RNA Methylation pp 211-229

Part of the Methods in Molecular Biology book series (MIMB, volume 1562)

In Silico Identification of RNA Modifications from High-Throughput Sequencing Data Using HAMR

  • Pavel P. Kuksa
  • Yuk Yee Leung
  • Lee E. Vandivier
  • Zachary Anderson
  • Brian D. Gregory
  • Li-San Wang
Protocol

Abstract

RNA molecules are often altered post-transcriptionally by the covalent modification of their nucleotides. These modifications are known to modulate the structure, function, and activity of RNAs. When reverse transcribed into cDNA during RNA sequencing library preparation, atypical (modified) ribonucleotides that affect Watson-Crick base pairing will interfere with reverse transcriptase (RT), resulting in cDNA products with mis-incorporated bases or prematurely terminated RNA products. These interactions with RT can therefore be inferred from mismatch patterns in the sequencing reads, and are distinguishable from simple base-calling errors, single-nucleotide polymorphisms (SNPs), or RNA editing sites. Here, we describe a computational protocol for the in silico identification of modified ribonucleotides from RT-based RNA-seq read-out using the High-throughput Analysis of Modified Ribonucleotides (HAMR) software. HAMR can identify these modifications transcriptome-wide with single nucleotide resolution, and also differentiate between different types of modifications to predict modification identity. Researchers can use HAMR to identify and characterize RNA modifications using RNA-seq data from a variety of common RT-based sequencing protocols such as Poly(A), total RNA-seq, and small RNA-seq.

Key words

RNA modification RNA posttranscriptional modification RNA covalent modification Small RNA Small RNA sequencing Messenger RNA RNA sequencing Machine learning Classification 

Abbreviations

3′

3-prime (3′)

5′

5-prime (5′)

bp

Base pair

cDNA

Complementary DNA

HAMR

High-throughput annotation of modified ribonucleotides

mRNA

Messenger RNA

mRNA-seq

messenger RNA sequencing

nt

Nucleotide

RNA-seq

RNA sequencing

RT

Reverse transcriptase

smRNA

Small RNA

smRNA-seq

Small RNA sequencing

SNP

single nucleotide polymorphism

tRNA

Transfer RNA

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Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Pavel P. Kuksa
    • 1
    • 2
  • Yuk Yee Leung
    • 1
    • 2
  • Lee E. Vandivier
    • 3
    • 4
  • Zachary Anderson
    • 4
  • Brian D. Gregory
    • 4
  • Li-San Wang
    • 1
    • 2
    • 5
  1. 1.Department of Pathology and Laboratory MedicineUniversity of PennsylvaniaPhiladelphiaUSA
  2. 2.Penn Institute for Biomedical InformaticsUniversity of PennsylvaniaPhiladelphiaUSA
  3. 3.Cell and Molecular Biology Graduate GroupUniversity of PennsylvaniaPhiladelphiaUSA
  4. 4.Department of BiologyUniversity of PennsylvaniaPhiladelphiaUSA
  5. 5.Institute on AgingUniversity of PennsylvaniaPhiladelphiaUSA

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