药用美洲大蠊全基因组测序分析

以美洲大蠊Periplaneta americana为原料生产的康复新液等药品临床疗效显著,得到了广泛应用。本文以四川好医生攀西药业有限责任公司饲养的药用美洲大蠊为材料,首次采用Illumina Hi Seq 2000和Pac Bio SMRT测序平台开展了全基因组测序,并进行基因组组装、注释和分析。原始测序数据经过滤后得到1.4 Tb的二代测序数据和33.81 Gb的三代测序数据。组装结果表明,美洲大蠊基因组大小为3.26 Gb,这在已报道的昆虫基因组中仅次于东亚飞蝗Locusta migratoria。基因组重复序列含量为62.38%,杂合度为0.635%,表明其为复杂基因组。组装的Contig N50和scaffold N50长度分别为28.2 kb、315 kb,单拷贝基因完整性为88.1%,小片段文库测序数据平均比对率为99.8%,测序和组装质量满足后续分析要求。采用De novo预测、同源预测和基于转录本预测3种方法共注释到14 568个基因,其中92.4%的基因获得了功能注释。本研究首次完成了美洲大蠊的全基因组测序,也是大蠊属Periplaneta昆虫的第一个基因组,为美洲大蠊遗传进化分析和药用基因资源挖掘打下了重要基础。     

黄唇鱼(Bahaba flavolabiata)的全基因组测序和基因组特征研究

黄唇鱼(Bahaba flavolabiata)为国家二级重点保护野生动物、IUCN(世界自然保护联盟)红色名录的极度濒危物种(CR)。基于其样本数量极其有限,全基因组研究可以提供大量与重要性状相关的功能基因和分子标记,从而揭示其重要生命现象的遗传机制。采用二代测序技术于2018年5月完成了黄唇鱼基因组精细图的测序,分析结果表明,测序得到约202 Gb的高质量数据,总测序深度约为317×;组装得到的基因组大小为637.43 Mb,Contig N50约为88 Kb,Scaffold N50约为4.65 Mb;重复序列约142.72 Mb,占比22.39%,预测得到23743个基因、920个t RNA、85个rRNA、176个假基因;98.46%的基因可以注释到NR、GO等数据库中;有67个基因家族是黄唇鱼所特有的。本研究从单碱基错误率、核心基因完整性及二代Reads比对分析3个方面对黄唇鱼基因组精细图的组装结果进行了评估,结果显示所组装的基因区的完整性较好。黄唇鱼基因组序列图谱的绘制完成,对于黄唇鱼自然资源的保护和种质资源挖掘具有极其重要的科学意义。     

基于三代测序技术的微生物组学研究进展

微生物在人类生活中无处不在, 过去人们对微生物的认识仅停留在单菌培养和定性研究上, 而测序技术的发展极大地促进了微生物组学的研究。越来越多的证据表明: 人体共生微生物、特别是肠道微生物与人类健康息息相关。 二代测序技术凭借其高通量、高准确率和低成本的特点, 成为微生物组学研究中的主流测序技术。但是随着研究的深入, 二代测序技术的短读长(< 450 bp)增加了后续数据分析和基因组拼接难度, 也限制了该技术在未来研究中的应用。在此背景下, 第三代测序技术应运而生。第三代测序技术又称单分子测序, 能够直接对单个DNA分子进行实时测序, 而不需要经过PCR扩增。第三代测序技术的平均读长在2-10 kb左右, 最高可以达到2.2 Mb, 实现了长序列的高通量测序。凭借其超长的测序读长、无GC偏好性等优势, 三代测序技术为微生物基因组全长测序, 组装完整可靠的基因组提供了新的方法。本文在描述三代测序的技术特点和原理的基础上, 重点介绍了三代测序技术在微生物16S/18S rRNA基因测序、单菌的基因组组装以及宏基因组中的研究应用和进展。     

