High-throughput sequencing with thermostable group II intron reverse transcriptases




Wu, Ching Kai Douglas

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Group II intron reverse transcriptases from bacterial thermophiles have high fidelity, processivity and thermostability. With these properties, thermostable group II intron reverse transcriptases are thought to be more advantageous in different biotechnological applications, such as RNA sequencing and qRT-PCR, than widely- adopted retroviral reverse transcriptases. In this dissertation, I begin with evaluating the usage of thermostable group II intron reverse transcriptase end-to-end template-switching activities in rapid preparations of strand-specific, low bias RNA sequencing libraries for the Illumina short-read sequencing platform. By comparing RNA sequencing results from thermostable group II intron reverse transcriptases to that of other widely-adopted commercially-available RNA-seq kits, I demonstrated that this method provided a more accurate and comprehensive measurement of RNA expressions in biological samples. Using RNA sequencing data generated with this novel class of reverse transcriptase, I also showed that current state-of-the-art bioinformatic tools were not optimal for analyzing all RNA classes and biasing against small RNA and RNA with high abundant of mutations and modifications. Finally, I also established an efficient method to prepare high-quality single-stranded DNA sequencing libraries by exploiting the end-to-end template-switching activity and the DNA-dependent, DNA-polymerase activity of this reverse transcriptase and demonstrated its use in liquid biopsies.


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