Construction of full-length Japanese reference panel of class I HLA genes with single-molecule, real-time sequencing

AbstractHematopoietic cells are continuously replenished from progenitor cells that reside in the bone marrow. To evaluate molecular changes during this process, we analyzed the transcriptomes of freshly harvested human bone marrow progenitor (lineage-negative) and differentiated (lineage-positive) cells by single molecule, real time (SMRT) full length RNA sequencing. This analysis revealed a ∼5-fold higher number of transcript isoforms than previously detected and showed a distinct composition of individual transcript isoforms characteristic for bone marrow subpopulations. A detailed analysis of mRNA isoforms transcribed from the ANXA1 and EEF1A1 loci confirmed their distinct composition. The expression of proteins predicted from the transcriptome analysis was validated by mass spectrometry and validated previously unknown protein isoforms predicted e.g. for EEF1A1. These protein isoforms distinguished the lineage negative cell population from the lineage positive cell population. Finally, transcript isoforms expressed from paralogous gene loci (e.g. CFD, GATA2, HLA-A, B & C) also distinguished cell subpopulations but were only detectable by full length RNA sequencing. Thus, qualitatively distinct transcript isoforms from individual genomic loci separate bone marrow cell subpopulations indicating complex transcriptional regulation and protein isoform generation during hematopoiesis.

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Full-length transcriptome sequencing of Heliocidaris crassispina using PacBio single-molecule real-time

Fish & Shellfish Immunology ◽

10.1016/j.fsi.2021.12.014 ◽

2021 ◽

Author(s):

Yongyu Huang ◽

Lili Zhang ◽

Shiyu Huang ◽

Guodong Wang

Keyword(s):

Real Time ◽

Single Molecule ◽

Transcriptome Sequencing ◽

Full Length

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The complexity of alternative splicing and landscape of tissue-specific expression in lotus (Nelumbo nucifera) unveiled by Illumina- and single-molecule real-time-based RNA-sequencing

DNA Research ◽

10.1093/dnares/dsz010 ◽

2019 ◽

Vol 26 (4) ◽

pp. 301-311 ◽

Cited By ~ 11

Author(s):

Yue Zhang ◽

Tonny Maraga Nyong'A ◽

Tao Shi ◽

Pingfang Yang

Keyword(s):

Alternative Splicing ◽

Real Time ◽

Single Molecule ◽

Intron Retention ◽

Critical Role ◽

Full Length ◽

Specific Expression ◽

Splice Isoforms ◽

Tissue Specific ◽

Genomic Studies

Abstract Alternative splicing (AS) plays a critical role in regulating different physiological and developmental processes in eukaryotes, by dramatically increasing the diversity of the transcriptome and the proteome. However, the saturation and complexity of AS remain unclear in lotus due to its limitation of rare obtainment of full-length multiple-splice isoforms. In this study, we apply a hybrid assembly strategy by combining single-molecule real-time sequencing and Illumina RNA-seq to get a comprehensive insight into the lotus transcriptomic landscape. We identified 211,802 high-quality full-length non-chimeric reads, with 192,690 non-redundant isoforms, and updated the lotus reference gene model. Moreover, our analysis identified a total of 104,288 AS events from 16,543 genes, with alternative 3ʹ splice-site being the predominant model, following by intron retention. By exploring tissue datasets, 370 tissue-specific AS events were identified among 12 tissues. Both the tissue-specific genes and isoforms might play important roles in tissue or organ development, and are suitable for ‘ABCE’ model partly in floral tissues. A large number of AS events and isoform variants identified in our study enhance the understanding of transcriptional diversity in lotus, and provide valuable resource for further functional genomic studies.

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Single-molecule real-time sequencing of the full-length transcriptome of loquat under low-temperature stress

PLoS ONE ◽

10.1371/journal.pone.0238942 ◽

2020 ◽

Vol 15 (9) ◽

pp. e0238942

Author(s):

Cuiping Pan ◽

Yongqing Wang ◽

Lian Tao ◽

Hui Zhang ◽

Qunxian Deng ◽

...

Keyword(s):

Low Temperature ◽

Real Time ◽

Temperature Stress ◽

Single Molecule ◽

Low Temperature Stress ◽

Full Length

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Single molecule studies reveal that p53 tetramers dynamically bind response elements containing one or two half sites

Scientific Reports ◽

10.1038/s41598-020-73234-6 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Elina Ly ◽

Jennifer F. Kugel ◽

James A. Goodrich

Keyword(s):

