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 tRNAseq 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 (i) rationally designing RNA-seq adapters that minimize adapter dimer formation, and (ii) developing biochemical and computational methods that remediate 5'and 3'-end 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.

Tau aggregates are RNA-protein assemblies that mis-localize multiple nuclear speckle components

“…S2D-L). As our library preparation protocol was not specifically designed to capture tRNAs, miRNAs, or rRNAs, our analyses may underestimate the abundance of these species (Motameny et al, 2010;Xu et al, 2019). The mechanisms by which specific RNAs are enriched in tau aggregates remains to be determined but could be due to tau's intrinsic RNA binding specificity, the structure of the tau conformers, and/or the presence of specific RNAs at sites of tau aggregation such as snRNAs in nuclear speckles or mRNAs at the centrosome (Sepulveda et al, 2018).…”

5´XP sRNA-seq: efficient identification of transcripts with and without 5´ phosphorylation reveals evolutionary conserved small RNA

“…Future research should therefore explore smaller sets of 5´P tag oligos with unrelated sequence compositions to tackle this problem. One must bear in mind, however, that some ligases also show preferences against specific nucleotides in the terminals of their targets [39]. We have tried to design the 5´P tag for optimal performance with the NEBNext 5´ Ligation Mix.…”

“…To better understand how 5P and 5X sRNA-seq vary in their enrichment for different RNA species, we first compared libraries generated using a popular commercial 5P-seq kit (NEBNext Multiplex Small RNA Library prep kit for Illumina; New England Biolabs) and an adaptation of published 5X-seq protocols [21,[37][38][39]. For comparative reasons, we modified the 5X-seq protocol to work with the reverse transcriptase of the 5P-seq kit.…”

Human cells contain myriad excised linear intron RNAs with links to gene regulation and potential utility as biomarkers

“…The long RNAs were then fragmented to 70-100 nt by using an NEBNext Magnesium RNA Fragmentation Module (94 °C for 7 min; New England Biolabs). After clean-up by using a Zymo RNA Clean & Concentrator kit (8X ethanol protocol), the fragmented long RNAs were combined with the unfragmented short RNAs and treated with T4 polynucleotide kinase (Epicentre) to remove 3’ phosphates (Xu et al 2019), followed by clean-up using a Zymo RNA Clean & Concentrator kit (8X ethanol protocol). After confirming the RNA fragment size range and RNA concentration by using an Agilent 2100 Bioanalyzer with a 6000 RNA Pico chip, the RNA was aliquoted into 4 ng portions for storage in −80 °C.…”

“…After cleanup by using a Zymo RNA Clean & Concentrator kit (8X ethanol protocol), the fragmented long RNAs were combined with the unfragmented short RNAs and treated with T4 polynucleotide kinase (Epicentre) to remove 3' phosphates(Xu et al 2019), followed by clean-up using a Zymo RNA Clean & Concentrator kit (8X ethanol protocol). After confirming the RNA fragment size range and RNA concentration by using an Agilent 2100 Bioanalyzer with a 6000 RNA Pico chip, the RNA was aliquoted into 4 ng portions for storage in -80 °C.TGIRT-seqTGIRT-seq libraries were prepared as described(Xu et al 2019) using 20-50 ng of ribodepleted unfragmented RNA or 4-10 ng of ribodepleted chemically fragmented RNA. The templateswitching and reverse transcription reactions were done with 1 μ M TGIRT-III (InGex) and 100 nM pre-annealed R2 RNA/R2R DNA starter duplex in 20 μ l of reaction medium containing 450…”