The Effect of Acceptor Oligoribonucleotide Sequence on the T₄ RNA Ligase Reaction

Abstract

In order to make efficient use of T4 RNA ligase in a program involving chemical‐enzymatic oligoribonucleotide synthesis, the sequence effects of the acceptor oligomer have been refined. The reaction of the donor molecules pCp and pUpUpUpCp with a series of trinucleoside diphosphates ApApN, CpNpA and NpCpA (where N = U, C, A or G) was examined. High‐performance liquid chromatography was used for simultaneous analysis of all substrates, intermediates and products. For all trinucleoside bisphosphates tested a substantial amount of the intermediate A(5′)pp(5′)Cp or A(5′)pp(5′)UpUpUpCp was observed. This indicated that the extent of ligation was dependent upon sequence of the acceptor molecule and not upon adenylation of the donor. Con‐ versely, examination of the four nucleoside 3′,5′‐bisphosphate donors p u p, pCp, pAp and pGp with a common acceptor ApApU indicated that in the case of poor ligation little of the intermediate adenylated donor was formed.

RNaseIII and T4 Polynucleotide Kinase sequence biases and solutions during RNA-seq library construction

“…Perhaps RNaseIII fragmentation causes a bias that has not been previously evaluated. However, the three steps following RNaseIII fragmentation (adaptor ligation, reverse transcription, and amplification) were already known to have sequence biases [4-6]. Therefore, we first inspected these three steps for potential problems.…”

“…For example, mapped read counts can over or underestimate true RNA abundances based on a variety of library construction steps, such as reverse transcription, adaptor ligation, or amplification [4-7]. In a comparison of multiple library preparation methods for RNA-seq, all methods introduced their own bias and showed different expression patterns from the same sample [8].…”

Small RNA Library Preparation Method for Next-Generation Sequencing Using Chemical Modifications to Prevent Adapter Dimer Formation

“…It is clear that many current library preparation protocols may not cover all small RNA species due to inherent secondary structures of miRNA [33, 34]. We investigated whether the chemically modified adapters would significantly alter miRNA signatures when compared to unmodified adapters in the same workflow.…”

Quantitative Bias in Illumina TruSeq and a Novel Post Amplification Barcoding Strategy for Multiplexed DNA and Small RNA Deep Sequencing

“…There are multiple sources of bias that can be introduced during sample purification and library preparation including ligation bias, secondary structures, PCR-bias created by amplification of differentially barcoded miRNAs and amplification bias introduced on the surface of the flow cell [11]–[13]. The important point in the context of the present work is that PALM and TruSeq barcoding, in contrast to the pre-PCR barcoding protocol we used, gives consistent and reproducible results allowing multiplexing and meaningful comparisons of differential miRNA and mRNA expression without the need for technical replicates with different barcodes.…”