蜜蜂球囊菌菌丝和孢子中全长转录本的差异表达分析

[目的]本研究旨在探究蜜蜂球囊菌Ascosphaera apis(简称“球囊菌”)菌丝(Aam)和孢子(Aas)中的全长转录本与菌丝生长、孢子萌发以及有性生殖的相关性.[方法]将前期获得的Aam和Aas的Nanopore长读段测序数据中的有效读段与球囊菌参考基因组注释的已知转录本进行序列比对,获取全长转录本与已知转录本...     

Characterization of 13 polymorphic microsatellite loci in the Japanese land leech

We developed 13 polymorphic microsatellite loci of the Japanese land leech (Haemadipsa japonica; Haemadipsidea) using an Illumina MiSeq sequencing approach. A total of 42,064 nuclear DNA contigs were filtered for microsatellite motifs, among which 30,873 simple sequence repeat loci were identified. From these sequences, we selected 30 primer sets, and 13 of these loci were successfully amplified. Polymorphism of the 13 loci was tested using 16 individuals sampled from sixteen populations across Japan. The number of alleles and polymorphism information content varied from 5 to 17 and 0.335 to 0.883, respectively, and observed and expected heterozygosity values ranged from 0.143 to 0.875 and 0.349 to 0.893, respectively, indicating that these loci are polymorphic. Furthermore, we established useful multiplex PCR using these loci. The 13 microsatellite loci described in this paper are the first nuclear microsatellite markers for a land leech species.     

高通量测序技术及其应用

高通量测序技术是DNA测序发展历程的一个里程碑,它为现代生命科学研究提供了前所未有的机遇。详细介绍了以454、Solexa和SOLiD为代表的第二代高通量测序技术,以HeliScope TIRM和Pacific Biosciences SMRT为代表的单分子测序技术,以及最近Life Science公司推出的Ion Personal Genome Machine (PGM)测序技术等高通量测序技术的最新进展。在此基础上,阐述了高通量测序技术在基因组测序、转录组测序、基因表达调控、转录因子结合位点的检测以及甲基化等研究领域的应用。最后,讨论了高通量测序技术在成本和后续数据分析等方面存在的问题及其未来的发展前景。     

Isolation and characterization of polymorphic microsatellite loci in Indian major carp,Catla catla using next-generation sequencing platform

Catla catla, the second most important Indian major carp, is gaining its popularity among Indian fish farmers due to its high growth rate and consumer preferences. Simple sequence repeats (SSRs) are rapidly evolving, versatile, co-dominant and highly informative molecular markers used in genetic research. However, the time and cost involved in developing such resources has limited their extensive use. Advent of massive parallel sequencing technology has considerably eased these limitations. In the present investigation, we used Ion Torrent sequencing platform to identify potentially amplifiable microsatellite loci for catla. A modest sequencing volume generated approximately 5.7 MB of sequence data. Out of 29,794 sequences generated, 21,477 contained simple sequence repeats. Only 81 sequences had enough flanking sequences for primer designing. Out of 81 loci, 51 were successfully PCR amplified in a panel of five unrelated individuals. Out of 15 loci randomly checked for polymorphism, 13 loci were polymorphic with allele number ranged from 3 to 6 and two loci were found to be monomorphic. The observed and expected heterozygosities ranged from 0.565 to 0.870 and 0.483–0.804, respectively. These markers will be useful for studying genetics of wild populations, breeding programs of C. catla and closely related species.     

二代测序在脓毒血症患者病原学诊断中的应用

脓毒血症是一种严重威胁生命的感染,精准、快速的病原学诊断可帮助临床医师优化抗菌药物的使用。目前,基于病原菌培养的方法仍是脓毒血症病原学诊断的主要手段,但具有耗时长、灵敏度低等不可忽视的缺点。近年来出现了一些不依赖培养的病原学诊断方法,其中基于聚合酶链反应(polymerase chain reaction,PCR)的方法已发展较为成熟。但PCR只能检测已知的特定病原体,临床定量PCR仅用于检测病毒及少数细菌,脓毒血症中的病原体PCR多仅为定性检测。目前,二代测序技术不断成熟并用于临床,成为病原学诊断的有力手段。与血培养等传统病原学检测方法相比,其具有快速、非选择性、可定量或半定量分析的优点。现阶段二代测序仍存在公认判读标准缺乏、测序结果与治疗关系不明确、耐药基因检测困难等不足,亦缺乏较大规模的二代测序与传统诊断方法比较验证的研究结果,尚有待更高级的循证医学证据支持。     

