天仙藤全长转录组测序及分析

目的:获得天仙藤全长转录组数据库,挖掘天仙藤功能基因。方法:采用PacBio SequeⅠ高通量测序系统,对天仙藤的茎、叶、果3个部位的混合样品进行全长转录组测序及分析。结果:共获得176354条环形一致性序列(CCS),获得184439个高质量isoforms,并注释了139826个isoforms,检测出3058个转录因子和135527个简单序列重复(SSR)位点,还预测到76862个长链非编码RNA(lncRNA)和36204个mRNAs。结论:获得了较可靠的天仙藤全长转录组数据,可为深入研究天仙藤基因组、生物学特性、相关代谢途径、信号通路及其分子机制提供数据支持。     

黄芪-丹参药对通过下调miR-466b-5p改善高血压肾损害

目的：探讨黄芪-丹参药对通过调节微小核糖核酸-466b-5P（miR-466b-5p）改善高血压肾损害的机制。方法：基于微RNA（miRNA）测序寻找自发性高血压大鼠与Wistar-京都鼠（WKY）的差异基因,构建腺相关病毒转染小鼠,24只C57BL/6小鼠,随机选取12只尾静脉注射小鼠腺相关病毒miR-466b-5P（rAAV-miR-466b-5P）构建miR-466b-5p过表达组,余12只作为空白对照组注射腺相关病毒-9（AAV-9）空载体,转染6周后再各随机分为2组,每组6只,A组（黄芪-丹参+miR-466b-5P过表达组）和C组（黄芪-丹参+空白对照组）以黄芪配方颗粒：2.036 g/kg+丹参配方颗粒：0.255 g/kg灌胃;B组（miR-466b-5P过表达组）和D组（空白对照组）以相同体积生理盐水灌胃;灌胃28 d后观察各组小鼠尿β2-微球蛋白（β2-MG）、N-乙酰-β-D-葡萄糖苷酶（NAG）、微量白蛋白（mALB）,血清胱抑素C（Cys-C）、血管紧张素Ⅱ（AngⅡ）、C反应蛋白（CRP）水平和肾脏病理,进行实时荧光定量(RT-qPCR)与蛋白质印迹法(Western Blotting)寻找其靶基因。结果：1）基于前期miRNA测序选定了miR-466b-5p;2）与D组比较,B组小鼠尿β2-MG、NAG、mALB、血清Cys-C、AngⅡ、CRP均显著升高（P<0.01）;与B组比较,A组小鼠血清Cys-C、CRP明显升高（P<0.05）,尿β2-MG、NAG、mALB、血清AngⅡ无明显改变（P>0.05）;3）与D组比较,B组小鼠出现明显肾小球硬化、小管纤维化,A组较B组有明显改善;4）与D组比较,B组小鼠透明质酸酶2（HAS2）明显下调,而经黄芪-丹参干预后有所回调。结论：miR-466b-5p的过表达会导致高血压肾损害的发生,黄芪-丹参药对通过靶向HAS2下调miR-466b-5p改善高血压肾损害。     

马鞭草全长转录组测序分析

目的:利用高通量测序技术获得马鞭草Verbena officinalis L.的全长转录组基因信息。方法:通过PacBio Sequel高通量测序平台,对马鞭草根、茎、叶3个部位的混合样品进行全长转录组测序,并基于序列同源性对转录本进行功能注释,得到马鞭草全长转录组的遗传信息。结果:测序数据经过质控后获得197542个高质量的转录本及169063条环形一致性序列(CCS),检测出4955个转录因子。其中161062个(81.53%)转录本在非冗余蛋白(NR)、真核生物相邻类的聚簇(KOG)数据库、京都基因与基因组百科全书(KEGG)、基因本体(GO)数据库、蛋白家族(Pfam)数据库和SwissProt蛋白序列数据库中均得到注释。MISA分析发现54063个简单重复序列(SSR)位点,还预测到70050个长链非编码RNAs(lncRNAs)和45499个信使核糖核酸(mRNAs),并在马鞭草全长转录组中共鉴定到了60个转录本可能参与单萜类化合物的生物合成。结论:利用高通量测序技术和生物信息学分析获得了马鞭草的全长转录组信息特征,可为后期开展马鞭草功能基因鉴定、解析萜类化合物次生代谢途径及其调控机制提供参考。     

