Food consumption and diet composition of the web-building spider Agelena limbata in two habitats

Summary Prey capture rate, food consumption, and diet composition of all developmental stages of the funnelweb spider Agelena limbata were estimated in woody and open habitats by a sight-count method. Prey availability was evaluated on the basis of two indices, i.e. the ratios of daily food consumption to dry weight of predator and to daily standard metabolic rate. These indices varied seasonally and between instars in this spider. Comparison of these indices between arthropod predators suggests that A. limbata live under conditions of relatively limited food supply. In the open habitat, the spiders reduced foraging activities to avoid heat stress at midday in summer because the sheet web was exposed to the direct rays of the sun and its temperature exceeded 40°C. The daily food consumption of adult spiders in the open habitat was about half of that in the woody habitat. The lower rate of energy intake of spiders in the open habitat may cause the observed smaller size of adults and lower fecundity. A. limbata captured a great range of prey comprising ten orders of arthropods and ate chemically defended insects, e.g. stink bugs, lady beetles, and ants which were rejected by many spiders. This generalistic foraging may be associated with limited and heterogeneous food supply in this spider.     

Next-generation sequencing approaches in genetic rodent model systems to study functional effects of human genetic variation

Rapid advances in DNA sequencing improve existing techniques and enable new approaches in genetics and functional genomics, bringing about unprecedented coverage, resolution and sensitivity. Enhanced toolsets can facilitate the untangling of connections between genomic variation, environmental factors and phenotypic effects, providing novel opportunities, but may also pose challenges in data interpretation, especially in highly heterogeneous human populations. Laboratory rodent strains, however, offer a variety of tailored model systems with controlled genetic backgrounds, facilitating complex genotype/phenotype relationship studies. In this review we discuss the advent of massively parallel sequencing, its methodological advantage for molecular analysis in model organisms and the expectation of increased understanding of biologically relevant consequences of human genetic variation.     

Application of Next-generation Sequencing Technology in Forensic Science

Next-generation sequencing （NGS） technology, with its high-throughput capacity and low cost, has developed rapidly in recent years and become an important analytical tool for many genomics researchers. New opportunities in the research domain of the forensic studies emerge by harnessing the power of NGS technology, which can be applied to simultaneously analyzing multi- ple loci of forensic interest in different genetic contexts, such as autosomes, mitochondrial and sex chromosomes. Furthermore, NGS technology can also have potential applications in many other aspects of research. These include DNA database construction, ancestry and phenotypic inference, monozygotic twin studies, body fluid and species identification, and forensic animal, plant and microbiological analyses. Here we review the application of NGS technology in the field of forensic science with the aim of providing a reference for future forensics studies and practice.     

Management of Incidental Findings in the Era of Next-generation Sequencing

Next-generation sequencing (NGS) technologies allow for the generation of whole exome or whole genome sequencing data, which can be used to identify novel genetic alterations associated with defined phenotypes or to expedite discovery of functional variants for improved patient care. Because this robust technology has the ability to identify all mutations within a genome, incidental findings (IF)- genetic alterations associated with conditions or diseases unrelated to the patient’s present condition for which current tests are being performed- may have important clinical ramifications. The current debate among genetic scientists and clinicians focuses on the following questions: 1) should any IF be disclosed to patients, and 2) which IF should be disclosed – actionable mutations, variants of unknown significance, or all IF? Policies for disclosure of IF are being developed for when and how to convey these findings and whether adults, minors, or individuals unable to provide consent have the right to refuse receipt of IF. In this review, we detail current NGS technology platforms, discuss pressing issues regarding disclosure of IF, and how IF are currently being handled in prenatal, pediatric, and adult patients.     

