人类粒细胞抗原系统基因测序方法的建立

目的人类粒细胞特异性抗原在不同人群中表现出高度的多态性,输注后由于个体抗原的差异,可以刺激不同的机体产生免疫反应,从而引发某些不良反应。本研究拟建立1种基于DNA扩增和测序分型(PCR sequencing-based typ-ing,PCR-SBT)的HNA-1、HNA-3到HNA-5和HNA-2a(G/C)抗原系统的基因分型技术。方法选择参与本单位献血人员的随机标本400例,采用DNA抽提试剂盒,抽提标本基因组DNA,严格按试剂说明书进行操作,参照数据库中人类HNA-1～HNA-5基因序列,采用计算机软件设计引物,并在引物5’端添加M13序列,5对引物分别扩增HNA-1～HNA-5基因序列,

沙门菌血清分型方法的比较

血清分型是沙门菌最基本和最重要的流行病学调查手段,不同血清型沙门菌引起的临床症状和造成的危害不同,快速准确地进行血清分型对于畜禽沙门菌病的防控和公共卫生安全意义重大。基于此,本试验选择我国1971—2020年分离的51株沙门菌(13种血清型),分别用全基因组测序(WGS)分型方法和液相芯片分型方法进行血清分型,并与玻片凝集法进行比较,分析3种分型方法的优劣。结果显示,玻片凝集法测得51株沙门菌的血清分型结果与原始结果一致;WGS分型方法和液相芯片分型方法分别测得34株和23株沙门菌血清分型与玻片凝集法结果一致。3种分型方法相比,WGS分型方法可鉴定出玻片凝集法和液相芯片分型方法无法分型的菌株,在操作时间和成本上更具优势。本试验可为沙门菌的血清分型研究提供参考。

PCR-SBT法HLA基因分型模棱两可结果的判读及解决对策

目的研究PCR-SBT法HLA基因分型结果判读中出现的模棱两可问题并提出解决策略。方法对306名随机抽取的组织配型患者的DNA采用PCR-序列特异性引物(Sequence Specific Primer,SSP)低分辨方法检测,同时采用PCR-SBT法进行HLA-A,B,DRB1高分辨基因分型。PCR-SBT法模棱两可结果用PCR-SSP高分辨方法复核确认。结果所有306例检测标本的高低分辨血清学抗原一致,经PCR-SBT基因测序方法检测有111份模棱两可结果,占36.3%(111/306)。其中HLA-A座位有30对等位基因存在模棱两可结果,占所有检测标本等位基因总数的3.3%;HLA-B座位有66对,占7.2%;HLA-DRB1座位有36对,占3.9%。所有模棱两可标本经PCR-SSP HLA基因高分辨分型试剂PCR-SSP等方法复核确认。结论针对PCR-SBT法进行HLA-A,B,DRB1高分辨基因分型模棱两可结果所建立的解决策略,对于提高HLA数据分型的速度和分型准确性有重要意义。

安徽省新型布尼亚病毒基因测序鉴定方法的建立

目的建立发热伴血小板减少综合征布尼亚病毒基因测序鉴定方法,对安徽省在2011年从病人血清中分离到的10株疑似发热伴血小板减少综合征布尼亚病毒进行基因测序鉴定,并分析其序列特征。方法设计引物,RT-PCR法扩增病毒核酸,对扩增产物进行测序分析。结果设计的三对引物可有效扩增核酸序列,10株疑似发热伴血小板减少综合征布尼亚病毒L、M、S三段基因序列和2010年我国发现毒株序列相似性93%以上,10株病毒和Phlebovirus AH12、Phlebovirus HN6两株病毒同属于一个进化分支,和白蛉病毒属的其它病毒株不在一个分枝上。结论 2011年从病人血清中分离到的10株病毒为发热伴血小板减少综合征布尼亚病毒,应加强安徽省对该病的监测,有效预防大范围传播。