基于Survey分析的濒危植物四合木基因组研究

评价濒危植物四合木（Tetraena mongolica）基因组的大小及复杂程度,开展基因组研究可揭示四合木的超旱生机制,进一步挖掘其特色基因资源。为更好破解四合木的全基因组信息,采用第二代高通量测序技术的基因组Survey分析技术开展四合木基因组大小估测研究,并利用生物信息学方法估计了四合木杂合率、重复序列和GC含量等基因组信息。结果表明：四合木基因组大小为1 079.25 Mb,修正后的基因组大小为1 065.84Mb,杂合率为0.76%,重复序列比例为75.25%,GC含量为33.57%。在经过四合木基因组初步组装后,获得3502 126条contigs,总计682 Mb,其N50为187 bp,推测四合木基因组属于同源四倍体复杂基因组,全基因组测序组装难度较大。由于四合木的高杂合率,后续可采用第三代高通量测序技术（单分子测序）同时结合染色质区域捕获技术,有望最终获得高质量的四合木全基因图谱。     

白芷全基因组测序分析及BGLU基因家族分析

白芷为常用的药食同源物种,既是临床常用中药,又是香料,用途十分广泛。为获取白芷全基因组序列信息,该研究首次以杭白芷叶片DNA为材料,采用Nanopore测序技术构建杭白芷全基因组数据库,并利用生物信息学方法对获得的核苷酸序列进行组装、功能注释以及进化分析研究。结果表明：(1)原始测序数据过滤后获得662 Gb三代数据,Read N50约为32 932 bp,经过组装得到杭白芷基因组大小为5.6 Gb, Contig N50约为806 638 bp。(2)组装后的序列通过与KOG、GO、KEGG等功能数据库比对,得到了功能注释的基因占66.47%,KOG功能注释结果表明杭白芷的蛋白功能主要集中在一般功能预测、翻译后修饰、蛋白质转换、伴侣以及信号转导机制;GO功能分类表明杭白芷的基因集中在生物学过程及细胞组分;KEGG通路注释表明参与代谢途径的基因占主要地位。(3)杭白芷中鉴定到45个BGLU家族基因。该研究首次利用第三代测序技术对杭白芷全基因组进行解析,为杭白芷的系统生物学研究和BGLU在杭白芷生长发育中的后续功能研究提供了重要的理论参考。     

基于Pacbio第三代测序技术的厚朴基因组测序分析

厚朴为著名的传统药用植物,归于木兰科、木兰属,于我国广泛种植,其树皮、根皮、枝皮、叶片、花、果实均能入药或食用。为获取厚朴全基因组序列信息,该文以厚朴叶片DNA为材料,采用Pacbio Sequel第三代测序技术构建厚朴全基因组数据库,并利用生物信息学方法对获得的核苷酸序列进行组装、功能注释以及进化分析研究。结果表明:(1)原始测序数据过滤后获得140.91 Gb三代数据,Read N50约为13 784bp,经过组装得到厚朴基因组大小为1.68 Gb,Contig N50约为222 069 bp,单拷贝基因完整性为81.0%。(2)组装后的序列通过与NR、KOG、KEGG等功能数据库比对,共有98.40%的基因得到了功能注释,其中KOG功能注释结果发现厚朴的蛋白功能主要集中在一般功能预测、翻译后修饰、蛋白质转换、伴侣以及信号转导机制; GO功能分类表明厚朴的基因集中在细胞组分及生物学过程; KEGG分析发现厚朴参与代谢通路的基因占主要地位。(3)通过与葡萄、拟南芥、水稻、杨树、银杏、无油樟、茶树及牛樟基因组的比对分析,发现厚朴23 424个基因中有20 801个基因可以分类到12 129个家族,其中有515个基因家族为厚朴所特有,而厚朴与牛樟(樟科)亲缘关系较近,两者的分化时间约在122.5百万年前(mya)。该研究首次利用第三代测序技术对厚朴全基因组解析,有利于对其进一步进行深入的开发与利用,也为研究其他药用植物全基因组奠定了基础。     