Promises and pitfalls of using high‐throughput sequencing for diet analysis

The application of high‐throughput sequencing‐based approaches to DNA extracted from environmental samples such as gut contents and faeces has become a popular tool for studying dietary habits of animals. Due to the high resolution and prey detection capacity they provide, both metabarcoding and shotgun sequencing are increasingly used to address ecological questions grounded in dietary relationships. Despite their great promise in this context, recent research has unveiled how a wealth of biological (related to the study system) and technical (related to the methodology) factors can distort the signal of taxonomic composition and diversity. Here, we review these studies in the light of high‐throughput sequencing‐based assessment of trophic interactions. We address how the study design can account for distortion factors, and how acknowledging limitations and biases inherent to sequencing‐based diet analyses are essential for obtaining reliable results, thus drawing appropriate conclusions. Furthermore, we suggest strategies to minimize the effect of distortion factors, measures to increase reproducibility, replicability and comparability of studies, and options to scale up DNA sequencing‐based diet analyses. In doing so, we aim to aid end‐users in designing reliable diet studies by informing them about the complexity and limitations of DNA sequencing‐based diet analyses, and encourage researchers to create and improve tools that will eventually drive this field to its maturity.     

Rapid identification and recovery of ENU-induced mutations with next-generation sequencing and Paired-End Low-Error analysis

Background

Targeting Induced Local Lesions IN Genomes (TILLING) is a reverse genetics approach to directly identify point mutations in specific genes of interest in genomic DNA from a large chemically mutagenized population. Classical TILLING processes, based on enzymatic detection of mutations in heteroduplex PCR amplicons, are slow and labor intensive.

Results

Here we describe a new TILLING strategy in zebrafish using direct next generation sequencing (NGS) of 250bp amplicons followed by Paired-End Low-Error (PELE) sequence analysis. By pooling a genomic DNA library made from over 9,000 N-ethyl-N-nitrosourea (ENU) mutagenized F1 fish into 32 equal pools of 288 fish, each with a unique Illumina barcode, we reduce the complexity of the template to a level at which we can detect mutations that occur in a single heterozygous fish in the entire library. MiSeq sequencing generates 250 base-pair overlapping paired-end reads, and PELE analysis aligns the overlapping sequences to each other and filters out any imperfect matches, thereby eliminating variants introduced during the sequencing process. We find that this filtering step reduces the number of false positive calls 50-fold without loss of true variant calls. After PELE we were able to validate 61.5% of the mutant calls that occurred at a frequency between 1 mutant call:100 wildtype calls and 1 mutant call:1000 wildtype calls in a pool of 288 fish. We then use high-resolution melt analysis to identify the single heterozygous mutation carrier in the 288-fish pool in which the mutation was identified.

Conclusions

Using this NGS-TILLING protocol we validated 28 nonsense or splice site mutations in 20 genes, at a two-fold higher efficiency than using traditional Cel1 screening. We conclude that this approach significantly increases screening efficiency and accuracy at reduced cost and can be applied in a wide range of organisms.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1263-4) contains supplementary material, which is available to authorized users.     

Evaluation of variant identification methods for whole genome sequencing data in dairy cattle

Background

Advances in human genomics have allowed unprecedented productivity in terms of algorithms, software, and literature available for translating raw next-generation sequence data into high-quality information. The challenges of variant identification in organisms with lower quality reference genomes are less well documented. We explored the consequences of commonly recommended preparatory steps and the effects of single and multi sample variant identification methods using four publicly available software applications (Platypus, HaplotypeCaller, Samtools and UnifiedGenotyper) on whole genome sequence data of 65 key ancestors of Swiss dairy cattle populations. Accuracy of calling next-generation sequence variants was assessed by comparison to the same loci from medium and high-density single nucleotide variant (SNV) arrays.

Results

The total number of SNVs identified varied by software and method, with single (multi) sample results ranging from 17.7 to 22.0 (16.9 to 22.0) million variants. Computing time varied considerably between software. Preparatory realignment of insertions and deletions and subsequent base quality score recalibration had only minor effects on the number and quality of SNVs identified by different software, but increased computing time considerably. Average concordance for single (multi) sample results with high-density chip data was 58.3% (87.0%) and average genotype concordance in correctly identified SNVs was 99.2% (99.2%) across software. The average quality of SNVs identified, measured as the ratio of transitions to transversions, was higher using single sample methods than multi sample methods. A consensus approach using results of different software generally provided the highest variant quality in terms of transition/transversion ratio.

