Vector cleaner:一种新的去除测序目的基因载体序列的方法

Sanger测序法测序目的基因常包含有目的基因和载体序列,为了快速去除测序目的基因载体序列,提出了一种新的目的基因载体序列去除方法并开发了程序Vector cleaner。首先利用该程序批量读取引物信息和目的基因测序序列;其次,程序在所读取的引物序列上建立引物半长的滑动窗口来产生种子,通过计数种子与测序序列的匹配次数,定位引物位置和删除引物两侧的载体序列;最后,程序通过比较上游引物序列和其反向互补序列分别与测序序列匹配种子数,判断和转换正义链。使用Vector cleaner对12条GhVIN1基因测序序列进行去载体测试,并与Seqclean和SeqMan软件相比较。结果表明:Vector cleaner能有效去除棉花GhVIN1基因测序载体序列,识别并翻译反义链序列。与Seqclean和SeqMan软件相比较,Vector cleaner正确率高,敏感性强。Vector cleaner、SeqMan和Seqclean所测试序列的总序列数正确率分别为100%、100%和91.6%,总碱基正确率分别为99.90%、99.00%和94.33%。与同类软件比较,Vector cleaner更适合实验人员批量去除测序目的基因载体序列,具有准确率高、敏感性强、自动翻译反义链的特点。

39例足癣复发后病原菌核糖体基因序列变化

目的:检测红色毛癣菌足癣复发后致病菌株基因型的变化。方法:对39例红色毛癣菌足癣复发后进行红色毛癣菌核糖体保守区(部分5.8s和ITS-2)和非转录间隔区(NTS)内的两个重复亚单位(TRS-1,TRS-2)进行PCR扩增,基因序列测定。结果:39株红色毛癣菌复发前后和标准株保守区基因序列测定比较,同源性均达99%,均为红色毛癣菌;(NTS)内的两个重复亚单位(TRS-1,TRS-2)基因序列测定比较,其中3株菌TRS-1区存在个别碱基突变,3株TRS-1区存在重复序列拷贝数变异,1株菌TRS-2区存在个别碱基突变。结论:复发后的红色毛癣菌基因型基本稳定,说明足癣复发大部分与原浅表真菌复燃有关。

婴儿双歧杆菌BI-G201的分离与鉴定

从由健康母乳喂养的婴儿肠道内容物中取得的5份样品中,利用选择性培养基和需氧条件分离筛选得到6株双歧杆菌。经菌落形态特征、生理生化特性及糖发酵试验,6株乳酸菌的鉴定结果为:2株长双歧杆菌(Bifidobacterium longum),2株乳双歧杆菌(Bifidobacterium lactis),2株婴儿双歧杆菌(Bifidobacterium infantis)。再经过16S r RNA基因序列测序分析对比,确认分别为长双歧杆菌、乳双歧杆菌和婴儿双歧杆菌BI-G201。

Detection and Sequence Analysis of Escherichia coli Virulence Genes

[Objective] This study aimed to explore the pathogenic mechanism of E., cherichia coll. [Method] An E. coil strain isolated from raccoon dogs in vivo was studied, which had been identified, PCR was used to detect the gene of irp2 （301 bp） and fyuA （953 bp） related to E. coil virulence and PCR products were s, quenced. [Result] The genes of irp2 and fyuA were successfully amplified in boi strains isolated from raccoon dogs. Compared with the GenBank, the identity of tTr irp2 gene sequence and the fyuA gene sequence of the strain reached 98.5% 99.2% and 98.9%-100% respectively. Compared with each other, the identity of tt- two gene sequences of irp2 was 99.3%, and that between the two fyuA gene se quences was 98.9%. [Conclusion] This study provided scientific experimental data fi E. coil pathogenicity, prevention, diagnosis and treatment.