Non-toxigenic tox gene-bearing Corynebacterium ulcerans in a traumatic ulcer from a human case and his asymptomatic dog

A non-toxigenic tox gene-bearing (NTTB) Corynebacterium ulcerans was grown from the wound of a 61-year-old gardener and in a nasal specimen from the patient's asymptomatic dog. The two isolates were similar in terms of antibiogram, multilocus sequence typing (ST341), virulence genes, and only three SNPs were found to differentiate the two NTTB C. ulcerans isolates supporting a zoonotic transmission to or between the patient and his dog. Of interest, we found that the two C. ulcerans isolates, although not expressing the diphtheria toxin tox, possessed 13 out of 14 recently described virulence candidate genes.     

exonsampler: a computer program for genome‐wide and candidate gene exon sampling for targeted next‐generation sequencing

The computer program exonsampler automates the sampling of thousands of exon sequences from publicly available reference genome sequences and gene annotation databases. It was designed to provide exon sequences for the efficient, next‐generation gene sequencing method called exon capture. The exon sequences can be sampled by a list of gene name abbreviations (e.g. IFNG, TLR1), or by sampling exons from genes spaced evenly across chromosomes. It provides a list of genomic coordinates (a bed file), as well as a set of sequences in fasta format. User‐adjustable parameters for collecting exon sequences include a minimum and maximum acceptable exon length, maximum number of exonic base pairs (bp) to sample per gene, and maximum total bp for the entire collection. It allows for partial sampling of very large exons. It can preferentially sample upstream (5 prime) exons, downstream (3 prime) exons, both external exons, or all internal exons. It is written in the Python programming language using its free libraries. We describe the use of exonsampler to collect exon sequences from the domestic cow (Bos taurus) genome for the design of an exon‐capture microarray to sequence exons from related species, including the zebu cow and wild bison. We collected ~10% of the exome (~3 million bp), including 155 candidate genes, and ~16 000 exons evenly spaced genomewide. We prioritized the collection of 5 prime exons to facilitate discovery and genotyping of SNPs near upstream gene regulatory DNA sequences, which control gene expression and are often under natural selection.     

Identification of a novel male germ cell-specific gene TESF-1 in mice

Mammalian spermatogenesis is precisely regulated by many germ cell-specific factors. In search for such a germ cell-specific factor, we have identified a novel mouse gene testis-specific factor 1 (TESF-1). Messenger RNA of TESF-1 was found only in the testis and its expression appeared to be regulated in a developmental manner. Further analysis demonstrated that the expression of TESF-1 was specifically in male germ cells, supported by the observation that we were not able to detect the TESF-1 mRNA from at/at homozygous mutant testes, which lack germ cells. The deduced amino acid sequence of TESF-1 contains a leucine-zipper motif, a potential nuclear localization signal, and two cAMP- and cGMP-dependent protein kinase phosphorylation sites. The green fluorescent protein (GFP)-tagged TESF-1 fusion protein was expressed in COS-7 cells and localized primarily in the nucleus. Taken together, these results indicate that TESF-1 is a novel male germ cell-specific gene, and its protein product may function as a nuclear factor involved in the regulation of spermatogenesis.     

乌鲁木齐市3~5岁儿童口腔微生物与肠道微生物的构成差异

目的 探索乌鲁木齐市3~5岁儿童口腔微生物与肠道微生物的菌群构成及多样性等差异。 方法 从本团队前期流行病学调查的乌鲁木齐市3~5岁儿童中,按调查时间顺序随机抽取12名儿童。分别收集唾液及粪便样本共计24份,分为口腔微生物组和肠道微生物组。利用16S V3-V4区设计引物来进行PCR扩增,使用MiSeq测序仪进行二代测序,比较两组的微生物构成及多样性差异。 结果 门水平上:放线菌门(t=5.98,P结论 放线菌门、变形菌门、TM7在口腔中丰度较高;厚壁菌门在肠道中丰度较高。口腔和肠道微生物在菌群功能上差异较大。奈瑟菌属、卟啉单胞菌属、Catenibacterium等可能为某些全身系统性疾病的标志性菌属。     

