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Tuesday, April 21, 2020

The Impact of cDNA Normalization on Long-Read Sequencing of a Complex Transcriptome

Normalization of cDNA is widely used to improve the coverage of rare transcripts in analysis of transcriptomes employing next-generation sequencing. Recently, long-read technology has been emerging as a powerful tool for sequencing and construction of transcriptomes, especially for complex genomes containing highly similar transcripts and transcript-spliced isoforms. Here, we analyzed the transcriptome of sugarcane, with a highly polyploidy plant genome, by PacBio isoform sequencing (Iso-Seq) of two different cDNA library preparations, with and without a normalization step. The results demonstrated that, while the two libraries included many of the same transcripts, many longer transcripts were removed and many new generally…

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Tuesday, April 21, 2020

Long-Read Sequencing Emerging in Medical Genetics

The wide implementation of next-generation sequencing (NGS) technologies has revolutionized the field of medical genetics. However, the short read lengths of currently used sequencing approaches pose a limitation for identification of structural variants, sequencing repetitive regions, phasing alleles and distinguishing highly homologous genomic regions. These limitations may significantly contribute to the diagnostic gap in patients with genetic disorders who have undergone standard NGS, like whole exome or even genome sequencing. Now, the emerging long-read sequencing (LRS) technologies may offer improvements in the characterization of genetic variation and regions that are difficult to assess with the currently prevailing NGS approaches. LRS…

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Tuesday, April 21, 2020

Reconstruction of the full-length transcriptome atlas using PacBio Iso-Seq provides insight into the alternative splicing in Gossypium australe.

Gossypium australe F. Mueller (2n?=?2x?=?26, G2 genome) possesses valuable characteristics. For example, the delayed gland morphogenesis trait causes cottonseed protein and oil to be edible while retaining resistance to biotic stress. However, the lack of gene sequences and their alternative splicing (AS) in G. australe remain unclear, hindering to explore species-specific biological morphogenesis.Here, we report the first sequencing of the full-length transcriptome of the Australian wild cotton species, G. australe, using Pacific Biosciences single-molecule long-read isoform sequencing (Iso-Seq) from the pooled cDNA of ten tissues to identify transcript loci and splice isoforms. We reconstructed the G. australe full-length transcriptome and…

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Tuesday, April 21, 2020

Analysis of Transcriptome and Epitranscriptome in Plants Using PacBio Iso-Seq and Nanopore-Based Direct RNA Sequencing.

Nanopore sequencing from Oxford Nanopore Technologies (ONT) and Pacific BioSciences (PacBio) single-molecule real-time (SMRT) long-read isoform sequencing (Iso-Seq) are revolutionizing the way transcriptomes are analyzed. These methods offer many advantages over most widely used high-throughput short-read RNA sequencing (RNA-Seq) approaches and allow a comprehensive analysis of transcriptomes in identifying full-length splice isoforms and several other post-transcriptional events. In addition, direct RNA-Seq provides valuable information about RNA modifications, which are lost during the PCR amplification step in other methods. Here, we present a comprehensive summary of important applications of these technologies in plants, including identification of complex alternative splicing (AS), full-length…

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Tuesday, April 21, 2020

Transcriptomic profiles of 33 opium poppy samples in different tissues, growth phases, and cultivars.

Opium poppy is one of the most important medicinal plants and remains the only commercial resource of morphinan-based painkillers. However, little is known about the regulatory mechanisms involved in benzylisoquinoline alkaloids (BIAs) biosynthesis in opium poppy. Herein, the full-length transcriptome dataset of opium poppy was constructed for the first time in accompanied with the 33 samples of Illumina transcriptome data from different tissues, growth phases and cultivars. The long-read sequencing produced 902,140 raw reads with 55,114 high-quality transcripts, and short-read sequencing produced 1,923,679,864 clean reads with an average Q30 rate of 93%. The high-quality transcripts were subsequently quantified using the…

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Tuesday, April 21, 2020

Full-length transcript sequencing and comparative transcriptomic analysis to evaluate the contribution of osmotic and ionic stress components towards salinity tolerance in the roots of cultivated alfalfa (Medicago sativa L.).

Alfalfa is the most extensively cultivated forage legume. Salinity is a major environmental factor that impacts on alfalfa’s productivity. However, little is known about the molecular mechanisms underlying alfalfa responses to salinity, especially the relative contribution of the two important components of osmotic and ionic stress.In this study, we constructed the first full-length transcriptome database for alfalfa root tips under continuous NaCl and mannitol treatments for 1, 3, 6, 12, and 24?h (three biological replicates for each time points, including the control group) via PacBio Iso-Seq. This resulted in the identification of 52,787 full-length transcripts, with an average length of…

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