High-throughput sequencing exclusively identified a novel Torque teno virus genotype in serum of a patient with fatal fever

Torque teno virus(TTV) has been found to be prevalent world-wide in healthy populations and in patients with various diseases, but its etiological role has not yet been determined. Using high-throughput unbiased sequencing to screen for viruses in the serum of a patient with persistent high fever who died of suspected viral infection and prolonged weakness, we identified the complete genome sequence of a TTV(isolate Hebei-1). The genome of TTV-Hebei-1 is 3649 bp in length, encoding four putative open reading frames, and it has a G+C content of 49%. Genomic comparison and a BLASTN search revealed that the assembled genome of TTV-Hebei-1 represented a novel isolate, with a genome sequence that was highly heterologous to the sequences of other reported TTV strains. A phylogenetic tree constructed using the complete genome sequence showed that TTV-Hebei-1 and an uncharacterized Taiwan Residents strain, TW53A37, constitute a new TTV genotype. The patient was strongly suspected of carrying a viral infection and died eventually without any other possible causes being apparent. No virus other than the novel TTV was identified in his serum sample. Although a direct causal link between the novel TTV genotype infection and the patient's disease could not be confirmed, the findings suggest that surveillance of this novel TTV genotype is necessary and that its role in disease deserves to be explored.

Genetic Characteristics of 2009 Pandemic H1N1 Influenza A Viruses Isolated from China's Mainland

A total of 100 H1N1 flu real-time-PCR positive throat swabs collected from fever patients in Zhejiang, Hubei and Guangdong between June and November 2009, were provided by local CDC laboratories. After MDCK cell culture, 57 Influenza A Pandemic （H1N1） viruses were isolated and submitted for whole genome sequencing. A total of 39 HA sequences, 52 NA sequences, 36 PB2 sequences, 31 PB1 sequences, 40 PA sequences, 48 NP sequences, 51 MP sequences and 36 NS sequences were obtained, including 20 whole genome sequences. Sequence comparison revealed they shared a high degree of homology （96%-99%） with known epidemic strains （A/Califomia/04/2009（H1N1）. Phylogenetic analysis showed that although the sequences were highly conserved, they clustered into a small number of groups with only a few distinct strains. Site analysis revealed three substitutions at loop 220 （221-228） of the HA receptor binding site in the 39 HA sequences： A/Hubei/86/2009 PKVRDQEG→PKVRDQEA, A/Zhejiang/08/2009 PKVRDQEG→PKVRDQER, A/Hubei/75/2009 PKVRDQEG→PKVRDQGG, the A/Hubei/75/2009 was isolated from an acute case, while the other two were from patients with mild symptoms. Other key sites such as 119, 274, 292 and 294 amino acids of NA protein,627 of PB2 protein were conserved. Meanwhile, all the M2 protein sequences possessed the Ser32Asn mutation, suggesting that these viruses were resistant to adamantanes. Comparison of these sequences with other H1N1 viruses collected from the NCBI database provides insight into H1N1 transmission and circulation patterns.

Preliminary Studies on Identification of Lycium Linn.Germplasm Resources by nrDNA ITS Sequencing

[Objective] The study aimed to identify woltberry （Lycium Linn.） germplasm resources at molecular level by analyzing the nrDNA ITS sequence. [Method] Genomic DNA from woltberry leaves extracted by modified CTAB method were regarded as templates for PCR amplification by specific primer, clone and sequencing. [Result] The nrDNA ITS sequences were obtained and then differentiated among three tested materials. [Conclusion] PCR amplification and sequencing on nrDNA ITS is a feasible approach to identify different woltberry germplasm resources.

Antigen Gene Cloning and Expression of HIV-1 Toward AIDS Vaccine Design Ⅱ.Subtype Classification and Quasi-species Identification of HIV-1

Objective: To analyze subtypes and quasi-species of isolatedviruses from HIV-1 infected individuals among the populationof Guangdong Province, for understanding the molecularepidemiological dynamics of local HIV-1 isolates, thus laying afoundation for designing a candidate AIDS vaccine. Methods: By hetero-duplex mobility assay (HMA) andsingle strand conformation poly-morphism (SSCP) analysison amplicons from single-primed polymerase chain reaction(SP-PCR), subtypes and quasi-species of tested HIV-1 isolateswere elucidated, and amplicons were sequenced forconfirmation. Results: Specific amplicons from different subtypes andquasi-species of HIV-1 could be discernible by HMA andSSCP analysis. Conclusion: HIV-1 isolates from different patients might beeither a different subtype or an identical subtype, and HIV-1isolates from an individual were present in a population ofquasi-species.