罗汉果全基因组Survey分析

罗汉果是广西特有药用及甜料植物,其主要成分之一甜苷V作为天然、非糖甜味剂,具有广阔的开发前景,但罗汉果目前完全来自于栽培,适生区狭窄,连作障碍严重,加之含量低导致甜苷V生产成本居高不下,严重限制了其应用。为了减少盲目性,在大规模全基因组深度测序之前,先做低覆盖度的基因组Survey测序,评价基因组的大小及复杂程度,以确定适合该植物全基因组的测序研究策略。该研究采用第二代高通量测序技术(Illumina Hiseq TM 2000)首次测定了罗汉果基因组大小,并利用生物信息学方法估计罗汉果杂合率、重复序列和GC含量等基因组信息。结果表明:(1)获得了18.1 Gb罗汉果基因组测序数据,基因组大小估计为344.95 Mb左右,测序深度为52×;(2)从K-mer分布曲线发现罗汉果基因组有明显的杂合峰,杂合率达1.5%,基因组高杂合导致组装的结果中Contig N50和Scaffold N50的长度比预期的要短很多,还造成GC平均深度及含量分布明显异常,存在一个低深度分布区域。基因组主峰后面有微弱的重复峰,说明罗汉果存在较多的重复序列;(3)由于罗汉果存在高杂合率和重复序列较多的特点,该基因组测序分析仅采用全基因组鸟枪法(WGS)策略不合适,为了更好地对全基因组进行序列拼接和组装,可尝试结合采用Fosmid-to-Fosmid或BAC-to-BAC策略。该研究结果对于揭示罗汉果产量、有效成分含量、发育及抗病虫的分子机制,以及通过分子育种来提高甜苷V含量和降低生产成本具有重要意义,为全基因组测序策略的选择提供了依据。     

植物基因组测序的研究进展

近年来,随着测序技术的不断发展,基因组测序技术渐趋成熟并在动物和植物基因组上获得了越来越多的成功,大量植物的基因组的草图和精细图不断地被公布出来。比较和分析了三代测序技术各自的特点,对测序前的准备、基因组组装、注释和比较基因组学等方面的研究进展进行了详细的评述,阐明了植物基因组研究的特点和难点。通过植物的全基因组测序,研究者不仅可以获得该植物基因组和重要功能基因的序列信息,为从分子水平研究植物的分子进化、基因组成和基因调控等提供了一定的依据,而且还对即将测序的植物基因组研究具有重要的借鉴意义。     

金针菇rDNA序列结构特征分析

rDNA序列中的ITS作为DNA barcoding广泛应用于真菌的系统发育与物种辅助鉴定,IGS被认为可以用于种内水平不同菌株的鉴别。食用菌中还没有完整的rDNA序列的报道。本研究采用二代和三代测序技术分别对金针菇单核菌株“6-3”进行测序,用二代测序的数据对三代测序组装得到的基因组序列进行修正,得到一个在基因完整性、连续性和准确性均较好的基因组序列,对比Fibroporia vaillantii rDNA序列,获得金针菇完整的rDNA序列。金针菇rDNA序列结构分析表明,它有8个rDNA转录单元,长度均为5 903bp,有9个基因间隔区,其长度有较大差异,3 909-4 566bp。rDNA转录单元中,各元件的序列长度分别为：18S rDNA 1 796bp、ITS1 234bp、5.8S rDNA 173bp、ITS2 291bp、28S rDNA 3 410bp。基因间间隔区中,IGS1 1 351-1 399bp、5S rDNA 124bp、IGS2 2 435-3 092bp。金针菇的5S、5.8S、18S、28S rDNA序列准确性得到转录组数据的验证,也得到系统发育分析结果的支持。多序列比对发现,不同拷贝的基因间间隔区序列（IGS1和IGS2）存在丰富的多态性,多态性来源于SNP、InDel和TRS（串联重复序列）,而TRS来源于重复单元的类型和数量。9个基因间间隔区之间,IGS1只有少量的SNP和InDel,IGS2不仅有SNP和InDel,还有TRS。本研究结果提示,在应用IGS进行种内水平不同菌株之间的鉴别时,需要选取不同拷贝之间的保守IGS序列。     

