Machine learning of reverse transcription signatures of variegated polymerases allows mapping and discrimination of methylated purines in limited transcriptomes

Abstract

Reverse transcription (RT) of RNA templates containing RNA modifications leads to synthesis of cDNA containing information on the modification in the form of misincorporation, arrest, or nucleotide skipping events. A compilation of such events from multiple cDNAs represents an RT-signature that is typical for a given modification, but, as we show here, depends also on the reverse transcriptase enzyme. A comparison of 13 different enzymes revealed a range of RT-signatures, with individual enzymes exhibiting average arrest rates between 20 and 75%, as well as average misincorporation rates between 30 and 75% in the read-through cDNA. Using RT-signatures from individual enzymes to train a random forest model as a machine learning regimen for prediction of modifications, we found strongly variegated success rates for the prediction of methylated purines, as exemplified with N1-methyladenosine (m1A). Among the 13 enzymes, a correlation was found between read length, misincorporation, and prediction success. Inversely, low average read length was correlated to high arrest rate and lower prediction success. The three most successful polymerases were then applied to the characterization of RT-signatures of other methylated purines. Guanosines featuring methyl groups on the Watson-Crick face were identified with high confidence, but discrimination between m1G and m22G was only partially successful. In summary, the results suggest that, given sufficient coverage and a set of specifically optimized reaction conditions for reverse transcription, all RNA modifications that impede Watson-Crick bonds can be distinguished by their RT-signature.

Survey and Validation of tRNA Modifications and Their Corresponding Genes in Bacillus subtilis sp Subtilis Strain 168

“…Deep sequencing-based identification of the B. subtilis tRNA modifications was performed using RiboMethSeq protocol, allowing us to map 2′-O-methylations by their protection to cleavage [ 41 , 42 ]. Reverse Transcriptase (RT) misincorporation signatures at RT-arresting nucleotides [ 43 , 44 ] were extracted from RiboMethSeq data and also from the RT-primer extension, using TTO-based ScriptSeq v2 (Illumina, San Diego, CA, USA) library preparation kit. RiboMethSeq analysis of tRNAs was performed using alkaline fragmentation of total B. subtilis RNA, followed by library preparation and sequencing [ 42 ].…”

Manganese Ions Individually Alter the Reverse Transcription Signature of Modified Ribonucleosides

“…The ability of polymerases to skip certain nucleotides in primer elongation is a rather uncharacterized appearance, which has only recently been described in a RNA modification context [16]. Here, we used the percentage of deletions occurring at, or shortly after the respective position, as another characterizing feature in RT signature, named jump rate.…”

“…This promotes incorporation of non-complementary deoxynucleoside triphosphates (dNTPs) in reverse transcription [10], or even arrest of primer elongation. According to the necessity of converting RNA into DNA for current RNA-seq methods, apart from direct RNA sequencing by nanopore technology [11,12], a combination of suitable library preparation protocols that allow capture of abortive cDNA fragments [13,14] and use of advanced bioinformatical pipelines [15], is needed for efficient analysis of modification prevalence in RNA species [16]. However, most modified nucleosides do not impede reverse transcription to a detectable extent, implying that information about modification type and sequence context may get lost due to the limited vocabulary of only four canonical deoxynucleotides in cDNA synthesis.…”

“…This feature, termed jump, is visible in cDNA as deletion of one or two nucleotides at or shortly after a modification-associated site and proved suitable as an influential parameter of RT signature in modification calling [16]. However, the RT signature is dependent on multiple factors, comprising the kind of bases preceding the modification site, type of modification, the polymerase used and reaction conditions [16,21]. Altered transcription conditions are commonly used for error-prone PCR, a method by which random mutations are introduced to the newly synthesized DNA strand by adding MnCl 2 to the reaction buffer, thus lowering sequence-fidelity of the polymerase [26].…”

“…In addition to primer extension arrest and dNTP misincorporation, an event occurring in reverse transcription of modified nucleosides that has only recently been described [ 16 ] focuses on the ability of polymerases to skip certain nucleotides, visible as deletion in sequencing data. While the occurrence in a modification context is rather uncharacterized, nucleotide skipping is well-known in the field of RNA splicing research [ 23 , 24 ] and probably based on a slipped-strand mispairing mechanism [ 25 ].…”