鸡T细胞受体γ链基因位点重新测序和组装

动物T细胞受体(T cell receptor,TCR)基因由多个不同的高度同源的基因家族组成,通过全基因组测序很难获得准确的基因序列和排列位置。文章通过在NCBI中发布的鸡TCR的γ链(TCRγ或TRG)基因片段序列定位了鸡TRG基因所在区域,并确定了与鸡TRG基因位点对应的细菌人工染色体(BAC)克隆(CH261-174P24)。对该克隆进行高通量的重新测序和组装后,得到含有10个scaffolds的基因组草图,较完整地覆盖了鸡TRG基因位点及两侧区域。通过PCR扩增和测序证明了scaffold内部结构的正确性,校正了鸡参考基因组TRG基因位点一个可变基因和一个缺口序列(gap)附近各一处错误序列,以及可变基因区多处序列错误。文章通过校正鸡参考基因组TRG基因位点的序列,为鸡TRA/D和TRB基因位点的基因组序列分析提供了新方法。     

鸡T细胞受体γ链基因位点重新测序和组装

动物T细胞受体(T cell receptor,TCR)基因由多个不同的高度同源的基因家族组成,通过全基因组测序很难获得准确的基因序列和排列位置。文章通过在NCBI中发布的鸡TCR的γ链(TCRγ或TRG)基因片段序列定位了鸡TRG基因所在区域,并确定了与鸡TRG基因位点对应的细菌人工染色体(BAC)克隆(CH261-174P24)。对该克隆进行高通量的重新测序和组装后,得到含有10个scaffolds的基因组草图,较完整地覆盖了鸡TRG基因位点及两侧区域。通过PCR扩增和测序证明了scaffold内部结构的正确性,校正了鸡参考基因组TRG基因位点一个可变基因和一个缺口序列(gap)附近各一处错误序列,以及可变基因区多处序列错误。文章通过校正鸡参考基因组TRG基因位点的序列,为鸡TRA/D和TRB基因位点的基因组序列分析提供了新方法。     

高通量测序序列比对研究综述

高通量测序技术的飞速发展,给生物信息学带来了新的机遇和挑战,第二代测序序列数量多、长度短使得原来的序列分析手段不再适用。近几年来,针对高通量测序的序列分析算法和软件日益增多,目前已有上百种,导致选择合适的软件成为一个难题。对第二代测序的测序类型、序列类型以及分析算法进行了总结和归纳,对现今常用的分析软件的序列的类型、长度以及软件应用算法、输入/输出格式、特点和功能等方面做了详细分析和比较并给出建议。分析了现今测序技术和序列分析存在的问题,预测了今后的发展方向。     

基孔肯雅病毒非结构蛋白基因合成中多位点缺失突变的校正方法

目的:建立一种PCR方法,以快速校正基孔肯雅病毒非结构蛋白基因合成过程中发生的多位点缺失突变。方法:用PCR方法合成基孔肯雅病毒非结构蛋白基因;对测序的克隆进行序列比对,分析不同克隆上缺失突变发生的位置,以保守区域互相重叠的寡核苷酸为上下游引物、以该区域测序正确的克隆为模板进行PCR扩增,得到所需片段,再将这些片段用PCR方法进一步组装成完整的基因序列并进行测序。结果:测序结果表明,经过2次PCR扩增,校正了基孔肯雅病毒非结构蛋白基因合成过程中发生的5个位点缺失突变。结论:得到序列正确的基孔肯雅病毒非结构蛋白基因。在进行基因合成过程中如发生多位点缺失突变,可利用该方法同时对以上突变进行校正,无须再合成引物,降低了实验操作难度,并提高了实验效率。     

快捷的定点突变效率近100%的大引物PCR方法

报道了一种新的PCR突变方法,它不需要纯化大引物或设计特别的旁侧引物．利用一个诱变引物和两个测序引物(T_m≤58℃)作为旁侧引物．第一轮PCR产物12.5 μl直接加入到50 μl的第二轮PCR反应体系作为模板和大引物,在开始第二轮PCR反应时,增加在68℃退火温度下进行10个循环的不对称PCR,这一步骤大大提高了通过600 bp或800 bp大引物所导致的突变效率．结果表明,该方法的产物能够达到高保真、97%～98%的突变效率和高产率．     