利用RNA-seq技术分析棘腹蛙编码区微卫星特征

棘腹蛙Paa boulengeri的遗传研究和基因组信息比较匮乏,致使可有效利用的分子标记非常有限。以棘腹蛙RNA-seq高通量测序数据为基础进行微卫星分子标记的大规模发掘和特征分析,结果显示:在121.6 Mb的棘腹蛙转录组序列中发现微卫星位点3165个,包含于3034条Contig序列中。在筛选到的1~6碱基重复核心的微卫星中,单碱基重复核心的比例最高,之后为三碱基、二碱基、四碱基、六碱基和五碱基重复核心,分别占29.0%、25.2%、21.7%、10.0%、10.0%和3.0%。其中A/T、AC/GT、AGG/CCT、ACAT/ATCT、AAAAT/ATTTT和AAAAAG/CTTTTT分别是单碱基、二碱基、三碱基、四碱基、五碱基、六碱基重复类型中对应的优势重复单元。棘腹蛙编码区微卫星多为重复长度小于24 bp的短序列,长度大于24 bp的微卫星仅占总数的0.92%。对编码区微卫星的侧翼序列分析发现,微卫星侧翼序列的GC含量显著低于转录组整体GC含量,且在含有微卫星上下游侧翼序列的Contig中,71.9%的序列可以设计特异引物扩增出含有微卫星序列的位点。研究结果为棘腹蛙的遗传研究和分子系统地理学研究提供了丰富的序列信息和标记资源。     

Chromosome‐level genome assembly of the greenfin horse‐faced filefish (Thamnaconus septentrionalis) using Oxford Nanopore PromethION sequencing and Hi‐C technology

The greenfin horse‐faced filefish, Thamnaconus septentrionalis, is a valuable commercial fish species that is widely distributed in the Indo‐West Pacific Ocean. This fish has characteristic blue–green fins, rough skin and a spine‐like first dorsal fin. Thamnaconus septentrionalis is of conservation concern because its population has declined sharply, and it is an important marine aquaculture fish species in China. Genomic resources for the filefish are lacking, and no reference genome has been released. In this study, the first chromosome‐level genome of T. septentrionalis was constructed using nanopore sequencing and Hi‐C technology. A total of 50.95 Gb polished nanopore sequences were generated and were assembled into a 474.31‐Mb genome, accounting for 96.45% of the estimated genome size of this filefish. The assembled genome contained only 242 contigs, and the achieved contig N50 was 22.46 Mb, a surprisingly high value among all sequenced fish species. Hi‐C scaffolding of the genome resulted in 20 pseudochromosomes containing 99.44% of the total assembled sequences. The genome contained 67.35 Mb of repeat sequences, accounting for 14.2% of the assembly. A total of 22,067 protein‐coding genes were predicted, 94.82% of which were successfully annotated with putative functions. Furthermore, a phylogenetic tree was constructed using 1,872 single‐copy orthologous genes, and 67 unique gene families were identified in the filefish genome. This high‐quality assembled genome will be a valuable resource for a range of future genomic, conservation and breeding studies of T. septentrionalis.     

De novo assembly of a chromosome‐level reference genome of red‐spotted grouper (Epinephelus akaara) using nanopore sequencing and Hi‐C

The red‐spotted grouper Epinephelus akaara (E. akaara) is one of the most economically important marine fish in China, Japan and South‐East Asia and is a threatened species. The species is also considered a good model for studies of sex inversion, development, genetic diversity and immunity. Despite its importance, molecular resources for E. akaara remain limited and no reference genome has been published to date. In this study, we constructed a chromosome‐level reference genome of E. akaara by taking advantage of long‐read single‐molecule sequencing and de novo assembly by Oxford Nanopore Technology (ONT) and Hi‐C. A red‐spotted grouper genome of 1.135 Gb was assembled from a total of 106.29 Gb polished Nanopore sequence (GridION, ONT), equivalent to 96‐fold genome coverage. The assembled genome represents 96.8% completeness (BUSCO) with a contig N50 length of 5.25 Mb and a longest contig of 25.75 Mb. The contigs were clustered and ordered onto 24 pseudochromosomes covering approximately 95.55% of the genome assembly with Hi‐C data, with a scaffold N50 length of 46.03 Mb. The genome contained 43.02% repeat sequences and 5,480 noncoding RNAs. Furthermore, combined with several RNA‐seq data sets, 23,808 (99.5%) genes were functionally annotated from a total of 23,923 predicted protein‐coding sequences. The high‐quality chromosome‐level reference genome of E. akaara was assembled for the first time and will be a valuable resource for molecular breeding and functional genomics studies of red‐spotted grouper in the future.     

