Complete genomic and transcriptional landscape analysis using third-generation sequencing: a case study of Saccharomyces cerevisiae CEN.PK113-7D

Abstract

Completion of eukaryal genomes can be difficult task with the highly repetitive sequences along the chromosomes and short read lengths of second-generation sequencing. Saccharomyces cerevisiae strain CEN.PK113-7D, widely used as a model organism and a cell factory, was selected for this study to demonstrate the superior capability of very long sequence reads for de novo genome assembly. We generated long reads using two common third-generation sequencing technologies (Oxford Nanopore Technology (ONT) and Pacific Biosciences (PacBio)) and used short reads obtained using Illumina sequencing for error correction. Assembly of the reads derived from all three technologies resulted in complete sequences for all 16 yeast chromosomes, as well as the mitochondrial chromosome, in one step. Further, we identified three types of DNA methylation (5mC, 4mC and 6mA). Comparison between the reference strain S288C and strain CEN.PK113-7D identified chromosomal rearrangements against a background of similar gene content between the two strains. We identified full-length transcripts through ONT direct RNA sequencing technology. This allows for the identification of transcriptional landscapes, including untranslated regions (UTRs) (5′ UTR and 3′ UTR) as well as differential gene expression quantification. About 91% of the predicted transcripts could be consistently detected across biological replicates grown either on glucose or ethanol. Direct RNA sequencing identified many polyadenylated non-coding RNAs, rRNAs, telomere-RNA, long non-coding RNA and antisense RNA. This work demonstrates a strategy to obtain complete genome sequences and transcriptional landscapes that can be applied to other eukaryal organisms.

Yeast Systems Biology: Model Organism and Cell Factory

“…One main advantage of RNAseq is that it is quantitative, and with standards it is now possible to measure >4000 mRNAs in yeast cells to absolute levels, with it being found that mRNA copy number per cell generally varies across two orders of magnitude, from 1 to 100 per cell. RNAseq is traditionally done using next‐generation sequencing, but recently it has been shown that is possible to use nanopore sequencing technology not only to obtain long DNA sequences required for genome assembly, but also for measuring mRNA levels …”

Current Trends in Diagnostics of Viral Infections of Unknown Etiology

“…Another great advantage of Nanopore technology is that unique protocols for direct RNA sequencing have been developed, meaning that RNA viruses could be analyzed right away, without reverse transcription [192,193]. Even more, depending on the experiment objective, either differential RNA sequencing (dRNA-Seq) or mRNA could be investigated, the latter being achieved by ligating an adapter only to the poly-A tail, thus allowing for transcriptome analysis [192,194]. Direct RNA-Seq helps avoid distortions of fragments' representation [195] in the sample, revolutionizing the approach to studying RNA-viruses and the intricacies of their life cycle [196].…”

Gene expression is encoded in all parts of a co-evolving interacting gene regulatory structure

“…To obtain mRNA counts that were uncorrelated to gene length, the residual of a linear model, based on ORF lengths as the response variable and mRNA counts as the explanatory variable, was used as the corrected response variable (Fig S1-4). By testing whether the introduced correction could potentially remove biological signal associated with gene length, using data from whole molecule RNA sequencing that does not rely on short-read assembly 80 , no correlation between gene length and its expression was found (Pearson's r = -0.08, p -value < 1e-6; Fig S1-8).…”