Comparison of Multiple Displacement Amplification (MDA) and Multiple Annealing and Looping-Based Amplification Cycles (MALBAC) in Single-Cell Sequencing

Single-cell sequencing promotes our understanding of the heterogeneity of cellular populations, including the haplotypes and genomic variability among different generation of cells. Whole-genome amplification is crucial to generate sufficient DNA fragments for single-cell sequencing projects. Using sequencing data from single sperms, we quantitatively compare two prevailing amplification methods that extensively applied in single-cell sequencing, multiple displacement amplification (MDA) and multiple annealing and looping-based amplification cycles (MALBAC). Our results show that MALBAC, as a combination of modified MDA and tweaked PCR, has a higher level of uniformity, specificity and reproducibility.     

Comparative analysis of single-cell genome sequencing techniques toward the characterization of germline and somatic genomes in ciliated protists

Ciliated protists contain both germline micronucleus (MIC) and somatic macronucleus (MAC) in a single cytoplasm. Programmed genome rearrangements occur in ciliates during sexual processes, and the extent of rearrangements varies dramatically among species, which lead to significant differences in genomic architectures. However, genomic sequences remain largely unknown for most ciliates due to the difficulty in culturing and in separating the germline from the somatic genome in a single cell. Single-cell whole genome amplification (WGA) has emerged as a powerful technology to characterize the genomic heterogeneity at the single-cell level. In this study, we compared two single-cell WGA, multiple displacement amplification (MDA) and multiple annealing and looping-based amplification cycles (MALBAC) in characterizing the germline and somatic genomes in ciliates with different genomic architectures. Our results showed that: 1) MALBAC exhibits strong amplification bias towards MAC genome while MDA shows bias towards MIC genome of ciliates with extensively fragmented MAC genome; 2) both MDA and MALBAC could amplify MAC genome more efficiently in ciliates with moderately fragmented MAC genome. Moreover, we found that more sample replicates could help to obtain more genomic data. Our work provides a reference for selecting the appropriate method to characterize germline and somatic genomes of ciliates.     

不同单细胞全基因组扩增方法的比较及MALBAC在辅助生殖中的应用

单细胞全基因组扩增(whole genome amplification, WGA)是指在单细胞水平对全基因组进行扩增的新技术,其原理是将分离的单个细胞的微量全基因组DNA进行扩增,获得高覆盖率的完整的基因组后进行高通量测序,用于揭示细胞异质性。目前,WGA方法主要包括引物延伸预扩增(primer extension preamplification PCR, PEP-PCR)、简并寡核苷酸引物PCR (degenerate oligonucleotide primed PCR, DOP-PCR)、多重置换扩增(multiple displacement amplification, MDA)、多次退火环状循环扩增(multiple annealing and looping-based amplification cycles, MALBAC)等。本文对不同的单细胞WGA方法的原理及应用情况分别进行了阐述,并对其扩增效率进行评价和比较,包括基因组覆盖度、均一性、重现性、SNV (single-nucleotide variants)和CNV (copy number variants)检测力等。综合对比不同单细胞WGA方法后发现,MALBAC的扩增均一性最高、等位基因脱扣率最低、重现性最好,且对于CNV和SNV的检测效果最好。本文还阐述了MALBAC技术在人类单精子减数重组、非整倍体分析以及人类卵细胞基因组研究中的应用。     

A Quantitative Comparison of Single-Cell Whole Genome Amplification Methods

Single-cell sequencing is emerging as an important tool for studies of genomic heterogeneity. Whole genome amplification (WGA) is a key step in single-cell sequencing workflows and a multitude of methods have been introduced. Here, we compare three state-of-the-art methods on both bulk and single-cell samples of E. coli DNA: Multiple Displacement Amplification (MDA), Multiple Annealing and Looping Based Amplification Cycles (MALBAC), and the PicoPLEX single-cell WGA kit (NEB-WGA). We considered the effects of reaction gain on coverage uniformity, error rates and the level of background contamination. We compared the suitability of the different WGA methods for the detection of copy-number variations, for the detection of single-nucleotide polymorphisms and for de-novo genome assembly. No single method performed best across all criteria and significant differences in characteristics were observed; the choice of which amplifier to use will depend strongly on the details of the type of question being asked in any given experiment.     

