前兩期我們介紹了Sage系列產品在Small RNA、ChiP-seq、cffDNA和ctDNA測序中的應用，本期我們將介紹Sage系列產品在RAD seq和甲基化測序中的應用。

RAD-seq 相關應用

RAD-seq技術，是廣泛用于遺傳多樣性測序技術的一種。利用限制性內切酶消化以降低基因組的復雜度，對特定酶切片段進行NGS高通量測序，實現基因分型的技術。

RAD-seq不僅實驗操作簡單，性價比高，更為重要的是他并不依賴參考基因組的信息，一次測序即可獲得數以萬計的多態性遺傳標記，已廣泛應用于生態學、遺傳學、基因組學等研究領域。

在ddRAD-seq實驗流程中，Pippin 系列全自動DNA片段回收儀，可以降低片段選擇過程對相同位點在文庫間代表性的影響，另外通過實驗初期的監測，可以評估樣品的酶切片段均勻度，排除轉座子等高頻出現的片段。

相關文獻：

1	Jiang, Ning, et al. "A highly robust and optimized sequence-based approach for genetic polymorphism discovery and genotyping in large plant populations." Theoretical and Applied Genetics 129.9 (2016): 1739-1757.
2	Auteri, Giorgia G., and L. Lacey Knowles. "Decimated little brown bats show potential for adaptive change." Scientific Reports 10.1 (2020): 3023.
3	Rubi, Tricia L., L. Lacey Knowles, and Ben Dantzer. "Museum epigenomics: Characterizing cytosine methylation in historic museum specimens." Molecular ecology resources 20.5 (2020): 1161-1170.
4	Kim, Han-Na, et al. "The Origin and Invasion Pathway of Brown Rats Rattus norvegicus on Dok-Do Island Revealed by Genome-Wide Markers from 3-RADseq Approach." Animals 13.7 (2023): 1243.
5	Gargiulo, Roberta, Tiiu Kull, and Michael F. Fay. "Effective double‐digest RAD sequencing and genotyping despite large genome size." Molecular Ecology Resources 21.4 (2021): 1037-1055.
6	Goodall, Jake, et al. "RAD sequencing of common whelk, Buccinum undatum, reveals fine‐scale population structuring in Europe and cryptic speciation within the North Atlantic." Ecology and Evolution 11.6 (2021): 2616-2629.

甲基化測序（MeD-seq）應用

甲基化測序是一類專門分析DNA分子中胞嘧啶（C）甲基化水平的測序分析技術。其主要原理是通過將DNA中非甲基化的胞嘧啶轉化為尿嘧啶（U），繼而進行測序的技術手段。由于正常情況下胞嘧啶的配對堿基是鳥嘌呤（G），而尿嘧啶的配對堿基是腺嘌呤（A）。這樣就可以在生成配對序列時產生不同的序列，進一步通過測序分析和參考鏈比較，以了解哪些部位發生了甲基化。

目前，減少代表性亞硫酸氫鹽測序（Reduced Representation Bisulfite Sequencing ，RRBS）覆蓋了大部分的基因啟動子和CpG島，但是對CpG周圍和其他有生物學意義的基因間區域的覆蓋卻不足。因此有越來越多的研究人員開發了增強減少代表性亞硫酸氫鹽測序技術（Enhanced Reduced Representation Bisulfite Sequencing ，ERRBS）。ERRBS技術可以在在產生的數據中有覆蓋更多的CpG，并增加了所有待測基因組區域的覆蓋率，因此改善了RRBS在甲基化測序中的缺陷。其中在Garrett-Bakelman等人設計的ERRBS方法，通過利用Pippin Prep回收135-410bp的DNA片段，可以高效回收25ng以上的DNA并用于構建DNA文庫，經過驗證是一種可行的甲基化測序技術。

1	Garrett-Bakelman, Francine E., et al. "Enhanced reduced representation bisulfite sequencing for assessment of DNA methylation at base pair resolution." JoVE (Journal of Visualized Experiments) 96 (2015): e52246.
2	Smit, Kyra N., et al. "Genome-wide aberrant methylation in primary metastatic UM and their matched metastases." Scientific Reports 12.1 (2022): 42.
3	Shankar, Anusha S., et al. "Kidney organoids are capable of forming tumors, but not teratomas." Stem Cells 40.6 (2022): 577-591.
4	Siamoglou, Stavroula, et al. "Genome-wide analysis toward the epigenetic aetiology of myelodysplastic syndrome disease progression and pharmacoepigenomic basis of hypomethylating agents drug treatment response." Human Genomics 17.1 (2023): 1-12.
5	Siamoglou, Stavroula, et al. "Genome-wide analysis toward the epigenetic aetiology of myelodysplastic syndrome disease progression and pharmacoepigenomic basis of hypomethylating agents drug treatment response." Human Genomics 17.1 (2023): 1-12.
6	Wang, Maojun, et al. "Multi-omics maps of cotton fibre reveal epigenetic basis for staged single-cell differentiation." Nucleic Acids Research 44.9 (2016): 4067-4079.
7	Wilbanks, Elizabeth G., et al. "Metagenomic methylation patterns resolve complex microbial genomes." bioRxiv (2021): 2021-01.