Journal Article

Poly(a) selection introduces bias and undue noise in direct RNA-sequencing

Viscardi, Marcus J., Arribere, Joshua A.

2022

Overview

Background Genome-wide RNA-sequencing technologies are increasingly critical to a wide variety of diagnostic and research applications. RNA-seq users often first enrich for mRNA, with the most popular enrichment method being poly(A) selection. In many applications it is well-known that poly(A) selection biases the view of the transcriptome by selecting for longer tailed mRNA species. Results Here, we show that poly(A) selection biases Oxford Nanopore direct RNA sequencing. As expected, poly(A) selection skews sequenced mRNAs toward longer poly(A) tail lengths. Interestingly, we identify a population of mRNAs (> 10% of genes’ mRNAs) that are inconsistently captured by poly(A) selection due to highly variable poly(A) tails, and demonstrate this phenomenon in our hands and in published data. Importantly, we show poly(A) selection is dispensable for Oxford Nanopore’s direct RNA-seq technique, and demonstrate successful library construction without poly(A) selection, with decreased input, and without loss of quality. Conclusions Our work expands the utility of direct RNA-seq by validating the use of total RNA as input, and demonstrates important technical artifacts from poly(A) selection that inconsistently skew mRNA expression and poly(A) tail length measurements.

• Publisher: BioMed Central,BioMed Central Ltd,Springer Nature B.V,BMC
• Subject: Adenosine / Analysis / Animal Genetics and Genomics / Background noise / Bias / Biomedical and Life Sciences / Direct RNA-sequencing / Gene expression / Gene sequencing / Genomes / Genomics / Life Sciences / Messenger RNA / Microarrays / Microbial Genetics and Genomics / Oxford Nanopore / Plant Genetics and Genomics / Poly(a) selection / Polyadenine / Polyadenylation / Proteomics / Protocol / Ribonucleic acid / RNA / RNA sequencing / Transcriptomes / Transcriptomics