用于蜜蜂和熊蜂肠道微生物分类的细菌16S rRNA数据库优化

蜜蜂和熊蜂是重要的传粉昆虫, 对农业生产及生态平衡的维持具有重要作用。近年来, 研究发现蜜蜂及熊蜂肠道内含有大量微生物, 其组成简单、特异。正常的肠道微生物群落对蜜蜂的生长、激素调节、致病菌抵抗等具有重要作用。随着高通量测序的发展, 研究者们也可快速获得传粉蜂肠道微生物组成, 这给生物多样性和物种保护及蜂类健康等的研究带来了便捷。但是由于蜜蜂和熊蜂肠道微生物群落均由特殊菌种组成, 目前的细菌16S rRNA数据库无法对其进行准确的分类, 并且部分东方蜜蜂(Apis cerana)特有的肠道微生物菌种缺乏16S rRNA序列信息。本文从来源于5个不同省份的东方蜜蜂肠道中分离得到在东方蜜蜂中普遍含有的Apibacter菌属纯菌, 获取其全长16S rRNA序列, 并对目前蜜蜂和熊蜂肠道的5个核心菌种的分类进行了综述, 对其分类和命名进行了修正。根据蜜蜂肠道微生物的明确分类, 在目前常用的SILVA细菌分类数据库基础之上对其进行了命名及分类优化, 并加入东方蜜蜂中普遍含有的Apibacter序列, 从而获得了优化数据库Bee Gut Microbiota-Database (BGM-Db)。通过1组东方蜜峰及1组西方蜜蜂(Apis mellifera)的肠道菌群高通量测序结果, 分析不同数据库的表现, 我们发现相比于SILVA和Ribosomal Database Project (RDP), BGM-Db对蜜蜂肠道16S rRNA高通量测序短序列实现了菌种级别的分类, 分辨率更高。     

基于Cytb基因的NCBI数据库序列可靠性分析

本研究以常用于动物种属鉴定的Cytb基因位点为研究对象,利用所测得的30种盗猎案件中常见的野生动物Cytb基因部分片段序列及NCBI数据库中下载的该物种序列及其近缘物种序列,构建系统进化树。根据进化树的聚类情况,判断NCBI数据库中的相关基因序列或物种名称的正确性,并对其中错误序列的登陆号进行标记,以防对后续涉案动物的准确鉴定造成影响。分别从30种常见涉案野生动物(共35份样本)中提取线粒体DNA,并利用通用引物扩增线粒体DNA上的Cytb基因部分片段并进行测序分析。通过NCBI数据库的Blast比对功能,筛选出与本研究物种同源性由高到低的物种,并从NCBI基因数据库中下载此类近缘物种的Cytb基因序列共482条,利用MEGA软件构建该物种的系统进化树。比对发现NCBI中登录号为DQ246798、KP202269、AY286434等3个序列所对应物种拉丁名错误。登录号为AY509634、AJ131639、AM072747等5个序列所对应物种拉丁名可能存在疑问。NCBI数据库数据可靠性有待进一步验证,只能作为涉案物种鉴定的参考数据之一,可借助构建系统进化树等方法来确认其结果的准确性。     

链霉菌看家基因引物的设计与验证

看家基因的扩增与测序是进行多基因系统进化分析首先需要解决的问题。针对链霉菌这一群高(G C)mol%革兰氏阳性细菌,选定4个看家基因:atpD、recA、rpoB和trpB,利用NCBI数据库中已有的2个链霉菌和3个分枝杆菌的全基因组序列,以及另两个链霉菌的recA基因序列,通过软件分析设计了各基因的扩增和测序引物,并优化了扩增反应条件。从所试验的55株链霉菌中,均特异地扩增出了上述4个基因的片段,并成功进行了序列测定,验证了所设计引物的实用性。所归纳的引物设计方法可用于高(G C)mol%革兰氏阳性细菌的其它看家基因,以促进多基因系统进化研究的开展。     

基于涉案兽类12S rRNA基因的NCBI数据库序列可靠性分析

本研究以常用于动物种属鉴定的12S rRNA基因位点为研究对象,利用所测得的17种常见涉案兽类12S rRNA基因部分片段序列及NCBI数据库中下载的该物种DNA序列及其近缘物种DNA序列,构建系统进化树。根据进化树的聚类情况,判断NCBI数据库中的相关基因序列或物种名称的正确性,并对其中错误序列的登陆号进行标记,以防对后续涉案动物的准确鉴定造成影响。分别从17种常见涉案兽类(共26份样本)中提取线粒体DNA,并利用通用引物扩增线粒体DNA上的12S rRNA基因部分片段并进行测序分析。通过NCBI数据库的Blast比对功能,筛选出与本研究物种同源性由高到低的物种,并从NCBI基因数据库中下载此类近缘物种的12S rRNA基因序列共351条,利用MEGA7.0软件构建该物种及其近缘物种系统进化树。通过比对发现NCBI中登录号为KP202279等3个序列所对应物种拉丁名错误。登录号为AY184436等11个序列所对应物种拉丁名可能存在疑问。GenBank中某些物种拉丁名有同种异名现象。因此,NCBI数据库数据可靠性有待进一步验证,只能作为涉案物种鉴定的参考数据之一,可借助构建系统进化树等方法来确认其结果的准确性。     

