Foundational drugs for HFrEF: the growing evidence for a rapid sequencing strategy

AbstractLarge-scale sequencing of RNAs from individual cells can reveal patterns of gene, isoform and allelic expression across cell types and states1. However, current single-cell RNA-sequencing (scRNA-seq) methods have limited ability to count RNAs at allele- and isoform resolution, and long-read sequencing techniques lack the depth required for large-scale applications across cells2,3. Here, we introduce Smart-seq3 that combines full-length transcriptome coverage with a 5’ unique molecular identifier (UMI) RNA counting strategy that enabled in silico reconstruction of thousands of RNA molecules per cell. Importantly, a large portion of counted and reconstructed RNA molecules could be directly assigned to specific isoforms and allelic origin, and we identified significant transcript isoform regulation in mouse strains and human cell types. Moreover, Smart-seq3 showed a dramatic increase in sensitivity and typically detected thousands more genes per cell than Smart-seq2. Altogether, we developed a short-read sequencing strategy for single-cell RNA counting at isoform and allele-resolution applicable to large-scale characterization of cell types and states across tissues and organisms.

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Novel Insights Into Genetic Responses for Waterlogging Stress in Two Local Wheat Cultivars in Yangtze River Basin

Frontiers in Genetics ◽

10.3389/fgene.2021.681680 ◽

2021 ◽

Vol 12 ◽

Author(s):

Mingmei Wei ◽

Xiu Li ◽

Rui Yang ◽

Liulong Li ◽

Zhuangzhi Wang ◽

...

Keyword(s):

Genome Annotation ◽

Yangtze River Basin ◽

Genetic Regulation ◽

Full Length ◽

Control Group ◽

Wheat Cultivars ◽

Waterlogging Stress ◽

Genes Encoding ◽

Sequencing Strategy ◽

Waterlogging Treatment

Wheat (Triticum aestivum L.), the most widely cultivated crop, is affected by waterlogging that limited the wheat production. Given the incompleteness of its genome annotation, PacBio SMRT plus Illumina short-read sequencing strategy provided an efficient approach to investigate the genetic regulation of waterlogging stress in wheat. A total of 947,505 full-length non-chimetric (FLNC) sequences were obtained with two wheat cultivars (XM55 and YM158) with PacBio sequencing. Of these, 5,309 long-non-coding RNAs, 1,574 fusion genes and 739 transcription factors were identified with the FLNC sequences. These full-length transcripts were derived from 49,368 genes, including 47.28% of the genes annotated in IWGSC RefSeq v1.0 and 40.86% genes encoded two or more isoforms, while 27.31% genes in the genome annotation of IWGSC RefSeq v1.0 were multiple-exon genes encoding two or more isoforms. Meanwhile, the individuals with waterlogging treatments (WL) and control group (CK) were selected for Illumina sequencing. Totally, 6,829 differentially expressed genes (DEGs) were detected from four pairwise comparisons. Notably, 942 DEGs were overlapped in the two comparisons (i.e., XM55-WL vs. YM158-WL and YM158-WL vs. YM158-CK). Undoubtedly, the genes involved in photosynthesis were downregulated after waterlogging treatment in two cultivars. Notably, the genes related to steroid biosynthesis, steroid hormone biosynthesis, and downstream plant hormone signal transduction were significantly upregulated after the waterlogging treatment, and the YM158 variety revealed different genetic regulation patterns compared with XM55. The findings provided valuable insights into unveiling regulation mechanisms of waterlogging stress in wheat at anthesis and contributed to molecular selective breeding of new wheat cultivars in future.

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Holistic and Affordable Analyses of MicroRNA Expression Profiles Using Tagged cDNA Libraries and a Multiplex Sequencing Strategy

Methods in Molecular Biology - Functional Genomics ◽

10.1007/978-1-4939-7231-9_12 ◽

2017 ◽

pp. 179-196 ◽

Cited By ~ 1

Author(s):

Patrick P. Weil ◽

Yan Jaszczyszyn ◽

Anne Baroin-Tourancheau ◽

Jan Postberg ◽

Laurence Amar

Keyword(s):

Expression Profiles ◽

Microrna Expression ◽

Cdna Libraries ◽

Sequencing Strategy ◽

Multiplex Sequencing

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Mapping of transgenic alleles in soybean using a nanopore-based sequencing strategy

Journal of Experimental Botany ◽

10.1093/jxb/erz202 ◽

2019 ◽

Vol 70 (15) ◽

pp. 3825-3833 ◽

Cited By ~ 2

Author(s):

Shengjun Li ◽

Shangang Jia ◽

Lili Hou ◽

Hanh Nguyen ◽

Shirley Sato ◽

...

Keyword(s):

High Throughput ◽

Soybean Genome ◽

Transgenic Technology ◽

Transgenic Soybean ◽

Pooled Dna ◽

Sequencing Strategy ◽

Ds Element ◽

Soybean Plants ◽

Cost Efficient ◽

Transgene Insertion

Abstract Transgenic technology was developed to introduce transgenes into various organisms to validate gene function and add genetic variations >40 years ago. However, the identification of the transgene insertion position is still challenging in organisms with complex genomes. Here, we report a nanopore-based method to map the insertion position of a Ds transposable element originating in maize in the soybean genome. In this method, an oligo probe is used to capture the DNA fragments containing the Ds element from pooled DNA samples of transgenic soybean plants. The Ds element-enriched DNAs are then sequenced using the MinION-based platform of Nanopore. This method allowed us to rapidly map the Ds insertion positions in 51 transgenic soybean lines through a single sequencing run. This strategy is high throughput, convenient, reliable, and cost-efficient. The transgenic allele mapping protocol can be easily translated to other eukaryotes with complex genomes.

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Genome Sequences of Lactococcus lactis MG1363 (Revised) and NZ9000 and Comparative Physiological Studies

Journal of Bacteriology ◽

10.1128/jb.00533-10 ◽

2010 ◽

Vol 192 (21) ◽

pp. 5806-5812 ◽

Cited By ~ 75

Author(s):

Daniel M. Linares ◽

Jan Kok ◽

Bert Poolman

Keyword(s):

Lactic Acid ◽

Lactic Acid Bacteria ◽

Glucose Uptake ◽

Lactococcus Lactis ◽

Point Mutations ◽

Genome Sequences ◽

Sugar Fermentation ◽

Sequencing Strategy ◽

Physiological Studies ◽

Transcriptomic Studies

ABSTRACT Lactococcus lactis NZ9000 and its parent MG1363 are the most commonly used lactic acid bacteria for expression and physiological studies. We noted unexpected but significant differences in the growth behaviors of both strains. We sequenced the entire genomes of the original NZ9000 and MG1363 strains using an ultradeep sequencing strategy. The analysis of the L. lactis NZ9000 genome yielded 79 differences, mostly point mutations, with the annotated genome sequence of L. lactis MG1363. Resequencing of the MG1363 strain revealed that 73 out of the 79 differences were due to errors in the published sequence. Comparative transcriptomic studies revealed several differences in the regulation of genes involved in sugar fermentation, which can be explained by two specific mutations in a region of the ptcC promoter with a key role in the regulation of cellobiose and glucose uptake.

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