Detecting Factor Ⅺ Deficiency in Holstein Cattle Using PCR Analysis

[Objective] This study established a method to detect Factor Ⅺ by polymerase chain reaction analysis.[Method]A pair of primers was designed and synthesized according to sequences of FⅪ gene in Holstein calves,published in Genbank. Polymerase chain reaction was used to analyze FⅪ deficiency of 576 Holstein calves in Henan,and the result was verified by DNA sequencing. [Result] We detect 576 cows,which include two carriers and one F Ⅺ deficiency,and the result was consistent with the DNA sequencing. The frequency of the FⅪ mutant allele was 0.3%,the carrier was 0.3%,the prevalence was 0.2%.[Conclusion]A method detecting FⅪ by polymerase chain reaction analysis was established. This method is not only simple and convenient,but also has a high accuracy and low cost,which is more suitable for large-scale FⅪ investigation.

41例5α-还原酶2型缺陷症患儿临床特点及SRD5A2基因突变分析

目的探讨41例5α-还原酶2型缺陷症患儿临床表型特点及SRD5A2基因突变类型。方法收集分析患儿临床资料,包括体格检查、病史、实验室检查、B超等;并提取患儿外周血基因组DNA,采用PCR扩增产物直接测序法或靶目的基因序列捕获二代测序法检测SRD5A2基因突变类型。结果41例患儿年龄从4个月至11岁不等,汉族。所有患儿染色体核型为46,XY,SRY基因检测结果阳性。病例临床主要表现为小阴茎和尿道下裂,其中单纯表现为小阴茎的有26例,占总病例人数63%;其余15例为尿道下裂合并小阴茎,占总病例人数37%。在39例患儿中检测到双等位基因突变,2例患儿只检测到1个等位基因突变。在41例患儿中,共检测出16种基因突变类型,其中c.725A>G(p.Tyr242Cys)、c.694C>G(p.His232Asp)和c.548-9T>G这3种突变类型为未报道过的新突变。在16种突变类型中,以c.680G>A(p.Arg227Gln)为主,占所有突变类型60%(48/80)。结论5α-还原酶2型缺陷症患儿临床表现以小阴茎或尿道下裂合并小阴茎为主;c.680G>A(p.Arg227Gln)突变是中国5α-还原酶2型缺陷症患儿SRD5A2基因的热点突变类型。

从真菌全基因组中筛选丛赤壳科的DNA条形码

将粗糙脉孢菌(Neurospora crassa)的注释基因分别与30种丝状子囊菌基因组比较,根据E值大小预测同源序列,从中选择Hsp90,AAC,CDC48和EF3作为候选基因,以丛赤壳科(Nectriaceae)13个属34个概念清晰的种为材料,对215个序列片段采用不同方法进行分析,筛选适合于该科的DNA条形码.将种内与种间序列差异以及序列获得的难易程度作为评价指标.结果表明,Hsp90和AAC基因虽然具有较高的PCR扩增与测序成功率,但种内、种间距离频率分布存在重叠,可能导致部分种的鉴定错误;CDC48基因可以恰当地区分种内与种间差异,但扩增与测序成功率相对较低;EF3基因不存在种内、种间遗传距离重叠,序列容易获得,适合作为该科真菌的DNA条形码.

乙型脑炎病毒减毒活疫苗株(SA14-14-2)神经毒力减弱的分子基础

目的将乙型脑炎病毒减毒活疫苗株SA14-14-2经乳鼠脑内回传毒力回复后,对回复后病毒测其神经毒力和全长基因序列并与减毒株SA14-14-2和母株SA14进行核苷酸和氨基酸序列比较。方法将回传一代SA14-14-2 M1和SA14-14-2 M3病毒在幼鼠脑内测神经毒力和全基因组序列。运用RT-PCR方法,分段扩增出覆盖基因组全长的基因片段,PCR产物纯化后直接测序,针对5′和3′端的片段TA克隆后进行测序。经核苷酸序列测定后,利用DNAStar软件,进行序列比对分析。结果实验结果表明SA14-14-2 M1病毒毒力未见增强,SA14-14-2 M3病毒的脑内致病力明显增强,但皮下接种仍无致病性。通过序列比对分析发现,M1和M3病毒均在非结构区的NS3-343、NS4A-57位发生氨基酸回复突变,但只有M3病毒在E区毒力相关的E107、E138位点发生回复突变。结论在SA14-14-2减毒株病毒结构基因中E蛋白区E107、E138位氨基酸是与毒力密切相关的重要位点,应该是神经毒力减弱的重要分子基础,非结构区的个别位点对神经毒力减弱也可能起一定的协同作用。

