去年Nature Methods盤點值得期待的技術(shù)中,，就有單細胞測序,，今年Science盤點更是將這一技術(shù)列為2013科研熱點的榜首,。近期兩個研究組分別在Science，Nature Methods雜志上公布了單細胞測序技術(shù)的最新進展,，分別介紹了這種“Solitary Sequencing”的兩種最新技術(shù),。
首先來自加拿大英屬哥倫比亞大學(xué)等處的研究人員研發(fā)出了一種稱為Strand-seq的新型測序方法，這種單細胞測序新方法能分別對單細胞的雙親DNA模板鏈進行測序,，獲得高分辨率的姊妹染色體交換圖譜,。
在細胞分裂過程中，當(dāng)雙螺旋解旋后,，兩條染色體上的遺傳信息偶然會出現(xiàn)交換,，如果這樣的交換水平不斷提高，也就是DNA損傷和癌癥的一個標志,，那么傳統(tǒng)的基因組測序方法就無法檢測出來,。
利用Strand-seq方法，研究人員可以完成單鏈DNA測序,，并發(fā)現(xiàn)了首個基因組壓力和不穩(wěn)定性的痕跡,。這種方法能來捕捉DNA一條鏈上的信息，并且能令研究人員對親本DNA模板鏈進行單細胞測序,。
這種方法的一大優(yōu)點在于其提供了測序的方向,。目前的方法都是在一個單細胞的DNA擴增和測序的時候，會丟失定向信息，Lansdorp說,。這種丟失導(dǎo)致了這些方法難以檢測到基因重排,。而通過Strand-seq方法，則有可能發(fā)現(xiàn)細胞復(fù)制過程中,，DNA序列的翻轉(zhuǎn)或交換,。
除此之外，謝曉亮教授領(lǐng)導(dǎo)的研究小組研發(fā)出了一種可避免周圍的擴增偏差問題的新方法：多重退火和成環(huán)循環(huán)擴增(Multiple Annealing and Looping-Based Amplification Cycles),，簡稱MALBAC,，這種方法能從一個細胞的基因組中，分離出來自單細胞的DNA,，然后添加稱作引物的短DNA分子,。這些引物可與DNA的隨意部分互補，從而使得它們能夠附著到DNA鏈上,，充當(dāng)DNA復(fù)制起點,。利用這種方法，進行與加入的引物的DNA復(fù)制時,，可以完成高達93%的基因組測序,。
為了驗證這種方法，研究人員將其應(yīng)用在三個關(guān)系緊密的細胞的DNA測序,，以及一個單一的亞洲男性精子的DNA測序,，結(jié)果證明這種方法能準確識別單個核苷酸的變化。比如d在單精子測序中,，研究人員首先對這一男性的99個精子進行了單細胞全基因組DNA,，然后利用HiSeq高通量測序技術(shù)對每個精子分別進行了一倍深度的測序，結(jié)果首次發(fā)現(xiàn),，基因區(qū)附近重組率的降低由分子機制所決定,，而非自然選擇的結(jié)果，從而一舉解決了多年來困擾學(xué)術(shù)界的生物學(xué)難題,。(生物谷Bioon.com)
doi:10.1038/nmeth.2206
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DNA template strand sequencing of single-cells maps genomic rearrangements at high resolution
Ester Falconer,1 Mark Hills,1 Ulrike Naumann,1 Steven S S Poon,1 Elizabeth A Chavez,1 Ashley D Sanders,1 Yongjun Zhao,2 Martin Hirst2, 3 & Peter M Lansdorp1, 4, 5
DNA rearrangements such as sister chromatid exchanges (SCEs) are sensitive indicators of genomic stress and instability, but they are typically masked by single-cell sequencing techniques. We developed Strand-seq to independently sequence parental DNA template strands from single cells, making it possible to map SCEs at orders-of-magnitude greater resolution than was previously possible. On average, murine embryonic stem (mES) cells exhibit eight SCEs, which are detected at a resolution of up to 23 bp. Strikingly, Strand-seq of 62 single mES cells predicts that the mm9 mouse reference genome assembly contains at least 17 incorrectly oriented segments totaling nearly 1% of the genome. These misoriented contigs and fragments have persisted through several iterations of the mouse reference genome and have been difficult to detect using conventional sequencing techniques. The ability to map SCE events at high resolution and fine-tune reference genomes by Strand-seq dramatically expands the scope of single-cell sequencing.
DOI: 10.1126/science.1229164
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Genome-Wide Detection of Single-Nucleotide and Copy-Number Variations of a Single Human Cell
Chenghang Zong1,*, Sijia Lu1,*,†, Alec R. Chapman1,2,*, X. Sunney Xie1,‡
Kindred cells can have different genomes because of dynamic changes in DNA. Single-cell sequencing is needed to characterize these genomic differences but has been hindered by whole-genome amplificationbias, resulting in low genome coverage. Here, we report on a new amplification method—multiple annealingand looping-based amplification cycles (MALBAC)—that offers high uniformity across the genome. Sequencing MALBAC-amplified DNA achieves 93% genome coverage ≥1x for a single human cell at 25x mean sequencing depth. We detected digitized copy-number variations (CNVs) of a single cancer cell. By sequencing three kindred cells, we were able to identify individual single-nucleotide variations (SNVs), with no false positives detected. We directly measured the genome-wide mutation rate of a cancer cell line and found that purine-pyrimidine exchanges occurred unusually frequently among the newly acquired SNVs.