The Performance of Whole Genome Amplification Methods and Next-Generation Sequencing for Pre-Implantation Genetic Diagnosis of Chromosomal Abnormalities

Reliable and accurate pre-implantation genetic diagnosis(PGD) of patient’s embryos by next-generation sequencing(NGS) is dependent on efficient whole genome amplification(WGA) of a representative biopsy sample. However, the performance of the current state of the art WGA methods has not been evaluated for sequencing. Using low template DNA(15 pg) and single cells, we showed that the two PCR-based WGA systems Sure Plex and MALBAC are superior to the REPLI-g WGA multiple displacement amplification(MDA) system in terms of consistent and reproducible genome coverage and sequence bias across the 24 chromosomes, allowing better normalization of test to reference sequencing data. When copy number variation sequencing(CNV-Seq) was applied to single cell WGA products derived by either Sure Plex or MALBAC amplification, we showed that known disease CNVs in the range of 3e15 Mb could be reliably and accurately detected at the correct genomic positions. These findings indicate that our CNV-Seq pipeline incorporating either Sure Plex or MALBAC as the key initial WGA step is a powerful methodology for clinical PGD to identify euploid embryos in a patient’s cohort for uterine transplantation.     

组织中少量细胞的微卫星长度精细分析

微卫星是基因组上的特殊短重复序列,微卫星不稳定的程度与一些肿瘤的分型、治疗和预后相关。目前,微卫星长度变化的检测往往是基于大量细胞,检测灵敏度低。本文整合激光显微切割技术,基于多次退火环状循环扩增（multiple annealing and looping-based amplification cycles, MALBAC）的单细胞全基因组放大技术和毛细管电泳长度测量方法,经过关键技术点的改进,建立了一套针对组织中少量细胞的多微卫星位点检测方法。研究结果表明,基于技术改进,HE染色的组织细胞经激光显微切割分选后,可成功用MALBAC技术进行全基因组放大,并在多微卫星位点的检测上,获得了高度的准确性和重复性。利用所建立的方法,发现肠型胃癌早期病变组织-肠上皮化生（intestinal metaplasia, IM）的单个腺体内部出现多种长度变化的微卫星改变,说明该癌前病变组织中DNA错配修复系统已经出现问题。本方法所获得的全基因组放大产物,也可以用于外显子组测序和全基因组测序。另外,该方法适用于任何组织的少量细胞,甚至单细胞的多微卫星位点检测,以及全基因组的研究,为精细研究少量病变细胞的基因组特征以及组织异质性提供了有效的方法。     

Genomic sequencing of single microbial cells from environmental samples

Recently developed techniques allow genomic DNA sequencing from single microbial cells [Lasken RS: Single-cell genomic sequencing using multiple displacement amplification. Curr Opin Microbiol 2007, 10:510-516]. Here, we focus on research strategies for putting these methods into practice in the laboratory setting. An immediate consequence of single-cell sequencing is that it provides an alternative to culturing organisms as a prerequisite for genomic sequencing. The microgram amounts of DNA required as template are amplified from a single bacterium by a method called multiple displacement amplification (MDA) avoiding the need to grow cells. The ability to sequence DNA from individual cells will likely have an immense impact on microbiology considering the vast numbers of novel organisms, which have been inaccessible unless culture-independent methods could be used. However, special approaches have been necessary to work with amplified DNA. MDA may not recover the entire genome from the single copy present in most bacteria. Also, some sequence rearrangements can occur during the DNA amplification reaction. Over the past two years many research groups have begun to use MDA, and some practical approaches to single-cell sequencing have been developed. We review the consensus that is emerging on optimum methods, reliability of amplified template, and the proper interpretation of 'composite' genomes which result from the necessity of combining data from several single-cell MDA reactions in order to complete the assembly. Preferred laboratory methods are considered on the basis of experience at several large sequencing centers where >70% of genomes are now often recovered from single cells. Methods are reviewed for preparation of bacterial fractions from environmental samples, single-cell isolation, DNA amplification by MDA, and DNA sequencing.     