Assembly and features of secondary metabolite biosynthetic gene clusters in Streptomyces ansochromogenes

A draft genome sequence of Streptomyces ansochromogenes 7100 was generated using 454 sequencing technology. In combination with local BLAST searches and gap filling techniques, a comprehensive antiSMASH-based method was adopted to assemble the secondary metabolite biosynthetic gene clusters in the draft genome of S. ansochromogenes. A total of at least 35 putative gene clusters were identified and assembled. Transcriptional analysis showed that 20 of the 35 gene clusters were expressed in either or all of the three different media tested, whereas the other 15 gene clusters were silent in all three different media. This study provides a comprehensive method to identify and assemble secondary metabolite biosynthetic gene clusters in draft genomes of Streptomyces, and will significantly promote functional studies of these secondary metabolite biosynthetic gene clusters.

High-throughput sequencing-based genome-wide identification of micro RNAs expressed in developing cotton seeds

Micro RNAs(mi RNAs) have been shown to play critical regulatory roles in gene expression in cotton. Although a large number of mi RNAs have been identified in cotton fibers, the functions of mi RNAs in seed development remain unexplored. In this study, a small RNA library was constructed from cotton seeds sampled at 15 days post-anthesis(DPA) and was subjected to high-throughput sequencing. A total of 95 known mi RNAs were detected to be expressed in cotton seeds. The expression pattern of these identified mi RNAs was profiled and 48 known mi RNAs were differentially expressed between cotton seeds and fibers at 15 DPA. In addition, 23 novel mi RNA candidates were identified in 15-DPA seeds. Putative targets for 21 novel and 87 known mi RNAs were successfully predicted and 900 expressed sequence tag(EST) sequences were proposed to be candidate target genes, which are involved in various metabolic and biological processes, suggesting a complex regulatory network in developing cotton seeds. Furthermore, mi RNA-mediated cleavage of three important transcripts in vivo was validated by RLM-5′ RACE. This study is the first to show the regulatory network of mi RNAs that are involved in developing cotton seeds and provides a foundation for future studies on the specific functions of these mi RNAs in seed development.

Genomes and evolutionary genomics of animals

Alongside recent advances and booming applications of DNA sequencing technologies, a great number of complete genome sequences for animal species are available to researchers. Hundreds of animals have been involved in whole genome se- quencing, and at least 87 non-human animal species＇ complete or draft genome sequences have been published since 1998. Based on these technological advances and the subsequent accumulation of large quantity of genomic data, evolutionary genomics has become one of the most rapidly advancing disciplines in biology. Scientists now can perform a number of comparative and evolu- tionary genomic studies for animals, to identify conserved genes or other functional elements among species, genomic elements that confer animals their own specific characteristics and new phenotypes for adaptation. This review deals with the current ge- nomic and evolutionary research on non-human animals, and displays a comprehensive landscape of genomes and the evolution- ary genomics of non-human animals. It is very helpful to a better understanding of the biology and evolution of the myriad forms within the animal kingdom [Current Zoology 59 （1）： 87-98, 2013].

Next generation sequencing under de novo genome assembly

The next generation sequencing （NGS） is an important process which assures inexpen- sive organization of vast size of raw sequence dataset over any traditional sequencing systems or methods. Various aspects of NGS such as template preparation, sequencing imaging and genome alignment and assembly outline the genome sequencing and align- ment. Consequently, de Bruijn graph （dBG） is an important mathematical tool that graphically analyzes how the orientations are constructed in groups of nucleotides. Basi- cally, dBG describes the formation of the genome segments in circular iterative fashions. Some pivotal dBG-based de novo algorithms and software packages such as T-IDBA, Oases, IDBA-tran, Euler, Velvet, ABYSS, AllPaths, SOAPde novo and SOAPde novo2 are illustrated in this paper. Consequently, overlap layout consensus （OLC） graph-based algorithms also play vital role in NGS assembly. Some important OLC-based algorithms such as MIRA3, CABOG, Newbler, Edena, Mosaik and SHORTY are portrayed in this paper. It has been experimented that greedy graph-based algorithms and software pack- ages are also vital for proper genome dataset assembly. A few algorithms named SSAKE, SHARCGS and VCAKE help to perform proper genome sequencing.