端粒長度與端粒功能障礙有關的衰老和遺傳疾病有相關聯,因此被視為一種生物標記。目前已經建立的實驗方法著重於平均端粒長度的測量,它們在特定染色體的端粒長度(chromosome-specific telomere length)評估上仍存在一些限制。然而少數端粒長度的缺陷就足以對細胞產生危害甚至引發疾病,所以迫切地需要一個可信賴的方法能夠分析單端粒長度。在此,我以第三代DNA序列定序技術——牛津奈米孔定序(Oxford Nanopore sequencing)為基礎建立一套實驗方法,包含樣本中端粒的富集與單端粒長度的測量。 在執行奈米孔定序前,透過限制酶HinfI與RsaI消化基因組DNA,再將其碎片通過AMPure XP磁珠進行純化,來富集樣本中端粒DNA的含量。獲得序列資料後,我利用BLAST演算法識別檔案中含有端粒序列的測序片段(TSCRs),並同時測量TSCRs的端粒長度。因為端粒重複序列TTAGGG與CCCTAA在奈米孔定序的鹼基識別(base-calling)中,被分別地錯誤判讀成TTAAAA與TGGCC,所以我將上述之非典型的重複序列納入考量,校正對端粒長度的測量結果。除此之外,我構思了兩個標準——AR比例(AR ratio)與端粒重複序列樣式百分比(Pattern percentage),來提升我的程式對判定TSCR的準確度。 我的實驗結果顯示,這套方法能夠有效的富集樣本中的端粒DNA含量,以及識別不同細胞株或者多重樣本定序(multiplex sequencing)資料中的TSCRs。最重要的是,它可以測定單個TSCR的端粒長度,有助益於未來對特定染色體的端粒以及端粒表觀遺傳學的探索。
Telomere length is a biomarker for aging and genetic disorders associated with telomere dysfunction. Established methods for assessing telomere length focus on the average telomere length among a pool of cells, which have some limitations on determining chromosome-specific telomere length. Given that defects in a few telomeres are sufficient to cause cellular effects or diseases, a reliable approach for single telomere analysis is sorely needed. Therefore, I established a method to enrich telomeres and to conduct single telomere length analysis based on third-generation DNA sequencing technology, Oxford Nanopore. Telomeric DNA for Nanopore sequencing was enriched by digesting genomic DNA with HinfI and RsaI and then subjecting it to AMPure XP Beads-based purification. Using a custom BLAST program, telomere sequence-containing reads (TSCRs) in the sequence data could be identified and measured their telomere length. Moreover, telomere lengths were calibrated by including the atypical repeats TTAAAA and TGGCC, which were Nanopore base-calling errors from TTAGGG and CCCTAA repeats, respectively. Additionally, I devised two criteria to improve the accuracy of telomeric read identification, namely the AR ratio and Pattern percentage. My results indicated that this method could effectively enrich telomeric fragments and identify TSCRs from different cell lines or even from multiplex sequencing. Moreover, I determined telomere length for individual TSCRs, which might be a useful approach for future explorations of chromosome-specific telomeres and telomere epigenetics.