Chromosome‐level genome assembly of a cyprinid fish Onychostoma macrolepis by integration of nanopore sequencing,Bionano and Hi‐C technology

Onychostoma macrolepis is an emerging commercial cyprinid fish species. It is a model system for studies of sexual dimorphism and genome evolution. Here, we report the chromosome‐level assembly of the O.macrolepis genome obtained from the integration of nanopore long‐read sequencing with physical maps produced using Bionano and Hi‐C technology. A total of 87.9 Gb of nanopore sequence provided approximately 100‐fold coverage of the genome. The preliminary genome assembly was 883.2 Mb in size with a contig N50 size of 11.2 Mb. The 969 corrected contigs obtained from Bionano optical mapping were assembled into 853 scaffolds and produced an assembly of 886.5 Mb with a scaffold N50 of 16.5 Mb. Finally, using the Hi‐C data, 881.3 Mb (99.4% of genome) in 526 scaffolds were anchored and oriented in 25 chromosomes ranging in size from 25.27 to 56.49 Mb. In total, 24,770 protein‐coding genes were predicted in the genome, and ~96.85% of the genes were functionally annotated. The annotated assembly contains 93.3% complete genes from the BUSCO reference set. In addition, we identified 409 Mb (46.23% of the genome) of repetitive sequence, and 11,213 non‐coding RNAs, in the genome. Evolutionary analysis revealed that O. macrolepis diverged from common carp approximately 24.25 million years ago. The chromosomes of O. macrolepis showed an unambiguous correspondence to the chromosomes of zebrafish. The high‐quality genome assembled in this work provides a valuable genomic resource for further biological and evolutionary studies of O. macrolepis.     

Chromosome‐level genome assembly of the predator Propylea japonica to understand its tolerance to insecticides and high temperatures

The ladybird beetle Propylea japonica is an important natural enemy in agro‐ecological systems. Studies on the strong tolerance of P. japonica to high temperatures and insecticides, and its population and phenotype diversity have recently increased. However, abundant genome resources for obtaining insights into stress‐resistance mechanisms and genetic intra‐species diversity for P. japonica are lacking. Here, we constructed the P. japonica genome maps using Pacific Bioscience (PacBio) and Illumina sequencing technologies. The genome size was 850.90 Mb with a contig N50 of 813.13 kb. The Hi‐C sequence data were used to upgrade draft genome assemblies; 4,777 contigs were assembled to 10 chromosomes; and the final draft genome assembly was 803.93 Mb with a contig N50 of 813.98 kb and a scaffold N50 of 100.34 Mb. Approximately 495.38 Mb of repeated sequences was annotated. The 18,018 protein‐coding genes were predicted, of which 95.78% were functionally annotated, and 1,407 genes were species‐specific. The phylogenetic analysis showed that P. japonica diverged from the ancestor of Anoplophora glabripennis and Tribolium castaneum ~ 236.21 million years ago. We detected that some important gene families involved in detoxification of pesticides and tolerance to heat stress were expanded in P. japonica, especially cytochrome P450 and Hsp70 genes. Overall, the high‐quality draft genome sequence of P. japonica will provide invaluable resource for understanding the molecular mechanisms of stress resistance and will facilitate the research on population genetics, evolution and phylogeny of Coccinellidae. This genome will also provide new avenues for conserving the diversity of predator insects.     

