Poly(A)-seq: A method for direct sequencing and analysis of the transcriptomic poly(A)-tails

PLoS One. 2020 Jun 16;15(6):e0234696. doi: 10.1371/journal.pone.0234696. eCollection 2020.

Abstract

Poly(A) tails at the 3' end of eukaryotic messenger RNAs control mRNA stability and translation efficiency. Facilitated by various NGS methods, alternative polyadenylation sites determining the 3'-UTR length of gene transcripts have been extensively studied. However, poly(A) lengths demonstrating dynamic and developmental regulation remain largely unexplored. The recently developed NGS-based methods for genome-wide poly(A) profiling have promoted the study of genom-wide poly(A) dynamics. Here we present a straight forward NGS-method for poly(A) profiling, which applies a direct 3'-end adaptor ligation and the template switching for 5'-end adaptor ligation for cDNA library construction. Poly(A) lengths are directly calculated from base call data using a self-developed pipeline pA-finder. The libraries were directly sequenced from the 3'-UTR regions into the followed poly(A) tails, firstly on NextSeq 500 to produce single-end 300-nt reads, demonstrating the method feasibility and that optimization of the fragmented RNA size for cDNA library construction could detecting longer poly (A) tails. We next applied Poly(A)-seq cDNA libraries containing 40-nt and 120-nt poly(A) tail spike-in RNAs on HiSeq X-ten and NovaSeq 6000 to obtain 150-nt and 250-nt pair-end reads. The sequencing profiles of the spike-in RNAs demonstrated both high accuracy and high quality score in reading poly(A) tails. The poly(A) signal bleeding into the 3' adaptor sequence and a sharp decreased quality score at the junction were observed, allowing the modification of pA-finder to remove homopolymeric signal bleeding. We hope that wide applications of Poly(A)-seq help facilitate the study of the development- and disease-related poly(A) dynamics and regulation, and of the recent emerging mixed tailing regulation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 3' Untranslated Regions / genetics
  • Gene Library
  • Genome
  • Humans
  • Poly A / genetics*
  • Polyadenylation / genetics
  • Sequence Analysis, RNA / methods*
  • Transcriptome / genetics

Substances

  • 3' Untranslated Regions
  • Poly A

Grants and funding

This work was supported by ABLife Inc. [grant number ABL2015-JK002]. The funder provided support in the form of salaries for authors [F.Y., Yu.Z., C.C., W.W., Z.Z, W.R., H.Y., Y.W., Q.W., and Yi.Z], but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.