Improved TGIRT-seq methods for comprehensive transcriptome profiling with decreased adapter dimer formation and bias correction

Abstract

Thermostable group II intron reverse transcriptases (TGIRTs) with high fidelity and processivity have been used for a variety of RNA sequencing (RNA-seq) applications, including comprehensive profiling of whole-cell, exosomal, and human plasma RNAs; quantitative tRNA-seq based on the ability of TGIRT enzymes to give full-length reads of tRNAs and other structured small ncRNAs; high-throughput mapping of post-transcriptional modifications; and RNA structure mapping. Here, we improved TGIRT-seq methods for comprehensive transcriptome profiling by rationally designing RNA-seq adapters that minimize adapter dimer formation. Additionally, we developed biochemical and computational methods for remediating 5′- and 3′-end biases, the latter based on a random forest regression model that provides insight into the contribution of different factors to these biases. These improvements, some of which may be applicable to other RNA-seq methods, increase the efficiency of TGIRT-seq library construction and improve coverage of very small RNAs, such as miRNAs. Our findings provide insight into the biochemical basis of 5′- and 3′-end biases in RNA-seq and suggest general approaches for remediating biases and decreasing adapter dimer formation.

Template switching by a group II intron reverse transcriptase: biochemical analysis and implications for RNA-seq

“…Under these conditions, we found larger differences in the amplitude of the reaction for acceptors with different 3' nucleotides in order G>C≈A>U ( Fig. 8A), which match the previously determined nucleotide sequence biases for TGIRT-seq (49). Additionally, when the reactions were carried out with lower concentrations of acceptor RNAs (10 nM) to more closely resemble the conditions during TGIRT-seq, the difference in amplitude for template switching to the most and least favored 3' nucleotides (G and U, respectively) increased ( Fig.…”

“…Nucleotide sequence biases in TGIRT-seq reflect the efficiency of template switching to different 3' nucleotides of acceptor RNAs at high salt concentration--Previous TGIRT-seq analysis of miRNA reference sets showed that 3'-sequence biases in TGIRT-seq are almost completely confined to the 3'-terminal nucleotide of the acceptor, with a marked preference for miR-NAs with a 3' G residue and against those with a 3' U residue (49). In light of our finding that template switching is slower at high salt concentrations ( Fig.…”

“…These 3' biases, which are restricted to 3' terminal nucleotide of the acceptor, account for about half the sequence bias in TGIRT-seq, the remainder coming from the 5' App RNA/DNA ligase used for R1R adapter ligation (see Fig. 1A) (49). Although we showed previously that these 3' biases could be remediated computationally or by changing the ratio of 3' DNA overhangs in the starter duplex (49), we found here that they can be more simply moderated by carrying out the template-switching reaction at lower salt concentrations (200 mM NaCl; Fig.…”

Systematic sequencing of chloroplast transcript termini fromArabidopsis thalianareveals >200 transcription initiation sites and the extensive imprints of RNA-binding proteins and secondary structures

“…In our case, transcript chemistry, size and secondary structure will all introduce biases to the dataset ultimately used to draw the main conclusions. In addition, factors such as ligation biasthe preferential ligation of adapters to certain sequences (83,84), undoubtedly impacted the selection and ratios of the transcript ends we have reported. Therefore, our conclusionsparticularly quantitative onesshould be taken with those caveats in mind.…”

Anomalous reverse transcription through chemical modifications in polyadenosine stretches

“…12 ). Other steps with potential bias include the synthesis of the RNA by in vitro transcription with T7 RNA polymerase, which is known to 'slip' on repeated sequences 20 ; similar slippage or biased termination of reverse transcriptases in the RTase step after chemical modification [32][33] ; the ligation of adapters onto the resulting cDNAs, which are known to have sequence biases 22,30,34 ; Illumina sequencing-by-synthesis of cDNAs used, which is also known to have sequence biases 29 ; or the bioinformatic workup of these sequencing data into inferred chemical modification profiles. 29,35 We first sought to test if the poly(A) SHAPE and DMS signatures were due to the many possible biases occurring any steps downstream of reverse transcription, by carrying out the simplest method of reading out the cDNA products, capillary electrophoresis with fluorescently labeled primers.…”