Conclusions

Our findings serve as a reference for variant identification pipeline development in non-human organisms and help assess the implication of preparatory steps in next-generation sequencing pipelines for organisms with incomplete reference genomes (pipeline code is included). Benchmarking this information should prove particularly useful in processing next-generation sequencing data for use in genome-wide association studies and genomic selection.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-948) contains supplementary material, which is available to authorized users.     

二代测序法深度分析水痘疫苗Oka株病毒基因特征

采用二代测序(next-generation sequencing,NGS)技术,对上海生物制品研究所有限责任公司(简称SIBP)2015-2017这3个年度生产的Oka株水痘疫苗进行深度测序,从分子水平分析疫苗质量的一致性,并与不同厂家疫苗/毒种进行比较,分析它们之间的遗传特征相关性。结果显示,3个年度生产的水痘疫苗中平均有77个位点的疫苗型位点检出频率(proportion of vaccine-type allele,Pv)≥10%,至少在两批疫苗中出现Pv≥10%的位点数共76个,两两之间Pv最大差值<10%,与平均值的最大差值为<5%。共有40个位点的Pv均值≥30%,其中19个位点导致氨基酸残基变异;15个位于ORF62区域。筛选出35个位点,比较5个公司水痘疫苗/毒种的Oka株病毒基因序列,结果显示它们之间存在相关性,同时存在一定的差异。研究表明,SIBP不同年度生产的水痘疫苗Oka株病毒具有良好的分子水平上的一致性,为疫苗质量管理与市场监督提供了方法和基础数据。     

A comparison of next-generation sequencing analysis methods for cancer xenograft samples

The application of next-generation sequencing (NGS) technology in cancer is influenced by the quality and purity of tissue samples. This issue is especially critical for patient-derived xenograft (PDX) models, which have proven to be by far the best preclinical tool for investigating human tumor biology, because the sensitivity and specificity of NGS analysis in xenograft samples would be compromised by the contamination of mouse DNA and RNA. This definitely affects downstream analyses by causing inaccurate mutation calling and gene expression estimates. The reliability of NGS data analysis for cancer xenograft samples is therefore highly dependent on whether the sequencing reads derived from the xenograft could be distinguished from those originated from the host. That is, each sequence read needs to be accurately assigned to its original species. Here, we review currently available methodologies in this field, including Xenome, Disambiguate, bamcmp and pdxBlacklist, and provide guidelines for users.     

二代测序技术在结核分枝杆菌研究中的应用进展

结核病是由结核分枝杆菌引起的全球第二大传染病。二代测序技术为从基因组水平研究结核分枝杆菌提供了重要的研究方法。本文从结核病流行病学、结核分枝杆菌耐药和进化及相关生物信息学等方面,介绍二代测序技术在结核分枝杆菌研究中的应用进展。     

Diversity of feeding habitats and diet composition in the turtle doves Streptopelia turtur to buffer loss and modification of natural habitats during breeding season

The approach of the birds to use physical and alimentary resources in degraded and modified natural habitats is an important aspect of their adaptation. This study was undertaken during 2016-2017 at forty habitats in the Moulouya plain, Morocco to examine behavioral diet, habitat use and foraging ecology of turtle dove, Streptopelia turtur. We monitored turtle doves in four major plots namely cereal plots, lucerne farms, apple orchards, and stations in the Ansgmir River covering 40 habitats. Digestive tract contents were also identified and evaluated for 68 Turtle Doves shot by hunters during two consecutive years. The results showed that the turtle doves use a variability of feeding habitats. The cereal cultivation seemed to be more preferable habitat for feeding especially in the month of May, the first breeding phase of the year. But, during the months of August and July, the riverbanks were the preferred habitat for turtle doves. The diet of this species is polyphagous and diverse with a granivorous tendency. Diet analysis showed that soft wheat and barley seeds constituted 44.53% and 38.74% respectively followed by barley seeds with 38.74% and sand stones (9.16%) of the seeds eaten by Turtle Doves. However, moderate proportion of elements (7.32%) remained undefined. All these aspects, including the variability of feeding habitats and the wide diet seem to be an adaptive strategy followed by turtle dove to counter the degradation and the modification of its natural feeding habitats.     