野生型工业酿酒酵母Miseq测序方法的建立

新一代测序方法在基因组学研究应用日趋广泛,已经成为工业酿酒酵母菌株基因组学研究的重要技术平台,但对工业酿酒酵母新一代测序技术方法缺乏详细报道。Miseq是小型新一代基因组测序仪,本文报道我们自建的野生型工业酿酒酵母基因组Miseq测序方法。该方法包括：制备分离a型和α型交配型的单倍体菌株、采用电泳和分光光度法方法监控基因组文库建立、优化上机文库浓度后测序。其中电泳和分光光度法方法为首创的文库质量监控简易方法,质控检测得到酿酒酵母工业菌株测序条件为：菌株的基因组文库DNA片段大小为250-850bp且主要集中在350-550bp,文库浓度范围为7-13ng/μL;上机测序文库的优化浓度为20pM。

Aberrant methylation of the 3q25 tumor suppressor gene PTX3 in human esophageal squamous cell carcinoma

AIM:To identify the novel methylation-silenced gene pentraxin 3(PTX3) in esophageal squamous cell carcinoma(ESCC).METHODS:PTX3 mRNA expression was examined in six human ESCC cell lines,one human immortalized normal esophageal epithelial cell line,primary ESCC tumor tissue,and paired adjacent nontumor tissue using reverse transcription polymerase chain reaction(RTPCR).Semi-quantitative immunohistochemistry was used to examine cellular localisation and protein levels.Methylation specific PCR and bisulphite genomic sequencing were employed to investigate the methylation of the candidate gene.RESULTS:In the majority of ESCC cell lines,we found that PTX3 expression was down-regulated due to gene promoter hypermethylation,which was further confirmed by bisulphite genomic sequencing.Demethylation treatment with 5-aza-2'-deoxycytidine restored PTX3 mRNA expression in ESCC cell lines.Methylation was more common in tumor tissues(85%) than in adjacent nontumor tissues(25%)(P < 0.01).CONCLUSION:PTX3 is down-regulated through promoter hypermethylation in ESCC,and could potentially serve as a biomarker of ESCC.

Sequence assembly using next generation sequencing data —challenges and solutions

Sequence assembling is an important step for bioinformatics study.With the help of next generation sequencing(NGS)technology,high throughput DNA fragment(reads)can be randomly sampled from DNA or RNA molecular sequence.However,as the positions of reads being sampled are unknown,assembling process is required for combining overlapped reads to reconstruct the original DNA or RNA sequence.Compared with traditional Sanger sequencing methods,although the throughput of NGS reads increases,the read length is shorter and the error rate is higher.It introduces several problems in assembling.Moreover,paired-end reads instead of single-end reads can be sampled which contain more information.The existing assemblers cannot fully utilize this information and fails to assemble longer contigs.In this article,we will revisit the major problems of assembling NGS reads on genomic,transcriptomic,metagenomic and metatranscriptomic data.We will also describe our IDBA package for solving these problems.IDBA package has adopted several novel ideas in assembling,including using multiple k,local assembling and progressive depth removal.Compared with existence assemblers,IDBA has better performance on many simulated and real sequencing datasets.

Antibiotic resistance mechanisms of Myroides sp.

Bacteria of the genus Myroides （Myroides spp.） are rare opportunistic pathogens. Myroides sp. infections have been reported mainly in China. Myroides sp. is highly resistant to most available antibiotics, but the resistance mechanisms are not fully elucidated. Current strain identification methods based on biochemical traits are unable to identify strains accurately at the species level. While 16S ribosomal RNA （rRNA） gene sequencing can accurately achieve this, it fails to give information on the status and mechanisms of antibiotic resistance, because the 16S rRNA sequence contains no information on resistance genes, resistance islands or enzymes. We hypothesized that ob- taining the whole genome sequence of Myroides sp., using next generation sequencing methods, would help to clarify the mechanisms of pathogenesis and antibiotic resistance, and guide antibiotic selection to treat Myroides sp. infec- tions. As Myroides sp. can survive in hospitals and the environment, there is a risk of nosocomial infections and pandemics. For better management of Myroides sp. infections, it is imperative to apply next generation sequencing technologies to clarify the antibiotic resistance mechanisms in these bacteria.