A chromosome‐level genome assembly of the parasitoid wasp Pteromalus puparum

Parasitoid wasps represent a large proportion of hymenopteran species. They have complex evolutionary histories and are important biocontrol agents. To advance parasitoid research, a combination of Illumina short‐read, PacBio long‐read and Hi‐C scaffolding technologies was used to develop a high‐quality chromosome‐level genome assembly for Pteromalus puparum, which is an important pupal endoparasitoid of caterpillar pests. The chromosome‐level assembly has aided in studies of venom and detoxification genes. The assembled genome size is 338 Mb with a contig N50 of 38.7 kb and a scaffold N50 of 1.16 Mb. Hi‐C analysis assembled scaffolds onto five chromosomes and raised the scaffold N50 to 65.8 Mb, with more than 96% of assembled bases located on chromosomes. Gene annotation was assisted by RNA sequencing for the two sexes and four different life stages. Analysis detected 98% of the BUSCO (Benchmarking Universal Single‐Copy Orthologs) gene set, supporting a high‐quality assembly and annotation. In total, 40.1% (135.6 Mb) of the assembly is composed of repetitive sequences, and 14,946 protein‐coding genes were identified. Although venom genes play important roles in parasitoid biology, their spatial distribution on chromosomes was poorly understood. Mapping has revealed venom gene tandem arrays for serine proteases, pancreatic lipase‐related proteins and kynurenine–oxoglutarate transaminases, which have amplified in the P. puparum lineage after divergence from its common ancestor with Nasonia vitripennis. In addition, there is a large expansion of P450 genes in P. puparum. These examples illustrate how chromosome‐level genome assembly can provide a valuable resource for molecular, evolutionary and biocontrol studies of parasitoid wasps.     

Finding Nemo’s Genes: A chromosome‐scale reference assembly of the genome of the orange clownfish Amphiprion percula

The iconic orange clownfish, Amphiprion percula, is a model organism for studying the ecology and evolution of reef fishes, including patterns of population connectivity, sex change, social organization, habitat selection and adaptation to climate change. Notably, the orange clownfish is the only reef fish for which a complete larval dispersal kernel has been established and was the first fish species for which it was demonstrated that antipredator responses of reef fishes could be impaired by ocean acidification. Despite its importance, molecular resources for this species remain scarce and until now it lacked a reference genome assembly. Here, we present a de novo chromosome‐scale assembly of the genome of the orange clownfish Amphiprion percula. We utilized single‐molecule real‐time sequencing technology from Pacific Biosciences to produce an initial polished assembly comprised of 1,414 contigs, with a contig N50 length of 1.86 Mb. Using Hi‐C‐based chromatin contact maps, 98% of the genome assembly were placed into 24 chromosomes, resulting in a final assembly of 908.8 Mb in length with contig and scaffold N50s of 3.12 and 38.4 Mb, respectively. This makes it one of the most contiguous and complete fish genome assemblies currently available. The genome was annotated with 26,597 protein‐coding genes and contains 96% of the core set of conserved actinopterygian orthologs. The availability of this reference genome assembly as a community resource will further strengthen the role of the orange clownfish as a model species for research on the ecology and evolution of reef fishes.     

Genome sequencing and analysis of two early-flowering cherry (Cerasus × kanzakura) varieties, ‘Kawazu-zakura’ and ‘Atami-zakura’

To gain genetic insights into the early-flowering phenotype of ornamental cherry, also known as sakura, we determined the genome sequences of two early-flowering cherry (Cerasus × kanzakura) varieties, ‘Kawazu-zakura’ and ‘Atami-zakura’. Because the two varieties are interspecific hybrids, likely derived from crosses between Cerasus campanulata (early-flowering species) and Cerasus speciosa, we employed the haplotype-resolved sequence assembly strategy. Genome sequence reads obtained from each variety by single-molecule real-time sequencing (SMRT) were split into two subsets, based on the genome sequence information of the two probable ancestors, and assembled to obtain haplotype-phased genome sequences. The resultant genome assembly of ‘Kawazu-zakura’ spanned 519.8 Mb with 1,544 contigs and an N50 value of 1,220.5 kb, while that of ‘Atami-zakura’ totalled 509.6 Mb with 2,180 contigs and an N50 value of 709.1 kb. A total of 72,702 and 69,528 potential protein-coding genes were predicted in the genome assemblies of ‘Kawazu-zakura’ and ‘Atami-zakura’, respectively. Gene clustering analysis identified 2,634 clusters uniquely presented in the C. campanulata haplotype sequences, which might contribute to its early-flowering phenotype. Genome sequences determined in this study provide fundamental information for elucidating the molecular and genetic mechanisms underlying the early-flowering phenotype of ornamental cherry tree varieties and their relatives.