肺癌循环肿瘤细胞的单细胞EGFR基因突变检测

循环肿瘤细胞（Circulating tumor cells,CTCs）是从肿瘤原发病灶脱落并侵入外周血循环的肿瘤细胞。由于CTCs存在较大的异质性,其与癌症发展转移密切相关,但目前尚缺乏有效的CTCs单细胞异质性检测方法。鉴于此,本文发展了在单细胞层面对CTCs进行基因突变的检测方法并用于单个肺癌CTC的EGFR（Epidermal growth factor receptor）基因突变检测。首先用集成式微流控系统完成血液中稀有CTCs的捕获,接着将CTCs释放入含有多个微孔的微阵列芯片中,得到含有单个CTC的微孔,通过显微操作将单个CTC转入PCR管内完成单细胞基因组的放大,并进行单细胞的EGFR基因突变检测。以非小细胞肺癌细胞系A549、NCI-H1650和NCI-H1975为样本,通过芯片与毛细管修饰、引物扩增条件（复性温度、循环次数）的优化,结果显示在复性温度59℃、30个循环次数的条件下,引物扩增效果最优。利用该方法成功地对非小细胞肺癌（Non-small cell lung cancer, NSCLC）患者的血液样本进行了测试。从患者2 mL血液中获取5个CTCs,分别对其EGFR基因的第18、19、20、21外显子进行测序,发现该患者CTCs均为EGFR野生型。研究结果证明此检测方法可以灵敏地用于单个CTC基因突变的检测,在临床研究上具有重要的指导意义。     

Monodisperse Picoliter Droplets for Low-Bias and Contamination-Free Reactions in Single-Cell Whole Genome Amplification

Whole genome amplification (WGA) is essential for obtaining genome sequences from single bacterial cells because the quantity of template DNA contained in a single cell is very low. Multiple displacement amplification (MDA), using Phi29 DNA polymerase and random primers, is the most widely used method for single-cell WGA. However, single-cell MDA usually results in uneven genome coverage because of amplification bias, background amplification of contaminating DNA, and formation of chimeras by linking of non-contiguous chromosomal regions. Here, we present a novel MDA method, termed droplet MDA, that minimizes amplification bias and amplification of contaminants by using picoliter-sized droplets for compartmentalized WGA reactions. Extracted DNA fragments from a lysed cell in MDA mixture are divided into 10⁵ droplets (67 pL) within minutes via flow through simple microfluidic channels. Compartmentalized genome fragments can be individually amplified in these droplets without the risk of encounter with reagent-borne or environmental contaminants. Following quality assessment of WGA products from single Escherichia coli cells, we showed that droplet MDA minimized unexpected amplification and improved the percentage of genome recovery from 59% to 89%. Our results demonstrate that microfluidic-generated droplets show potential as an efficient tool for effective amplification of low-input DNA for single-cell genomics and greatly reduce the cost and labor investment required for determination of nearly complete genome sequences of uncultured bacteria from environmental samples.     

Single-cell isolation from cell suspensions and whole genome amplification from single cells to provide templates for CGH analysis

A comprehensive genomic analysis of single cells is instrumental for numerous applications in tumor genetics, clinical diagnostics and forensic analyses. Here, we provide a protocol for single-cell isolation and whole genome amplification, which includes the following stages: preparation of single-cell suspensions from blood or bone marrow samples and cancer cell lines; their characterization on the basis of morphology, interphase fluorescent in situ hybridization pattern and antibody staining; isolation of single cells by either laser microdissection or micromanipulation; and unbiased amplification of single-cell genomes by either linker-adaptor PCR or GenomePlex library technology. This protocol provides a suitable template to screen for chromosomal copy number changes by conventional comparative genomic hybridization (CGH) or array CGH. Expected results include the generation of several micrograms of DNA from single cells, which can be used for CGH or other analyses, such as sequencing. Using linker-adaptor PCR or GenomePlex library technology, the protocol takes 72 or 30 h, respectively.     

High resolution array-CGH analysis of single cells

Heterogeneity in the genome copy number of tissues is of particular importance in solid tumor biology. Furthermore, many clinical applications such as pre-implantation and non-invasive prenatal diagnosis would benefit from the ability to characterize individual single cells. As the amount of DNA from single cells is so small, several PCR protocols have been developed in an attempt to achieve unbiased amplification. Many of these approaches are suitable for subsequent cytogenetic analyses using conventional methodologies such as comparative genomic hybridization (CGH) to metaphase spreads. However, attempts to harness array-CGH for single-cell analysis to provide improved resolution have been disappointing. Here we describe a strategy that combines single-cell amplification using GenomePlex library technology (GenomePlex^® Single Cell Whole Genome Amplification Kit, Sigma-Aldrich, UK) and detailed analysis of genomic copy number changes by high-resolution array-CGH. We show that single copy changes as small as 8.3 Mb in single cells are detected reliably with single cells derived from various tumor cell lines as well as patients presenting with trisomy 21 and Prader–Willi syndrome. Our results demonstrate the potential of this technology for studies of tumor biology and for clinical diagnostics.     

