Pippin全自動DNA片段回收儀在RAD-seq和MeD-seq文庫制備中的應(yīng)用

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

RAD-seq 相關(guān)應(yīng)用

RAD-seq技術(shù)，是廣泛用于遺傳多樣性測序技術(shù)的一種。利用限制性內(nèi)切酶消化以降低基因組的復(fù)雜度，對特定酶切片段進(jìn)行NGS高通量測序，實(shí)現(xiàn)基因分型的技術(shù)。

RAD-seq不僅實(shí)驗(yàn)操作簡單，性價(jià)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(shù)以萬計(jì)的多態(tài)性遺傳標(biāo)記，已廣泛應(yīng)用于生態(tài)學(xué)、遺傳學(xué)、基因組學(xué)等研究領(lǐng)域。

在ddRAD-seq實(shí)驗(yàn)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(diǎn)在文庫間代表性的影響，另外通過實(shí)驗(yàn)初期的監(jiān)測，可以評估樣品的酶切片段均勻度，排除轉(zhuǎn)座子等高頻出現(xiàn)的片段。

相關(guān)文獻(xiàn)：

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(yīng)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(shù)。其主要原理是通過將DNA中非甲基化的胞嘧啶轉(zhuǎn)化為尿嘧啶（U），繼而進(jìn)行測序的技術(shù)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(shí)產(chǎn)生不同的序列，進(jìn)一步通過測序分析和參考鏈比較，以了解哪些部位發(fā)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(xué)意義的基因間區(qū)域的覆蓋卻不足。因此有越來越多的研究人員開發(fā)了增強(qiáng)減少代表性亞硫酸氫鹽測序技術(shù)（Enhanced Reduced Representation Bisulfite Sequencing ，ERRBS）。ERRBS技術(shù)可以在在產(chǎn)生的數(shù)據(jù)中有覆蓋更多的CpG，并增加了所有待測基因組區(qū)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(shè)計(jì)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(gòu)建DNA文庫，經(jīng)過驗(yàn)證是一種可行的甲基化測序技術(shù)。

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.