台湾早熟蜜桃优良芽变新种质'五月红'的鉴定

桃是我国重要的果树种类,分布十分广泛,在水果生产中排第四位,但适合南方栽培的品种却稀少。该研究通过资源调查在广西大学标本园内的一棵台湾早熟蜜桃大枝上发现一个芽变新种质,命名为‘五月红'',并进一步对其主要生物学特性、植物学性状以及果实经济性状等进行观测分析,同时还通过SRAP分子标记检测与母株台湾早熟蜜桃的差异性。结果表明:‘五月红''芽变新种质成熟期比台湾早熟蜜桃提前20 d左右,5月上中旬成熟,果变大,核变小,可食率提高,且颜色均匀,可溶性固形物含量显著提高,保持了台湾早熟蜜桃需冷量低、品质优良和风味极佳的优点,属于特早熟优质种质资源; 通过琼脂糖电泳检测SRAP扩增组合引物me9/em5在‘五月红''DNA样品中能够扩增到1 200 bp大小的特异性条带,表明‘五月红''在DNA遗传水平上与母株台湾早熟蜜桃发生了变异,属于新的特早熟种质资源。‘五月红''芽变新种质的发现将为选育适合南方栽培的特早熟桃品种提供重要材料,有助于优化不同熟期品种的栽培结构,进而促进南方桃种植产业经济效益的提高。     

Dilute-‘N’-Go dideoxy sequencing of all DNA strands generated in multiplex LATE-PCR assays

We have recently described a Dilute-‘N’-Go protocol that greatly simplifies preparation and sequencing of both strands of an amplicon generated using linear-after-the-exponential (LATE)-PCR, an advanced form of asymmetric PCR . The same protocol can also be used to sequence all limiting primer strands in a multiplex LATE-PCR, by adding back each of the depleted limiting primers to a separate aliquot of the multiplex reaction. But, Dilute-‘N’-Go sequencing cannot be used directly to sequence each of the excess primer strands in the same multiplex reaction, because all of the excess primers are still present at high concentration. This report demonstrates for the first time that it is possible to sequence each of the excess primer strands using a modified Dilute-‘N’-Go protocol in which blockers are added to prevent all but one of the excess primers serving as the sequencing primer in separate aliquots. The optimal melting temperatures, positions and concentrations of blockers relative to their corresponding excess primers are defined in detail. We are using these technologies to measure DNA sequence changes in mitochondrial genomes that accompany aging and exposure to certain drugs.     

马铃薯瓢虫与茄二十八星瓢虫SCAR标记的建立及应用

为了探索快速鉴定马铃薯瓢虫Henosepilachna vigintioctomaculata(Motschulsky)和茄二十八星瓢虫Henosepilachna vigintioctopunctata(Fabricius)的分子生物学方法,本研究在随机扩增多态性DNA(random amplified polymorphic DNA,RAPD)的基础上,分别设计了可以鉴别两个物种的序列特征扩增区域(sequence characterized amplified regions,SCAR)标记。从随机合成的60条引物中筛选出来2条特异性引物(分别为OPI-6和OPJ-15),引物OPI-6在马铃薯瓢虫中扩增出约750 bp的特异性条带,引物OPJ-15在茄二十八星中扩增出约750 bp的特异性条带,根据测序结果设计了两对SCAR引物对筛选结果进行验证,发现根据OPI-6的测序结果所设计的SCAR引物(OPI-6 test)仅能在马铃薯瓢虫中扩增出645 bp的条带,而根据OPJ-15的测序结果所设计的SCAR引物(OPJ-15 test)仅能在茄二十八星瓢虫中扩增出436 bp的条带。这两对SCAR引物能够准确、稳定且快速地区分马铃薯瓢虫与茄二十八星瓢虫,对这两种害虫的精准防控具有重要意义。     

An improved method for utilizing high‐throughput amplicon sequencing to determine the diets of insectivorous animals

