Quantifying the Benefit Offered by Transcript Assembly on Single-Molecule Long Reads

Tung, Laura H.; Shao, Mingfu; Kingsford, Carl

doi:10.1101/632703

Cited by 2 publications

(1 citation statement)

References 34 publications

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“…For short-reads RNA-seq data (typically Illumina RNA-seq data), Cufflinks [2], Scripture [3], Traph [4], CLASS2 [5], TransComb [6], Scallop [7], StringTie [8], and RefShannon [9] have been developed. For assembling long-reads RNA-seq data (e.g., generated by PacBio Iso-Seq and Oxford Nanopore direct RNA and cDNA sequencing), StringTie2 [10] and Scallop-LR [11] have been released.…”

Section: Introductionmentioning

confidence: 99%

Scallop2 enables accurate assembly of multiple-end RNA-seq data

Zhang

Shi

Shao

2021

Preprint

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Transcript assembly (i.e., to reconstruct the full-length expressed transcripts from RNA-seq data) has been a critical but yet unsolved step in RNA-seq analysis. Modern RNA-seq protocols can produce paired-/multiple-end RNA-seq reads, where information is available that two or more reads originate from the same transcript. The long-range constraints implied in these paired-/multiple-end reads can be much beneficial in correctly phasing the complicated spliced isoforms. However, there often exist gaps among individual ends, which may even contain junctions, making the efficient use of such constraints algorithmically challenging. Here we introduce Scallop2, a new reference-based transcript assembler optimized for multiple-end (including paired-end) RNA-seq data. Scallop2 uses an algorithmic framework that first represents reads from the same molecule as the so-called multiple-end phasing paths in the context of a splice graph, then bridges each multiple-end phasing path into a long, single-end phasing path, and finally decomposes the splice graph into paths (i.e., transcripts) guided by the bridged phasing paths. An efficient bridging algorithm is designed to infer the true path connecting two consecutive ends following a novel formulation that is robust to sequencing errors and transcript noises. By observing that failing to bridge two ends is mainly due to incomplete splice graphs, we propose a new method to determine false starting/ending vertices of the splice graphs which has been showed efficient in reducing false positive rate. Evaluations on both (multiple-end) single-cell RNA-seq datasets from Smart-seq3 protocol and Illumina paired-end RNA-seq samples demonstrate that Scallop2 vastly outperforms recent assemblers including StringTie2, Scallop, and CLASS2 in assembly accuracy.

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Section: Introductionmentioning

confidence: 99%

Scallop2 enables accurate assembly of multiple-end RNA-seq data

Zhang

Shi

Shao

2021

Preprint

Self Cite

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Utilizing PacBio Iso-Seq for Novel Transcript and Gene Discovery of Abiotic Stress Responses in Oryza sativa L.

Schaarschmidt

Fischer

Lawas

et al. 2020

IJMS

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The wide natural variation present in rice is an important source of genes to facilitate stress tolerance breeding. However, identification of candidate genes from RNA-Seq studies is hampered by the lack of high-quality genome assemblies for the most stress tolerant cultivars. A more targeted solution is the reconstruction of transcriptomes to provide templates to map RNA-seq reads. Here, we sequenced transcriptomes of ten rice cultivars of three subspecies on the PacBio Sequel platform. RNA was isolated from different organs of plants grown under control and abiotic stress conditions in different environments. Reconstructed de novo reference transcriptomes resulted in 37,500 to 54,600 plant-specific high-quality isoforms per cultivar. Isoforms were collapsed to reduce sequence redundancy and evaluated, e.g., for protein completeness (BUSCO). About 40% of all identified transcripts were novel isoforms compared to the Nipponbare reference transcriptome. For the drought/heat tolerant aus cultivar N22, 56 differentially expressed genes in developing seeds were identified at combined heat and drought in the field. The newly generated rice transcriptomes are useful to identify candidate genes for stress tolerance breeding not present in the reference transcriptomes/genomes. In addition, our approach provides a cost-effective alternative to genome sequencing for identification of candidate genes in highly stress tolerant genotypes.

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Quantifying the Benefit Offered by Transcript Assembly on Single-Molecule Long Reads

Cited by 2 publications

References 34 publications

Scallop2 enables accurate assembly of multiple-end RNA-seq data

Scallop2 enables accurate assembly of multiple-end RNA-seq data

Utilizing PacBio Iso-Seq for Novel Transcript and Gene Discovery of Abiotic Stress Responses in Oryza sativa L.

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