微生物单细胞基因组技术及其在环境微生物研究中的应用

单细胞及单细胞基因组学研究是近年生命科学研究的热点之一,微生物单细胞基因组学研究是继微生物元基因组学(又称宏基因组学,Metagenomics)之后新发展起来的,可有效获取环境中大量无法培养的微生物遗传信息的技术。微生物单细胞基因组技术包括单细胞获取、全基因组扩增、全基因组测序以及数据分析等步骤,目前该技术在环境微生物研究中的应用主要集中于探索未被元基因组技术或其它常规技术探测到的新型功能基因,或是对环境中物种丰度极小的未培养微生物的发现,以及对微生物细胞生命进化过程的研究等。本文对微生物单细胞基因组技术中单细胞获取和全基因组扩增所涉及到的不同方法以及应用此技术对环境微生物取得的主要研究进展进行综述。     

单染色体靶向富集方法的建立

靶向富集技术具有良好的均一性、特异性和灵敏性,只需较低的测序覆盖度即可满足后续的分析。该技术被广泛应用于特定位点重测序和染色体构象等研究。为了靶向富集测序文库中特定的染色体DNA片段,本研究以21号染色体为例,采用人胚肝二倍体细胞CCC-HEL-1建立了一种单染色体靶向富集方法。具体步骤如下：（1）首先将人正常核型二倍体细胞同步化到G2/M 期,制备染色体悬液;然后流式分选出21号单染色体。（2）采用多重退火环形扩增技术（MALBAC）对21号染色体DNA进行扩增;用扩增的DNA产物构建文库。（3）利用体外转录合成、生物素标记的RNA探针与文库进行液相杂交,捕获并验证捕获效率。荧光定量PCR检测结果证明,特定的21号染色体呈上百倍的富集,而靶标文库则富集了近60 倍。结果提示,特异单染色体靶向富集方法成功建立。该法不仅可实现任一单染色体的靶向富集,用于单条染色体功能组学研究,而且还可能用于查找特定疾病相关的染色体异常（如DNA突变、异位、重复及倒置等）,具有一定的实用意义。     

单细胞转录组高通量测序分析新进展

细胞异质性是生物组织的普遍特征。常规转录组测序(RNA-Seq)技术需要上万个细胞,所测结果实际上是一群细胞基因表达的平均值,所以难以鉴别细胞之间基因表达的异质性。单细胞RNA-Seq技术的分辨率精确至单个细胞,为辨别异质性群体中各种细胞类型的转录组特征提供了有力的工具。近年来单细胞RNA-Seq技术发展迅速,在方法学上包括cDNA扩增方法的多样化、对灵敏度和技术噪声的定量分析、浅覆盖高通量单细胞RNA-Seq方法和原位RNA-Seq技术等;在技术应用方面应用范围从早期胚胎发育扩大到组织器官发育、免疫和肿瘤等多个领域。文章对单细胞RNA-Seq在方法学和技术应用两方面的研究进展进行了详细阐述。     

Detection of single-copy functional genes in prokaryotic cells by two-pass TSA-FISH with polynucleotide probes

In situ detection of functional genes with single-cell resolution is currently of interest to microbiologists. Here, we developed a two-pass tyramide signal amplification (TSA)-fluorescence in situ hybridization (FISH) protocol with PCR-derived polynucleotide probes for the detection of single-copy genes in prokaryotic cells. The mcrA gene and the apsA gene in methanogens and sulfate-reducing bacteria, respectively, were targeted. The protocol showed bright fluorescence with a good signal-to-noise ratio and achieved a high efficiency of detection (> 98%). The discrimination threshold was approximately 82-89% sequence identity. Microorganisms possessing the mcrA or apsA gene in anaerobic sludge samples were successfully detected by two-pass TSA-FISH with polynucleotide probes. The developed protocol is useful for identifying single microbial cells based on functional gene sequences.     

Whole-genome multiple displacement amplification from single cells

Multiple displacement amplification (MDA) is a recently described method of whole-genome amplification (WGA) that has proven efficient in the amplification of small amounts of DNA, including DNA from single cells. Compared with PCR-based WGA methods, MDA generates DNA with a higher molecular weight and shows better genome coverage. This protocol was developed for preimplantation genetic diagnosis, and details a method for performing single-cell MDA using the phi29 DNA polymerase. It can also be useful for the amplification of other minute quantities of DNA, such as from forensic material or microdissected tissue. The protocol includes the collection and lysis of single cells, and all materials and steps involved in the MDA reaction. The whole procedure takes 3 h and generates 1-2 microg of DNA from a single cell, which is suitable for multiple downstream applications, such as sequencing, short tandem repeat analysis or array comparative genomic hybridization.