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Author Spotlight: Exploring the Frontier of mRNA Research with Poly A Tail Analysis Techniques
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Poly(A)-seq: A method for direct sequencing and analysis of the transcriptomic poly(A)-tails.

Fengyun Yu1,2, Yu Zhang3, Chao Cheng2,3

  • 1Laboratory for Genomics Regulation and Human Health, ABLife Inc., Wuhan, PR China.

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|June 17, 2020
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Summary

This study introduces Poly(A)-seq, a new NGS method for profiling poly(A) tail lengths. This technique enables deeper investigation into dynamic and developmental regulation of poly(A) tails in messenger RNA.

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Area of Science:

  • Molecular Biology
  • Genomics
  • RNA Biology

Background:

  • Poly(A) tails regulate mRNA stability and translation.
  • Alternative polyadenylation is well-studied, but poly(A) length dynamics are less explored.
  • Genome-wide poly(A) profiling methods are advancing RNA biology research.

Purpose of the Study:

  • To present a straightforward NGS method for genome-wide poly(A) profiling.
  • To enable the study of dynamic and developmental regulation of poly(A) tail lengths.
  • To develop and validate a computational pipeline for accurate poly(A) length calculation.

Main Methods:

  • Developed a direct 3'-end adaptor ligation and template switching for 5'-end adaptor ligation for cDNA library construction.
  • Utilized a self-developed pipeline, pA-finder, for calculating poly(A) lengths from base call data.
  • Sequenced libraries on NextSeq 500, HiSeq X-ten, and NovaSeq 6000, including spike-in RNAs for validation.

Main Results:

  • Demonstrated the feasibility of the Poly(A)-seq method for detecting poly(A) tails.
  • Confirmed high accuracy and quality scores in reading poly(A) tails using spike-in RNAs.
  • Modified the pA-finder pipeline to address signal bleeding and quality score decrease at adaptor junctions.

Conclusions:

  • Poly(A)-seq is a robust method for genome-wide poly(A) profiling.
  • This method will facilitate research into development- and disease-related poly(A) dynamics.
  • The technique supports the study of emerging mixed tailing regulations.