Real Time ◽

Single Molecule ◽

Target Genes ◽

Population Distribution ◽

Kinetic Stability ◽

Full Length ◽

Dna Complexes ◽

Response Elements ◽

Cell Fate Decisions ◽

Half Site

Abstract The tumor suppressor protein p53 is critical for cell fate decisions, including apoptosis, senescence, and cell cycle arrest. p53 is a tetrameric transcription factor that binds DNA response elements to regulate transcription of target genes. p53 response elements consist of two decameric half-sites, and data suggest one p53 dimer in the tetramer binds to each half-site. Despite a broad literature describing p53 binding DNA, unanswered questions remain, due partly to the need for more quantitative and structural studies with full length protein. Here we describe a single molecule fluorescence system to visualize full length p53 tetramers binding DNA in real time. The data revealed a dynamic interaction in which tetrameric p53/DNA complexes assembled and disassembled without a dimer/DNA intermediate. On a wild type DNA containing two half sites, p53/DNA complexes existed in two kinetically distinct populations. p53 tetramers bound response elements containing only one half site to form a single population of complexes with reduced kinetic stability. Altering the spacing and helical phasing between two half sites affected both the population distribution of p53/DNA complexes and their kinetic stability. Our real time single molecule measurements of full length p53 tetramers binding DNA reveal the parameters that define the stability of p53/DNA complexes, and provide insight into the pathways by which those complexes assemble.

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Single-molecule real-time sequencing facilitates the analysis of transcripts and splice isoforms of anthers in Chinese cabbage (Brassica rapa L. ssp. pekinensis)

BMC Plant Biology ◽

10.1186/s12870-019-2133-z ◽

2019 ◽

Vol 19 (1) ◽

Cited By ~ 2

Author(s):

Chong Tan ◽

Hongxin Liu ◽

Jie Ren ◽

Xueling Ye ◽

Hui Feng ◽

...

Keyword(s):

Real Time ◽

Single Molecule ◽

Chinese Cabbage ◽

Anther Development ◽

Full Length ◽

Transcriptional Level ◽

Systematic Analysis ◽

Splice Isoforms ◽

A Genome ◽

Novel Isoforms

Abstract Background Anther development has been extensively studied at the transcriptional level, but a systematic analysis of full-length transcripts on a genome-wide scale has not yet been published. Here, the Pacific Biosciences (PacBio) Sequel platform and next-generation sequencing (NGS) technology were combined to generate full-length sequences and completed structures of transcripts in anthers of Chinese cabbage. Results Using single-molecule real-time sequencing (SMRT), a total of 1,098,119 circular consensus sequences (CCSs) were generated with a mean length of 2664 bp. More than 75% of the CCSs were considered full-length non-chimeric (FLNC) reads. After error correction, 725,731 high-quality FLNC reads were estimated to carry 51,501 isoforms from 19,503 loci, consisting of 38,992 novel isoforms from known genes and 3691 novel isoforms from novel genes. Of the novel isoforms, we identified 407 long non-coding RNAs (lncRNAs) and 37,549 open reading frames (ORFs). Furthermore, a total of 453,270 alternative splicing (AS) events were identified and the majority of AS models in anther were determined to be approximate exon skipping (XSKIP) events. Of the key genes regulated during anther development, AS events were mainly identified in the genes SERK1, CALS5, NEF1, and CESA1/3. Additionally, we identified 104 fusion transcripts and 5806 genes that had alternative polyadenylation (APA). Conclusions Our work demonstrated the transcriptome diversity and complexity of anther development in Chinese cabbage. The findings provide a basis for further genome annotation and transcriptome research in Chinese cabbage.

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Single molecule studies reveal that p53 tetramers dynamically bind response elements containing one or two half sites

10.1101/2019.12.19.883140 ◽

2019 ◽

Author(s):

Elina Ly ◽

Jennifer F. Kugel ◽

James A. Goodrich

Keyword(s):

Real Time ◽

Cell Fate ◽

Single Molecule ◽

Target Genes ◽

Full Length ◽

Forward Rate ◽

Kinetic Measurements ◽

Response Elements ◽

Cell Fate Decisions ◽

Half Site

AbstractThe tumor suppressor protein p53 is at the nexus of cell fate decisions, including apoptosis, senescence, and cell cycle arrest. p53 is a tetrameric transcription factor that binds to DNA response elements to regulate transcription of its target genes, a process activated by cellular stress. p53 response elements consist of two decameric half-sites, and most data suggest one p53 dimer in the tetramer binds to each half-site. Despite a broad literature describing p53 binding to DNA, unanswered questions remain, due in part to the need for more quantitative and structural studies with the full length protein. Here we describe a single molecule fluorescence system to visualize full length p53 tetramers binding to DNA in real time. The data reveal a dynamic interaction with many p53 binding and dissociation events occurring on single DNA molecules over minutes. We found that p53 tetramers bound to response elements containing only a single half site. The kinetic stability of tetramer/DNA complexes depended on the number of half sites and the helical phasing between them, with the most stable complexes forming on DNA containing two adjacent half sites. The forward rate of binding was not strongly impacted when one half site was mutated. These studies provide real time kinetic measurements of full length p53 tetramers binding to single molecules of DNA, and reveal new insight into the mechanisms by which this nucleoprotein complex forms.

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