Next-generation sequencing technologies for detection of modified nucleotides in RNAs

Abstract

Our ability to map and quantify RNA modifications at a genome-wide scale have revolutionized our understanding of the pervasiveness and dynamic regulation of diverse RNA modifications. Recent efforts in the field have demonstrated the presence of modified residues in almost any type of cellular RNA. Next-generation sequencing (NGS) technologies are the primary choice for transcriptome-wide RNA modification mapping. Here we provide an overview of approaches for RNA modification detection based on their RT-signature, specific chemicals, antibody-dependent (Ab) enrichment, or combinations thereof. We further discuss sources of artifacts in genome-wide modification maps, and experimental and computational considerations to overcome them. The future in this field is tightly linked to the development of new specific chemical reagents, highly specific Ab against RNA modifications and use of single-molecule RNA sequencing techniques.

Keywords: Bioinformatics; RNA maturation; RNA modifications; epitranscriptome; next-generation sequencing.

Figure 1.

Second- and third-generation sequencing technologies (NGS and NNGS). Second generation sequencing uses cluster amplification of DNA strands prior to fluorescent or potentiometric sequencing (A). Multiple molecules generate detectable signal. Third-generation sequencing technologies use single molecule sequencing with specially designed fluorescent detection systems (Zero-mode Waveguides, ZMW), or nanopore sequencing using exonuclease or DNA polymerase activities (B).

Figure 2.

Library preparation issues. Detection of RNA modification relies on exact determination of cDNA 3′-end (A). Priming at the 3′-end of RNA depends on its size. Short RNAs (typically < 100 - 200 nt) require adapter ligation step prior to primer annealing (left), while random priming (right) can be used for long RNA species (B). Methods for treatment of cDNA 3′-end for exact determination of RT stop. Four common techniques have been developed. One - single-strand DNA adapter ligation, 2 - oligonucleotide tailing terminal transferase followed by dsDNA primer ligation, 3 - CircLigase protocol with 5′-phosphorylated cDNA followed by second strand synthesis, and 4 - templated cDNA extension with 3′-blocked NNNNNN primer. In all cases the final step includes PCR amplification and appropriate barcoding.

Figure 3.

Three classes of NGS-based techniques for detection and mapping of RNA modifications. Class I methods are based on “natural” RT-signature generated by the modified nucleotide. Such signature may include RT-arrest (interpreted as a coverage drop) at the modification site or/and nucleotide misincorporation at the same position (left). Class II methods are similar to Class I, but the RNA modification is RT-silent and the visible signature is induced via appropriate chemical treatment (middle). Class III techniques are based on enrichment of RNA fragments containing the RNA modification with a specific Ab. In these cases, the position of modification is typically not determined at single nucleotide resolution, but rather as an enriched region (right).

Figure 4.

Applications of Class (I)techniques to A-to-(I)editing and m¹A. Inosine, which is derived from A by RNA editing, generates “misincorporation” into cDNA, due to its base-pairing to C. The RT-signature contains only the “misincorporated” nucleotide compared to the reference sequence (left). m¹A and other modified nucleotides with altered Watson-Crick edge generate complex RT-signatures composed of both RT-arrest and misincorporation at the modification site (right).

Figure 5.

Applications of Class II techniques to RT-silent pseudouridine (Ψ) and m⁵C. Pseudouridine is reactive with water-soluble carbodiimide (CMCT) and forms stable adduct, while U-CMC adducts are removed by alkaline treatment. The resulting Ψ-CMC generates RT-arrest, detectable in the sequencing profile (left). 5-methylcytosine (m⁵C) is RT silent, but its detection is based on its insensitivity to bisulfite deamination. All C residues in RNA are deaminated by bisulfite, while m⁵C remains non-deaminated after treatment. The presence of residual C is thus detected by sequencing (right).

Figure 6.

Complex cases of Class II protocols. Application for 2′-O-methylated residues, m¹A and Inosine. Left: Detection of 2′-O-methylation is based on the 2′-O-Me-mediated protection of 3′-adjacent phosphodiester bond from nucleophilic cleavage. RNA is first randomly fragmented and a sequencing library is prepared from all generated fragments. Calculation of 5′-end (or cumulated 5′- and 3′-end) coverage reveals characteristic “gaps” one nucleotide downstream of the 2′-O-methylated site. The depth of the gap is proportional to methylation level of the nucleotide. Center: Conversion of m¹A to m⁶A by Dimroth rearrangement. m¹A residues present in RNA generate characteristic RT-signature, which disappears after alkaline treatment catalyzing m¹A-to-m⁶A conversion. Comparison of 2 profiles allows identification of m¹A site. Right: Specific Inosine detection by reaction with acrylonitrile (ICE-Seq). As discussed above, Inosine generates a simple RT-signature, which can be confounded with SNPs or with sequencing error. Acrylonitrile treatment converts Inosine into an RT-arresting residue, detected by characteristic coverage drop and misincorporation, thus confirming the presence of modified residue (right).

Figure 7.

Approaches combining pulldown and specific chemistries. Left: Nucleotide-resolution detection of m¹A residues by combination of MeRIP and iCLIP. m¹A-containing RNA fragments are enriched after covalent cross-linking with specific anti m¹A-Ab. After Ab removal by protease, the resulting covalent adducts are used for primer extension. Abortive RT products correspond to neighboring nucleotides around m¹A site. Right: Enrichment of pseudouridine-containing RNA fragments using bi-functional “clickable” CMCT. RNA is first randomly fragmented and subjected to reaction with soluble carbodiimide CMCT bearing “clickable” azide (N₃) group. After “click” reaction with alkyne-modified biotin, modified fragments are enriched by avidin-beads pull-out. Modified residues are detected based on their RT-arrest signature.

Figure 8.

Sources of artifacts in Class I, II and III techniques. Left: Identification of RT-signatures in Class I methods may be affected by the presence of genomic SNPs, by possible mis-aligned nucleotides at exon-intron borders and by errors in the sequencing data set. Center: Class II approaches may generate false-positive signals due to strong non-specific cleavage sites in RNAs, due to mis-alignment of some reads to repetitive RNA sequences, due to unannotated transcription sites. Right: Class III approaches based on Ab enrichment may suffer from non-specific enrichment signals, from Ab promiscuity or mis-alignments. Important controls for these approaches include the coverage profile in the input (top trace), and the enrichment profile for KO or deleted strain (middle). Only specific peaks present in WT sample and absent in control traces should be considered as candidates. The insert shows that the position of the modified residue does not always correspond to the maximal enrichment.