DNA analysis of predator faeces using high‐throughput amplicon sequencing (HTS) enhances our understanding of predator–prey interactions. However, conclusions drawn from this technique are constrained by biases that occur in multiple steps of the HTS workflow. To better characterize insectivorous animal diets, we used DNA from a diverse set of arthropods to assess PCR biases of commonly used and novel primer pairs for the mitochondrial gene, cytochrome oxidase C subunit 1 (COI). We compared diversity recovered from HTS of bat guano samples using a commonly used primer pair “ZBJ” to results using the novel primer pair “ANML.” To parameterize our bioinformatics pipeline, we created an arthropod mock community consisting of single‐copy (cloned) COI sequences. To examine biases associated with both PCR and HTS, mock community members were combined in equimolar amounts both pre‐ and post‐PCR. We validated our system using guano from bats fed known diets and using composite samples of morphologically identified insects collected in pitfall traps. In PCR tests, the ANML primer pair amplified 58 of 59 arthropod taxa (98%), whereas ZBJ amplified 24–40 of 59 taxa (41%–68%). Furthermore, in an HTS comparison of field‐collected samples, the ANML primers detected nearly fourfold more arthropod taxa than the ZBJ primers. The additional arthropods detected include medically and economically relevant insect groups such as mosquitoes. Results revealed biases at both the PCR and sequencing levels, demonstrating the pitfalls associated with using HTS read numbers as proxies for abundance. The use of an arthropod mock community allowed for improved bioinformatics pipeline parameterization.     

Efficient mutagenesis method for producing the templates of single nucleotide polymorphisms

DNA templates harboring specific single nucleotide polymorphism (SNP) sites are largely needed as positive controls in practical SNP analysis and in determination of the reliability of newly developed methods in high-throughput screening assays. Here we report a one-step method to produce SNP templates by amplifying a wild-type sequence with primers having single nucleotide mismatches at or near their 3′ ends. A short amplicon harboring an EcoRI site was used to evaluate the feasibility of our strategy. Perfectly matched primers and primers with a single base mismatch occurring from the first base to the sixth base of the EcoRI site were used for primer extension. By using polymerase without a proofreading function, we kept mismatched nucleotides from occurring in extended primer products, as confirmed by EcoRI digestion and sequencing analysis. The strategy of using primers with a single mismatched base and exo- polymerase was shown to be an efficient one-step method for preparing SNP templates, either for application in the development of SNP screening assays or as positive controls in practical SNP assays.     

Denaturing high-performance liquid chromatography in the detection of ABCA1 gene mutations in familial HDL deficiency

Mutations in the ABCA1 gene are the cause of familial high density lipoprotein deficiency (FHD). Because these mutations are spread over the entire gene, their detection requires the sequencing of all 50 exons. The aim of this study was to validate denaturing high-performance liquid chromatography (DHPLC) in mutation detection as an alternative to systematic sequencing. Exons of the ABCA1 gene were amplified using primers employed for sequencing. Temperatures for DHPLC were deducted from a software and empirically defined for each amplicon. To assess DHPLC reliability, we tested 30 sequence variants found in FHD patients and controls. Combined DHPLC and sequencing was applied to the genotyping of new FHD patients. Most of the amplicons required from two to five temperature conditions to obtain partially denatured DNA over the entire amplicon length. Twenty-nine of the variants found by sequencing were detected by DHPLC (97% sensitivity). The detection of the last variant (in exon 40) required different primers and amplification conditions. DHPLC and sequencing analysis of new FHD patients revealed that all amplicons showing a heteroduplex DHPLC profile contained sequence variants. No variants were detected in amplicons with a homoduplex profile. DHPLC is a sensitive and reliable method for the detection of ABCA1 gene mutations.     

Monoplex/multiplex linear-after-the-exponential-PCR assays combined with PrimeSafe and Dilute-'N'-Go sequencing

This protocol describes the design and execution of monoplex and multiplex linear-after-the-exponential (LATE)-PCR assays using a novel reagent, PrimeSafe, that suppresses all forms of mispriming. LATE-PCR is an advanced form of asymmetric amplification that uses a limiting primer and an excess primer for efficient exponential amplification of double-stranded DNA, followed by linear amplification of one strand. Each single-stranded amplicon can be quantitatively detected in real time or at end point. By separating primer annealing from product detection, LATE-PCR enables product analysis at low temperatures. Alternatively, each single strand can be sequenced by a convenient Dilute-'N'-Go procedure. Amplified samples are diluted with individual sequencing primers without the use of columns or spins. We have amplified and then sequenced 15 different single-stranded products generated in a single multiplexed LATE-PCR comprised of 15 pairs of unrelated primers. Dilute-'N'-Go dideoxy sequencing is more convenient, faster and less expensive than sequencing double-stranded amplicons generated via conventional symmetric PCR. The preparation of LATE-PCR products for Dilute-'N'-Go sequencing takes only 30 seconds.     