箭叶淫羊藿不同时期根际微生物群落组成及其与有效成分累积的相关性分

目的 通过分析箭叶淫羊藿Epimediumsagittatum不同生育期根际微生物群落结构及其与主要药用有效成分累积之间的相关性,探讨箭叶淫羊藿根际土壤微生物对其药材有效成分的影响,为箭叶淫羊藿的优质高产栽培提供科学依据。方法 以三年生箭叶淫羊藿的根际土为研究对象,采用高通量测序技术对根际细菌和真菌群落结构进行分析,同时测定根际土壤理化性质、酶活性及不同生育期药材总黄酮、淫羊藿苷等有效成分含量,通过皮尔逊相关性分析探究土壤生态因子与有效成分之间的关系。结果 高通量测序结果显示,箭叶淫羊藿根际细菌优势菌属包括Candidatus＿Solibacter、苔藓杆菌属、嗜酸栖热菌属、芽单胞菌属等,其中,Candidatus＿Solibacter属在5个生长时期的平均丰度值最高。根际真菌优势菌属中被孢霉属相对丰度占比最大,在花蕾期样品中的丰度值高达44.27%。UPGMA聚类和非度量多维标定法（NMDS）分析表明,花蕾期、盛花期、果实膨大期及盛果期的根际土壤细菌和真菌结构相似,而药材质量稳定期与前4个时期的根际微生物群落结构存在明显差异。同时,皮尔逊相关性分析结果显示：总黄酮含量与有效磷呈显著正...     

黄背草与2种菅属植物叶绿体基因组特征比较及系统发育分析

目的 以黄背草Themeda japonica为试验材料,比较分析其与阿拉伯黄背草T. triandra、中华菅T. quadrivalvis2种同属植物的叶绿体基因组特征及其与近缘物种的系统发育关系。方法 采用Illumina HiSeq高通量测序平台首次对黄背草叶绿体基因组进行测序,使用SPAdes和CpGAVAS2分别对其进行组装和注释,并用Codon W、DnaSP和MISA等对其与2种同属植物进行一系列比较基因组分析,利用最大似然法（maximum likelihood,ML）构建系统进化树。结果 3个叶绿体基因组全长为138 735～138 961 bp,具有典型的四分体结构,共注释出129个基因;黄背草与其同属的2个物种相比,反向重复区（inverted repeats,IR）收缩了2132 bp,大单拷贝区（large single copy region,LSC）扩张了约4000 bp,而小单拷贝区（small single copy region,SSC）变化不大。密码子偏好性分析显示,3个叶绿体基因组相对丰度最大和最小的密码子都相同,同义密码子相对使用丰度略有不同...     

Hand-foot-genital syndrome due to a duplication variant in the GC-rich region of HOXA13

BackgroundHand-Foot-Genital Syndrome (HFGS) is an autosomal dominant disorder characterized by a broad phenotypic spectrum. Variants in HOXA13 gene were associated with HFGS. To date, only twenty families with HFGS have been reported. However, the challenge in HFGS is the limited sample sizes and phenotypic heterogeneity. The advent of next-generation sequencing has permitted the identification of patients with HOXA13 variants who do not manifest with the full HFGS syndromic features.MethodsTrio (parents-proband) Whole-exome sequence(WES) and whole-genome sequencing(WGS) was carried out in this study to investigate the underlying pathogenic genetic factor of the neonate with a wide variety of clinical abnormalities.ResultsNo possible pathogenetic variation was detected by trio-WES, and a duplication variant in HOXA13 (c.360_377dup, p.Ala128_Ala133dup), inherited from her mother, was identified by the subsequent WGS in the proband with malnutrition, feeding difficulties, electrolyte disorders, metabolic acidosis, recurrent urinary tract infections, hydronephrosis, nephrolithiasis, abnormal ureter morphology, cholelithiasis, uterus didelphys. Sequence analysis of the variant region (exon1) indicated a high GC content of 73.92%. In addition, further enquiry of the family history revealed that 5 members of the family in 4 generations had hand and foot anomalies.ConclusionThe neonate was diagnosed with HFGS by genetic analysis. GC content had less influence on sequence coverage in WGS than WES analysis. This was the first report of trio-WGS study for HFGS genetic diagnosis, revealed that subsequent WGS was necessary for identification of potentially pathogenic variants in unexplained genetic disorders.     

