RATTLE: reference-free reconstruction and quantification of transcriptomes from Nanopore sequencing

Abstract

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.

Soil metatranscriptomics: An improved RNA extraction method toward functional analysis using nanopore direct RNA sequencing

“…Sodium acetate has been used previously to separate DNA from RNA (Chomczynski and Sacchi 2006;Xu et al 2019), however, its efficiency when added to the aqueous phases of soil RNA extraction has not yet been reported. Mettel et al (Mettel et al 2010) used an acidic phenol (pH = 4.5) to separate DNA from RNA in soil samples, but our results, as well as those of a recently published study (de la Rubia et al 2022), show that aqueous pH is more important than the pH of the added phenol solution when separating DNA from RNA (de la Rubia et al 2022). Interestingly, we observed that the addition of low-pH sodium acetate in this step also slightly reduced the amount of humic substances (Supplementary Table 1).…”

“…To answer this need, we designed in this study an in-house bioinformatics Abdonaser Poursalavati workflow for taxonomy analysis at different levels. Notably, using the RATTLE Pipeline (de la Rubia et al 2022), it was possible to avoid misclassifying the reads of species with the least sequencing coverage, one of the common issues when working with long-read assemblers. Thus, using this in-house workflow we analyzed the consensus sequences obtained from the clustering step in the total RNA datasets in terms of rRNA sequences and we completed the taxonomic profiling of the microbial population.…”

“…Seqkit (Shen et al 2016) fq2fa option was used to convert the trimmed fastq files to fasta files. RATTLE pipeline with a reference-free algorithm (de la Rubia et al 2022) was used for read clustering, error correction of reads, and read polishing. From each cluster, a consensus transcript was extracted, and the abundance was determined by counting the total reads in each cluster.…”

Dataset from de novo transcriptome assembly of Myristica fatua leaves using MinION nanopore sequencer

“…Then, polyA-tails and the remaining SSP adapters were removed using Cutadapt [14] . De novo assembly was then performed on clean full-length reads using the RATTLE program [15] ( Table 1 ).…”

Biochemical-free enrichment or depletion of RNA classes in real-time during direct RNA sequencing with RISER

“…Nanopore DRS signals for human heart were obtained from the European Nucleotide Archive (ENA) under accession number PRJEB40410 25 . Nanopore DRS signals for HEK293 cells were obtained from the ENA under accession number ENA PRJEB40872 26 and from the NCBI Gene Expression Omnibus (GEO) under accession number TBD 15 .…”