惠康信托基金会桑格研究所的研究人员宣布，他们在不建库的情况下成功地完成了对单分子DNA的测序。这项研究的主要负责人Paul Coupland博士表示：“这是首次以这种方式完成对单分子DNA的直接测序。”
桑格测序法作为测序技术的“金标准”是由Frederick Sanger博士发明的，这一技术让他获得了两次诺贝尔奖。
在桑格测序方法中，以待测DNA为模板生产一系列的在长度上有1个碱基差别的DNA片段。这些片段然后依据大小被分离开，对其末端的碱基进行识别，从而重建出原始DNA的序列。
到目前为止，这种技术以及其他的测序技术都需要费时费力地进行DNA文库构建，或者利用测序技术的特定技术从基因组样本中进行DNA片段收集。
而利用这种新的技术，研究者无需建库即可直接从DNA片段获得测序数据，与传统标准方法相比，所需的DNA量也相当少，只需要1纳克即可，相当于不到标准方法的1/500。这种新颖的技术在小基因组测序上很有潜力，既省时，所需样本也相当少。
研究人员开发的这种直接测序法使用的是Pacific Biosciences公司的RS测序系统（PacBio RS）。PacBio RS的SMRTCell可实现在高标记核苷酸背景下对单个荧光团的单分子实时观察，产出35000至50000个读长，100至160Mb的碱基数据。
据研究者的论文介绍，这种直接测序法每个SMRTCell可产生高达3000个读长。虽然目前其应用还限于小基因组，但是这种无需建库的方法可以在样本少于1纳克的条件下即得到所需的序列数据，在收到样本后8个小时内即可完成。
研究人员报告称，他们利用这种直接测序法得到了小型环状单链及双链DNA的病毒基因组序列，以及覆盖金黄色葡萄球菌耐甲氧西林菌株的全部基因组的线性片段的序列数据。
研究人员还尝试了利用只有800皮克的DNA对某生物的基因组进行分析，该样本量只有标准方法的1/600。在这个例子中，PacBio至产出了70个读长，只有基因组的少部分。尽管这只是标准方法产生读长的一部分，但是也足以实现对特定生物的鉴定，也可以对实现对以前红基因组样本中不可测定生物的鉴定。
“要对微生物进行测序，首先需要在实验室对其进行培养，”该方法的研究者之一Tamir Chandra博士说，“这不仅费时，而且有的微生物是无法在实验室培养的，使得很难对它们的基因组进行测序。”
“有了这种方法，我们可以直接对这些生物进行测序，在很短的时间内对它们进行身份鉴定。”
Direct sequencing of small genomes on the Pacific Biosciences RS without library preparation
Paul Coupland, Tamir Chandra, Mike Quail, Wolf Reik, and Harold Swerdlow
Pacific Biosciences (Menlo Park, CA, USA) have developed a platform that will sequence a single molecule of DNA in real-time via the polymerization of that strand with a single polymerase . This technique has many benefits over multi-molecule (clonal) sequencing technologies; one such potential advantage is that it may not be absolutely necessary to make a library (i.e., create SMRT bells to generate sequence data. The only input (molecular) requirements to enable sequencing are a primed piece of DNA; both single-stranded and double-stranded molecules will work. The polymerase is necessarily highly processive starting with a location on the DNA at which it can bind, i.e., a free 3′-OH group. We decided to test whether any primed DNA molecules, lacking any other features of a PacBio SMRT bell, could be used directly in a sequencing reaction. The bound complex (DNA-primer-polymerase), although lacking PacBio adapter sequences, can still be sequenced on the PacBio platform. The present efficiency of this process, in terms of the numbers of reads generated and Mb yield per SMRT cell, is considerably less than that using standard libraries. With standard methods a typical SMRT cell will yield 35,000–50,000 reads and 100–160 Mb of mapped bases. The direct sequencing method described here has generated up to 3000 reads per SMRT cell and therefore its utility is limited to small genomes. However, this approach enables one to acquire sequence data from comparatively low amounts of DNA, even less than 1 ng of input, and within eight hours from receiving the sample. There is a slight time saving, compared with the 12 h required for standard library prep. This is not the main advantage, though it does now offer a route from sample to sequence within an average working day. This protocol may be of benefit to the direct sequencing of plasmids, single-standed or double-stranded viruses, mitochondrial DNA, and microbial pathogens in a clinical setting.
文献链接：Direct sequencing of small genomes on the Pacific Biosciences RS without library preparation
