Stimulation of reverse transcriptase generated cDNAs with specific indels by template RNA structure: retrotransposon, dNTP balance, RT-reagent usage

Penno, Christophe; Kumari, Romika; Baranov, Pavel V.; van Sinderen, Douwe; Atkins, John F.

Thermostable RTs encoded by group II introns from thermophilic bacteria are proving very useful for next generation RNA sequencing (49) and one of them, TGIRT, is commercially available and becoming widely used because of its thermostability (60 • C) and advantageous template switching. TGIRT was first tested using the constructs specifying the WT or mutated 'model' stem-loop 5 to the A7 slippage motif. As described more fully in Methods, the experimental conditions involved attachment of a preformed 41 bp DNA:RNA hybrid that is utilized as primer for reverse transcription of the test template by the TGIRT enzyme. The hybrid contained a one base overhang at the 3 end of the DNA. It is complementary to the base at the 3 end of the RNA test construct. The overhang base is utilized by the RT enzyme to switch from the RNA of the hybrid to the RNA test template. Such template switching (48) is utilized in preparation of samples for deep sequencing. The buffer conditions used for the preparation of the cDNA for deep sequencing were the same as used here for the study of TGIRT reagent slippage.