去年Nature Methods盤點(diǎn)值得期待的技術(shù)中，就有單細(xì)胞測(cè)序,，今年Science盤點(diǎn)更是將這一技術(shù)列為2013科研熱點(diǎn)的榜首,。近期兩個(gè)研究組分別在Science，Nature Methods雜志上公布了單細(xì)胞測(cè)序技術(shù)的最新進(jìn)展,，分別介紹了這種“Solitary Sequencing”的兩種最新技術(shù),。
首先來(lái)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(cè)序方法,，這種單細(xì)胞測(cè)序新方法能分別對(duì)單細(xì)胞的雙親DNA模板鏈進(jìn)行測(cè)序，獲得高分辨率的姊妹染色體交換圖譜,。
在細(xì)胞分裂過(guò)程中,，當(dāng)雙螺旋解旋后，兩條染色體上的遺傳信息偶然會(huì)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(gè)標(biāo)志，那么傳統(tǒng)的基因組測(cè)序方法就無(wú)法檢測(cè)出來(lái),。
利用Strand-seq方法,，研究人員可以完成單鏈DNA測(cè)序，并發(fā)現(xiàn)了首個(gè)基因組壓力和不穩(wěn)定性的痕跡,。這種方法能來(lái)捕捉DNA一條鏈上的信息,，并且能令研究人員對(duì)親本DNA模板鏈進(jìn)行單細(xì)胞測(cè)序。
這種方法的一大優(yōu)點(diǎn)在于其提供了測(cè)序的方向,。目前的方法都是在一個(gè)單細(xì)胞的DNA擴(kuò)增和測(cè)序的時(shí)候,，會(huì)丟失定向信息，Lansdorp說(shuō),。這種丟失導(dǎo)致了這些方法難以檢測(cè)到基因重排,。而通過(guò)Strand-seq方法，則有可能發(fā)現(xiàn)細(xì)胞復(fù)制過(guò)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(kuò)增偏差問(wèn)題的新方法：多重退火和成環(huán)循環(huán)擴(kuò)增(Multiple Annealing and Looping-Based Amplification Cycles),，簡(jiǎn)稱MALBAC,，這種方法能從一個(gè)細(xì)胞的基因組中，分離出來(lái)自單細(xì)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(bǔ),，從而使得它們能夠附著到DNA鏈上,，充當(dāng)DNA復(fù)制起點(diǎn)。利用這種方法,，進(jìn)行與加入的引物的DNA復(fù)制時(shí),，可以完成高達(dá)93%的基因組測(cè)序。
為了驗(yàn)證這種方法,，研究人員將其應(yīng)用在三個(gè)關(guān)系緊密的細(xì)胞的DNA測(cè)序,，以及一個(gè)單一的亞洲男性精子的DNA測(cè)序，結(jié)果證明這種方法能準(zhǔn)確識(shí)別單個(gè)核苷酸的變化,。比如d在單精子測(cè)序中,，研究人員首先對(duì)這一男性的99個(gè)精子進(jìn)行了單細(xì)胞全基因組DNA,，然后利用HiSeq高通量測(cè)序技術(shù)對(duì)每個(gè)精子分別進(jìn)行了一倍深度的測(cè)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(jī)制所決定,，而非自然選擇的結(jié)果，從而一舉解決了多年來(lái)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.
