◽

Yan Long ◽

Zhuoqing Yang ◽

...

Keyword(s):

High Throughput ◽

High Efficiency ◽

Microfluidic Channel ◽

Trapping Efficiency ◽

Cell Trapping ◽

Research Fields ◽

Mouse Breast Cancer ◽

A Cell ◽

The Many ◽

Microfluidic Structure

Cell trapping is a very useful technique in a variety of cell-based assays and cellular research fields. It requires a high-throughput, high-efficiency operation to isolate cells of interest and immobilize the captured cells at specific positions. In this study, a dentate spiral microfluidic structure is proposed for cell trapping. The structure consists of a main spiral channel connecting an inlet and an out and a large number of dentate traps on the side of the channel. The density of the traps is high. When a cell comes across an empty trap, the cell suddenly makes a turn and enters the trap. Once the trap captures enough cells, the trap becomes closed and the following cells pass by the trap. The microfluidic structure is optimized based on the investigation of the influence over the flow. In the demonstration, 4T1 mouse breast cancer cells injected into the chip can be efficiently captured and isolated in the different traps. The cell trapping operates at a very high flow rate (40 μL/s) and a high trapping efficiency (>90%) can be achieved. The proposed high-throughput cell-trapping technique can be adopted in the many applications, including rapid microfluidic cell-based assays and isolation of rare circulating tumor cells from a large volume of blood sample.

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Communication Design of Collaborative Virtual Maintenance Training System Based on JMS

Applied Mechanics and Materials ◽

10.4028/www.scientific.net/amm.556-562.4871 ◽

2014 ◽

Vol 556-562 ◽

pp. 4871-4874 ◽

Cited By ~ 1

Author(s):

Chao Zhao ◽

Xing Xin Li ◽

Kong Xia

Keyword(s):

High Efficiency ◽

Data Communication ◽

Training System ◽

Virtual Reality Technology ◽

Training Systems ◽

Whole Process ◽

Research Fields ◽

Message Service ◽

Virtual Maintenance ◽

Collaborative Training

Taken the problem of data communication in collaborative virtual maintenance training system into consideration, the technology of JMS was used to transmission of information, which solved the asynchronous communication between each client in system, and ensured the timeliness, accuracy and high efficiency during the whole process. Forward In recent years, using virtual reality technology for maintenance training is one of the popular research fields, and many virtual maintenance training systems have been developed [1,2]. However, none of these systems could be used to collaborative training for multi-users. One difficulty is that how to share the information between clients. In other words, the way of communication is considerable to enable to share information as reasonably, timely and efficiently as possible and do not affect the training. For this reason, JMS (Java Message Service) technology is presented to solve the communication problem in collaborative training in the article.

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