Combining single-molecule sequencing and Illumina RNA sequencing to elucidate flowering induction of pineapple (Ananas comosus (L.) Merr.) treated with exogenous ethylene

Heretofore, little is known about the mechanism underlying the genotype-dependence of embryonic callus (EC) induction, which has severely inhibited the development of maize genetic engineering. Here, we report the genome sequence and annotation of a maize inbred line with high EC induction ratio, A188, which is assembled from single-molecule sequencing and optical genome mapping. We assembled a 2,210 Mb genome with a scaffold N50 size of 11.61 million bases (Mb), compared to those of 9.73 Mb for B73 and 10.2 Mb for Mo17. Comparative analysis revealed that ~30% of the predicted A188 genes had large structural variations to B73, Mo17 and W22 genomes, which caused considerable protein divergence and might lead to phenotypic variations between the four inbred lines. Combining our new A188 genome, previously reported QTLs and RNA sequencing data, we reveal 8 large structural variation genes and 4 differentially expressed genes playing potential roles in EC induction.

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Single molecule real time (SMRT) full length RNA-sequencing reveals novel and distinct mRNA isoforms in human bone marrow cell subpopulations

10.1101/555326 ◽

2019 ◽

Cited By ~ 1

Author(s):

Anne Deslattes Mays ◽

Marcel O. Schmidt ◽

Garrett T. Graham ◽

Elizabeth Tseng ◽

Primo Baybayan ◽

...

Keyword(s):

Bone Marrow ◽

Real Time ◽

Rna Sequencing ◽

Single Molecule ◽

Marrow Cell ◽

Full Length ◽

Protein Isoforms ◽

Mrna Isoforms ◽

Transcript Isoforms ◽

Cell Subpopulations

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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Nanopore direct RNA sequencing maps the complexity of Arabidopsis mRNA processing and m6A modification

eLife ◽

10.7554/elife.49658 ◽

2020 ◽

Vol 9 ◽

Cited By ~ 43

Author(s):

Matthew T Parker ◽

Katarzyna Knop ◽

Anna V Sherwood ◽

Nicholas J Schurch ◽

Katarzyna Mackinnon ◽

...

Keyword(s):

Rna Sequencing ◽

Single Molecule ◽

Tail Length ◽

Transcript Abundance ◽

Mrna Processing ◽

Full Length ◽

Transcription Start Sites ◽

Model Plant Arabidopsis Thaliana ◽

Defective Rna ◽

A Site

Understanding genome organization and gene regulation requires insight into RNA transcription, processing and modification. We adapted nanopore direct RNA sequencing to examine RNA from a wild-type accession of the model plant Arabidopsis thaliana and a mutant defective in mRNA methylation (m6A). Here we show that m6A can be mapped in full-length mRNAs transcriptome-wide and reveal the combinatorial diversity of cap-associated transcription start sites, splicing events, poly(A) site choice and poly(A) tail length. Loss of m6A from 3’ untranslated regions is associated with decreased relative transcript abundance and defective RNA 3′ end formation. A functional consequence of disrupted m6A is a lengthening of the circadian period. We conclude that nanopore direct RNA sequencing can reveal the complexity of mRNA processing and modification in full-length single molecule reads. These findings can refine Arabidopsis genome annotation. Further, applying this approach to less well-studied species could transform our understanding of what their genomes encode.

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2. How to read the book of life

Genomics: A Very Short Introduction ◽

10.1093/actrade/9780198786207.003.0002 ◽

2018 ◽

pp. 12-27

Author(s):

John Archibald

Keyword(s):

Dna Sequencing ◽

Human Genome ◽

Single Molecule ◽

Human Genome Project ◽

Genome Project ◽

Chain Termination ◽

Single Molecule Sequencing ◽

Semiconductor Sequencing ◽

The Cost ◽

The Human Genome Project

For all its biological importance, DNA is a fragile molecule so extracting it is a difficult process. ‘How to read the book of life’ explains the techniques required to sequence DNA. It begins by explaining the techniques developed for protein and RNA sequencing by Frederick Sanger, Robert Holley, and Carl Woese that were then developed further for DNA sequencing. Following the success of the Human Genome Project, the next generation of DNA sequencing was developed in the mid-2000s. Pyrosequencing was capable of generating orders of magnitude more data at a fraction of the cost, but was superceded within a decade by semiconductor sequencing, reversible chain-termination sequencing, and single-molecule sequencing.

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Massively parallel single molecule sequencing

The Dictionary of Genomics, Transcriptomics and Proteomics ◽

10.1002/9783527678679.dg07176 ◽

2015 ◽

pp. 1-1

Keyword(s):

Single Molecule ◽

Massively Parallel ◽

Single Molecule Sequencing

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A spatially restricted fibrotic niche in pulmonary fibrosis is sustained by M-CSF/M-CSFR signalling in monocyte-derived alveolar macrophages

European Respiratory Journal ◽

10.1183/13993003.00646-2019 ◽

2019 ◽

Vol 55 (1) ◽

pp. 1900646 ◽

Cited By ~ 30

Author(s):

Nikita Joshi ◽

Satoshi Watanabe ◽

Rohan Verma ◽

Renea P. Jablonski ◽

Ching-I Chen ◽

...

Keyword(s):

Pulmonary Fibrosis ◽

Single Cell ◽

Rna Sequencing ◽

Single Molecule ◽

Alveolar Macrophages ◽

Lineage Tracing ◽

Single Cell Rna Sequencing ◽

Pharmacological Blockade ◽

Macrophage Colony ◽

Druggable Targets

Ontologically distinct populations of macrophages differentially contribute to organ fibrosis through unknown mechanisms.We applied lineage tracing, single-cell RNA sequencing and single-molecule fluorescence in situ hybridisation to a spatially restricted model of asbestos-induced pulmonary fibrosis.We demonstrate that tissue-resident alveolar macrophages, tissue-resident peribronchial and perivascular interstitial macrophages, and monocyte-derived alveolar macrophages are present in the fibrotic niche. Deletion of monocyte-derived alveolar macrophages but not tissue-resident alveolar macrophages ameliorated asbestos-induced lung fibrosis. Monocyte-derived alveolar macrophages were specifically localised to fibrotic regions in the proximity of fibroblasts where they expressed molecules known to drive fibroblast proliferation, including platelet-derived growth factor subunit A. Using single-cell RNA sequencing and spatial transcriptomics in both humans and mice, we identified macrophage colony-stimulating factor receptor (M-CSFR) signalling as one of the novel druggable targets controlling self-maintenance and persistence of these pathogenic monocyte-derived alveolar macrophages. Pharmacological blockade of M-CSFR signalling led to the disappearance of monocyte-derived alveolar macrophages and ameliorated fibrosis.Our findings suggest that inhibition of M-CSFR signalling during fibrosis disrupts an essential fibrotic niche that includes monocyte-derived alveolar macrophages and fibroblasts during asbestos-induced fibrosis.

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