Phenotypic specificity in patients with neurodevelopmental delay does not correlate with diagnostic yield of trio-exome sequencing

In this study, we aimed to examine the diagnostic yield achieved by applying a trio approach in exome sequencing (ES) and the interdependency between the clinical specificity in families with neurodevelopmental delay. Thirty-seven families were recruited and trio-ES as well as three criteria for estimating the clinical phenotypic specificity were suggested and applied to the underaged children. All our patients showed neurodevelopmental delay and most of them a large spectrum of congenital anomalies. Applying the pathogenicity guidelines of the American College of Medical Genetics (ACMG), likely pathogenic (29.7%) and pathogenic variants (8.1%) were found in 40,5% of our index patients. Additionally, we found four variants of uncertain significance (VUS; according to ACMG) and two genes of interest (GOI; going beyond ACMG classification) (GLRA4, NRXN2). Spastic Paraplegia 4 (SPG4) caused by a formerly known SPAST variant was diagnosed in a patient with a complex phenotype, in whom a second genetic disorder may be present. A potential pathogenic variant linked to severe intellectual disability in GLRA4 requires further investigation. No interdependency between the diagnostic yield and the clinical specificity of the phenotypes could be observed. In consequence, trio-ES should be used early in the diagnostic process, independently from the specificity of the patient.     

Mutation profile and its correlation with clinicopathology in Chinese hepatocellular carcinoma patients

BackgroundHepatocellular carcinoma (HCC) is one of the most common causes of cancer worldwide. Although many studies have focused on oncogene characteristics, the genomic landscape of Chinese HCC patients has not been fully clarified.MethodsA total of 165 HCC patients, including 146 males and 19 females, were enrolled. The median age was 55 years (range, 27–78 years). Corresponding clinical and pathological information was collected for further analysis. A total of 168 tumor tissues from these patients were selected for next-generation sequencing (NGS)-based 450 panel gene sequencing. Genomic alterations including single nucleotide variations (SNV), short and long insertions and deletions (InDels), copy number variations, and gene rearrangements were analyzed. Tumor mutational burden (TMB) was measured by an algorithm developed in-house. The top quartile of HCC was classified as TMB high.ResultsA total of 1,004 genomic alterations were detected from 258 genes in 168 HCC tissues. TMB values were identified in 160 HCC specimens, with a median TMB of 5.4 Muts/Mb (range, 0–28.4 Muts/Mb) and a 75% TMB of 7.7 Muts/Mb. The most commonly mutated genes were TP53, TERT, CTNNB1, AXIN1, RB1, TSC2, CCND1, ARID1A, and FGF19. SNV was the most common mutation type and C:G>T:A and guanine transformation were the most common SNVs. Compared to wild-type patients, the proportion of Edmondson grade III–IV and microvascular invasion was significantly higher in TP53 mutated patients (P<0.05). The proportion of tumors invading the hepatic capsule was significantly higher in TERT mutated patients (P<0.05). The proportion of Edmondson grade I-II, alpha fetoprotein (AFP) <25 µmg/L, and those without a history of hepatitis B was significantly higher in CTNNB1 mutated patients (P<0.05). CTNNB1 mutations were associated with TMB high in HCC patients (P<0.05). Based on correlation analysis, the mutation of TP53 was independently correlated with microvascular invasion (P=0.002, OR =3.096) and Edmondson grade III–IV (P=0.008, OR =2.613). The mutation of TERT was independently correlated with tumor invasion of the liver capsule (P=0.001, OR =3.030), and the mutation of CTNNB1 was independently correlated with AFP (<25 µmg/L) (P=0.009, OR =3.414).ConclusionsThe most frequently mutated genes of HCC patients in China were TP53, TERT, and CTNNB1, which mainly lead to the occurrence and development of HCC by regulating the P53 pathway, Wnt pathway, and telomere repair pathway. There were more patients with microvascular invasion and Edmondson III–IV grade in TP53 mutated patients and more patients with hepatic capsule invasion in TERT mutated patients, while in CTNNB1 mutated patients, there were more patients with Edmondson I–II grade, AFP <25 µmg/L, and a non-hepatitis B background. Also, the TMB values were significantly higher in CTNNB1 mutated patients than in wild type patients.