Biased Diversity Metrics Revealed by Bacterial 16S Pyrotags Derived from Different Primer Sets

In recent years, PCR-based pyrosequencing of 16S rRNA genes has continuously increased our understanding of complex microbial communities in various environments of the Earth. However, there is always concern on the potential biases of diversity determination using different 16S rRNA gene primer sets and covered regions. Here, we first report how bacterial 16S rRNA gene pyrotags derived from a series of different primer sets resulted in the biased diversity metrics. In total, 14 types of pyrotags were obtained from two-end pyrosequencing of 7 amplicon pools generated by 7 primer sets paired by 1 of 4 forward primers (V1F, V3F, V5F, and V7F) and 1 of 4 reverse primers (V2R, V4R, V6R, and V9R), respectively. The results revealed that: i) the activated sludge exhibited a large bacterial diversity that represented a broad range of bacterial populations and served as a good sample in this methodology research; ii) diversity metrics highly depended on the selected primer sets and covered regions; iii) paired pyrotags obtained from two-end pyrosequencing of each short amplicon displayed different diversity metrics; iv) relative abundance analysis indicated the sequencing depth affected the determination of rare bacteria but not abundant bacteria; v) the primer set of V1F and V2R significantly underestimated the diversity of activated sludge; and vi) the primer set of V3F and V4R was highly recommended for future studies due to its advantages over other primer sets. All of these findings highlight the significance of this methodology research and offer a valuable reference for peer researchers working on microbial diversity determination.     

A method to type the potential angucycline producers in actinomycetes isolated from marine sponges

Angucyclines are aromatic polyketides with antimicrobial, antitumor, antiviral and enzyme inhibition activities. In this study, a new pair of degenerate primers targeting the cyclase genes that are involved in the aromatization of the first and/or second ring of angucycline, were designed and evaluated in a PCR protocol targeting the jadomycin cyclase gene of Streptomyces venezuelae ISP5230. The identity of the target amplicon was confirmed by sequencing. After validation, the primers were used to screen 49 actinomycete isolates from three different marine sponges to identify putative angucycline producers. Seven isolates were positively identified using this method. Sequence analysis of the positive amplicons confirmed their identity as putative angucycline cyclases with sequence highly similar to known angucycline cyclases. Phylogenetic analysis clustered these positives into the angucycline group of cyclases. Furthermore, amplifications of the seven isolates using ketosynthase-specific primers were positive, backing the results using the cyclase primers. Together these results provided strong support for the presence of angucycline biosynthetic genes in these isolates. The specific primer set targeting the cyclase can be used to identify putative angucycline producers among marine actinobacteria, and aid in the discovery of novel angucyclines.     

Does peptide-nucleic acid (PNA) clamping of host plant DNA benefit ITS1 amplicon-based characterization of the fungal endophyte community?

Fungal endophyte community amplicon sequencing can lose a significant number of informative reads due to host-plant co-amplification. Blocking of plant-specific sequences with peptide nucleic acid (PNA) clamps has been shown to improve metrics of detected microbial diversity in studies targeting 16S and 18S regions of rRNA genes. However, PNA clamping has not been applied to the plant ITS region of rRNA gene – a widely accepted fungal marker. By applying PNA clamping technique to ITS amplicon sequencing of the endophytic fungal community of elderberry this study shows that PNA clamping significantly reduces host-plant co-amplification with the universal ITS1/ITS4 primer set. However, PNA clamping in combination with the discriminatory ITS1F/ITS2 primer set did not improve the metrics of fungal endophyte community ITS amplicon Illumina sequencing. This study shows that PNA clamping does not add practical benefit to taxonomic profiling of plant-associated fungal communities if the primers are already specific enough to exclude amplification of host DNA.     

Ordered catenation of sequence-tagged sites and multiplexed SNP genotyping by sequencing

We describe a method for the efficient genotyping of SNPs, involving sequencing of ordered and catenated sequence-tagged sites (OCS). In OCS, short genomic segments, each containing an SNP, are amplified by PCR using primers that carry specially designed extra nucleotides at their 5′-ends. Amplification products are then combined and converted to a concatamer in a defined order by a second round of thermal cycling. The concatenation takes place because the 5′-ends of each amplicon are designed to be complementary to the ends of the presumptive neighboring amplicons. The primer sequences for OCS are chosen using newly developed dedicated software, OCS Optimizer. Using sets of SNPs, we show that at least 10 STSs can be concatenated in a predefined order and all SNPs in the STSs are accurately genotyped by one two-way sequencing reaction.