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Nanopore sequencing enables the efficient and unbiased measurement of transcriptomes. Current methods for transcript identification and quantification rely on mapping reads to a reference genome, which precludes the study of species with a partial or missing reference or the identification of disease-specific transcripts not readily identifiable from a reference. We present RATTLE, a tool to perform reference-free reconstruction and quantification of transcripts using only Nanopore reads. Using simulated data and experimental data from isoform spike-ins, human tissues, and cell lines, we show that RATTLE accurately determines transcript sequences and their abundances, and shows good scalability with the number of transcripts.
The direct interrogation of transcriptome has proven effective to study species without an available genome reference [1] or non-model organisms of ecological relevance [2], or to identify cancer-specific RNAs with diagnostic relevance [3]. However, these approaches have suffered from the limitations of the short sequencing reads, which lead to uncertainties in the reconstruction of transcripts [4]. Single-molecule long-read sequencing with Oxford Nanopore Technologies (ONT) enables the direct measurement of native RNA molecules, often identify complete known and novel transcripts [5], and accurately estimate transcript abundances [6]. These advantages coupled with the ease of sample preparation and the availability of portable platforms make this technology very compelling for the cost-effective and unbiased measurement of transcriptomes from any sample and species. However, current analysis methods rely on the comparison with a reference genome [5, 7,8,9] or on the use of multiple sequencing technologies [10, 11], which precludes the possibility of cost-effective studies. Methods for de novo DNA assembly [12,13,14] cannot be directly transferred to transcriptomes, since they do not model genes with multiple transcript isoforms. Similarly, methods developed for Illumina [1, 15] rely on their high sequence accuracy, which is currently lacking in individual Nanopore reads. To address these challenges, we have developed RATTLE, a tool to perform reference-free reconstruction and quantification of transcripts using only Nanopore long reads. Using simulated data, isoform spike-ins, and sequencing data from human tissues and cell lines, we show that RATTLE is competitive at recovering transcript sequences and their abundances despite not using any information from the reference. RATTLE lays the foundation for a multitude of potential new applications of Nanopore transcriptomics. 59ce067264
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