宏基因组测序在传染性疾病中的研究进展

微生物和人类已经共同进化几百万年了,微生物在维持宿主健康方面起到了非常重要的作用。随着下一代测序技术的进步,可以获得人体在不同环境下微生物群落的特征。本文综述了当前感染各种不同病原菌时复杂微生物群落的变化,病原菌包括HIV、乙肝病毒、流感病毒和结核分支杆菌,以及不同的身体部位。我们相信,增加对传染性疾病和微生物群落变化之间关系的认识,能够更好地管理疾病进展。然而,将来的研究可能需要更加整体化,通过分析人体宿主微生物与传染性疾病的发生机制之间的关系,以建立疾病的确切因果关系。     

四种常用的生物序列比对软件比较

随着高通量测序技术的快速发展,下一代测序技术也迅速发展为生物领域中的主流技术,而理解下一代测序数据最重要的一步是比对。比对是进行后续生物信息分析的基石,也因此催生了很多比对软件。本文主要选取了四种常用的比对软件Bowtie2、BWA、MAQ和SOAP2,对这四种软件及算法进行综述,并通过实际测序数据对四种软件进行比较和评估,为生物学研究者选择最佳的短序列比对软件提供理论和实践依据。     

基于第二代测序数据识别肿瘤基因突变的工具比较

使用第二代测序数据来发现癌细胞中的基因组突变,一直是很重要的科学应用问题。此研究使用一个癌症病人的大量数据,评估了甄别基因组突变的几个现有工具。经过比较各工具的方法和正确率,本文发现各自都有自己的优点和缺点。针对这些优缺点,本文提供一些建议,让工具使用者能更好地选择合适的工具。     

Paired analysis of tumor mutation burden calculated by targeted deep sequencing panel and whole exome sequencing in non-small cell lung cancer

Owing to rapid advancements in NGS (next generation sequen-cing), genomic alteration is now considered an essential pre-dictive biomarkers that impact the treatment decision in many cases of cancer. Among the various predictive biomarkers, tumor mutation burden (TMB) was identified by NGS and was con-sidered to be useful in predicting a clinical response in cancer cases treated by immunotherapy. In this study, we directly com-pared the lab-developed-test (LDT) results by target sequencing panel, K-MASTER panel v3.0 and whole-exome sequencing (WES) to evaluate the concordance of TMB. As an initial step, the reference materials (n = 3) with known TMB status were used as an exploratory test. To validate and evaluate TMB, we used one hundred samples that were acquired from surgically resected tissues of non-small cell lung cancer (NSCLC) patients. The TMB of each sample was tested by using both LDT and WES methods, which extracted the DNA from samples at the same time. In addition, we evaluated the impact of capture re-gion, which might lead to different values of TMB; the evalu-ation of capture region was based on the size of NGS and target sequencing panels. In this pilot study, TMB was evalu-ated by LDT and WES by using duplicated reference samples; the results of TMB showed high concordance rate (R² = 0.887). This was also reflected in clinical samples (n = 100), which showed R² of 0.71. The difference between the coding sequence ratio (3.49%) and the ratio of mutations (4.8%) indicated that the LDT panel identified a relatively higher number of mutations. It was feasible to calculate TMB with LDT panel, which can be useful in clinical practice. Furthermore, a customized approach must be developed for calculating TMB, which differs according to cancer types and specific clinical settings.     

Presence and distribution of insect-associated and entomopathogenic fungi in a temperate pine forest soil: An integrated approach

For decades entomopathogenic fungi have garnered interest as possible alternatives to chemical pesticides. However, their ecology outside of agroecosystems demands further study. We assessed the diversity and abundance of entomopathogenic and insect-associated fungi at a loblolly pine forest in North Carolina, USA using culture-dependent and next-generation sequencing libraries. Fungi were isolated using Galleria mellonella larvae, as well as from soil dilutions plated on a selective medium. Isolates were identified using Sanger sequencing of the ITS and LSU rRNA gene regions, and represented 36 OTUs including Metarhizium, Lecanicillium, and Paecilomyces. Additionally, we assessed the chitinolytic potential of isolates and found widespread, variable ability to degrade chitin within and between genera. Phylogenetic analyses resolved several isolates to genus, with some forming clades with other insect-associated taxa, as well as with fungi associated with plant tissues. Saprophytes were widely distributed in soil, while entomopathogens were less abundant and present primarily in the top two cm of the soil. The similarity between culture-dependent and next-generation sequencing results demonstrates that both methods can be used concurrently in this system to study the ecology of entomopathogenic fungi.