新一代测序技术在植物转录组研究中的应用

随着DNA测序技术的发展,新一代测序技术以其高通量、低成本的特点,成为越来越多的生物学研究者在开展工作时的首选。在所有的新一代测序技术中,454测序系统是最早实现商业化且发展相对成熟的一种,目前被广泛的应用于各个领域的生物学研究中。文章以454测序系统为例,综述了新一代测序系统的原理、优缺点,及其在植物转录组研究中的应用,并对其在植物研究领域中可能的发展应用方向进行了展望。     

454测序法在环境微生物生态研究中的应用

传统的Sanger测序技术虽已成熟,但其速度和成本的限制满足不了大规模测序的要求。第二代高通量测序技术结合了乳胶微粒和皮升级反应的454焦磷酸测序法,作为一种高通量测序技术,具有分析结果准确、高速、高灵敏度和高自动化的特点。对454测序法的技术原理和操作步骤进行了介绍,对近年来运用该方法在环境微生物生态研究领域的进展进行了综述。     

454测序技术在微生物生态学研究中的应用

以Sanger法(双脱氧核苷酸末端终止法)为代表的第1代测序技术由于其成本高、速度慢、通量低等不足,满足不了大规模测序的要求.进入21世纪后,以Roche 454为代表的第2代测序技术诞生了,454测序法作为一种高通量的测序方法,近年来已被广泛应用于微生物生态学研究中.介绍了该测序技术的原理和操作步骤,结合本实验室的研...     

Discovering genetic polymorphisms in next-generation sequencing data

The ongoing revolution in DNA sequencing technology now enables the reading of thousands of millions of nucleotide bases in a single instrument run. However, this data quantity is often compromised by poor confidence in the read quality. The identification of genetic polymorphisms from this data is therefore problematic and, combined with the vast quantity of data, poses a major bioinformatics challenge. However, once these difficulties have been addressed, next-generation sequencing will offer a means to identify and characterize the wealth of genetic polymorphisms underlying the vast phenotypic variation in biological systems. We describe the recent advances in next-generation sequencing technology, together with preliminary approaches that can be applied for single nucleotide polymorphism discovery in plant species.     

Commercial high-throughput sequencing and its applications in DNA analysis

DNA sequences can be used for the analysis of genetic variation and gene function. The high-throughput sequencing techniques that have been developed over the past three years can read as many as one billion bases per run, and are far less expensive than the traditional Sanger sequencing method. Therefore, the high-throughput sequencing has been applied extensively to genomic analyses, such as screening for mutations, construction of genomic methylation maps, and the study of DNA-protein interactions. Although they have only been available for a short period, high-throughput sequencing techniques are profoundly affecting many of the life sciences, and are opening out new potential avenues of research. With the highly-developed commercial high-throughput sequencing platforms, each laboratory has the opportunity to explore this research field. Therefore, in this paper, we have focused on commercially-popular high-throughput sequencing techniques and the ways in which they have been applied over the past three years.     

Targeted single nucleotide polymorphism (SNP) discovery in a highly polyploid plant species using 454 sequencing

Discovering single nucleotide polymorphisms (SNPs) in specific genes in a heterozygous polyploid plant species, such as sugarcane, is challenging because of the presence of a large number of homologues. To discover SNPs for mapping genes of interest, 454 sequencing of 307 polymerase chain reaction (PCR) amplicons (> 59 kb of sequence) was undertaken. One region of a four-gasket sequencing run, on a 454 Genome Sequencer FLX, was used for pooled PCR products amplified from each parent of a quantitative trait locus (QTL) mapping population (IJ76-514 × Q165). The sequencing yielded 96 755 (IJ76-514) and 86 241 (Q165) sequences with perfect matches to a PCR primer used in amplification, with an average sequence depth of approximately 300 and an average read length of 220 bases. Further analysis was carried out on amplicons whose sequences clustered into a single contig using an identity of 80% with the program cap 3. In the more polymorphic sugarcane parent (Q165), 94% of amplicons (227/242) had evidence of a reliable SNP – an average of one every 35 bases. Significantly fewer SNPs were found in the pure Saccharum officinarum parent – with one SNP every 58 bases and SNPs in 86% (213/247) of amplicons. Using automatic SNP detection, 1632 SNPs were detected in Q165 sequences and 1013 in IJ76-514. From 225 candidate SNP sites tested, 209 (93%) were validated as polymorphic using the Sequenom MassARRAY system. Amplicon re-sequencing using the 454 system enables cost-effective SNP discovery that can be targeted to genes of interest and is able to perform in the highly challenging area of polyploid genomes.     

Patterns of fungal communities among and within decaying logs, revealed by 454 sequencing

Owing to previous methodological limitations, knowledge about the fine-scale distribution of fungal mycelia in decaying logs is limited. We investigated fungal communities in decaying Norway spruce logs at various spatial scales at two environmentally different locations in Sweden. On the basis of 454 pyrosequencing of the ITS2 region of rDNA, 1914 operational taxonomic units (OTUs) were detected in 353 samples. The communities differed significantly among logs, but the physical distance between logs was not found to have a significant effect on whether fungal communities had any resemblance to each other. Within a log, samples that were closer together generally had communities that showed more resemblance to each other than those that were further apart. OTUs characteristic for particular positions on the logs could be identified. In general, these OTUs did not overlap with the most abundant OTUs, and their ecological role was often unknown. Only a few OTUs were detected in the majority of logs, whereas numerous OTUs were rare and present in only one or a few logs. Wood-decaying Basidiomycetes were often represented by higher sequence reads in individual logs than Ascomycete OTUs, suggesting that Basidiomycete mycelia spread out more rapidly when established. OTU richness tended to increase with the decay stage of the sample; however, the known wood decayers were most abundant in less-decomposed samples. The fungi identified in the logs represented different ecological strategies. Our findings differ from previously published sporocarp studies, indicating that the highly abundant fruiting species may respond to environment in different ways than the rest of the fungal community.     

SNP discovery and genotyping using restriction‐site‐associated DNA sequencing in chickens

Single nucleotide polymorphisms (SNPs) are essential to the understanding of population genetic variation and diversity. Here, we performed restriction‐site‐associated DNA sequencing (RAD‐seq) on 72 individuals from 13 Chinese indigenous and three introduced chicken breeds. A total of 620 million reads were obtained using an Illumina Hiseq2000 sequencer. An average of 75 587 SNPs were identified from each individual. Further filtering strictly validated 28 895 SNPs candidates for all populations. When compared with the NCBI dbSNP (chicken_9031), 15 404 SNPs were new discoveries. In this study, RAD‐seq was performed for the first time on chickens, implicating the remarkable effectiveness and potential applications on genetic analysis and breeding technique for whole‐genome selection in chicken and other agricultural animals.     

高通量转录组测序技术及其在鳞翅目昆虫上的应用

转录组是指组织或者细胞在某一特定状态下转录出来的所有RNA的集合。高通量第2代测序技术使转录组学的研究模式发生了巨大的改变,所衍生出的转录组测序迅速成为研究非模式生物的先进技术。转录组测序能够在整体水平上探究细胞内基因表达的种类和数量,揭示在特定条件下机体生理生化发生过程以及其中的分子机理。本文简要阐述了转录组测序技术的基本概念、技术流程与原理,详细介绍了转录组测序在解决鳞翅目昆虫的分类、毒理、发育、与寄主互作以及非编码RNA调控等问题上做出的贡献,并对该技术现存的困难进行了系统的阐述并对其未来的发展趋势作出了简要的预测与剖析。