高通量测序技术在野生动物食性分析中的应用

食性研究是动物生态学颇受关注的一个重要内容,而食性分析方法由于受到技术和适用范围的限制,也在不断改进和更新。随着高通量测序技术的发展,该技术逐渐扩展到野生动物的食性分析,使食性分析的效率得到极大提升,并拓宽了食性分析的应用范围。尽管高通量测序应用于食性分析在数据量、灵敏度和分辨率方面的优势较为明显,但由于涉及到的步骤较多,受到的影响因素较为复杂,目前高通量测序应用于食性分析还属于研究比较薄弱的领域。概述了高通量测序技术应用于食性分析的基本流程,总结了该技术在食物组成分析、种内和种间食性关系、食物与栖息地、行为关系方面的研究动态,分析了PCR、污染和定量分析对该技术应用性的影响,提出了相应的解决对策和建议,并对其应用前景进行了展望。     

The Role of Quality Control in Targeted Next-generation Sequencing Library Preparation

Next-generation sequencing (NGS) is getting routinely used in the diagnosis of hereditary diseases, such as human cardiomyopathies. Hence, it is of utter importance to secure high quality sequencing data, enabling the identification of disease-relevant mutations or the conclusion of neg-ative test results. During the process of sample preparation, each protocol for target enrichment library preparation has its own requirements for quality control (QC); however, there is little evi-dence on the actual impact of these guidelines on resulting data quality. In this study, we analyzed the impact of QC during the diverse library preparation steps of Agilent SureSelect XT target enrichment and Illumina sequencing. We quantified the parameters for a cohort of around 600 sam-ples, which include starting amount of DNA, amount of sheared DNA, smallest and largest frag-ment size of the starting DNA; amount of DNA after the pre-PCR, and smallest and largest fragment size of the resulting DNA;as well as the amount of the final library, the corresponding smallest and largest fragment size, and the number of detected variants. Intriguingly, there is a high tolerance for variations in all QC steps, meaning that within the boundaries proposed in the current study, a considerable variance at each step of QC can be well tolerated without compromising NGS quality.     

Next-generation sequencing in two cases of de novo acute basophilic leukaemia

Acute basophilic leukaemia (ABL) is a rare subtype of acute myeloid leukaemia (AML); therefore, few data are available about its biology. Herein, we analysed two ABL patients using flow cytometry and next-generation sequencing (NGS). Two cell populations were detected by flow cytometry in both patients. In Case no. 1, blasts (CD34⁺, CD203c⁻, CD117⁺, CD123dim⁺) and basophils (CD34⁻, CD203c⁺, CD117^±, CD123⁺) were identified, both of which were found by NGS to harbour the 17p deletion and have loss of heterozygosity of TP53. In Case no. 2, blasts (CD33⁺, CD34⁺, CD123⁻) and basophils (CD33⁺, CD34⁺, CD123⁺) were identified. NGS detected NPM1 mutations in either blasts or basophils, and TET2 in both. These data suggest an overlap of the mutational landscape of ABL and AML, including TP53 and TET2 mutations. Moreover, additional mutations or epigenetic factors may contribute for the differentiation into basophilic blasts.     

Toward next-generation sequencing of mitochondrial genomes — Focus on parasitic worms of animals and biotechnological implications

Helminths (worms) include parasitic nematodes (roundworms) and platyhelminths (flatworms). These worms are abundant, and many of them are of agricultural, aquacultural, veterinary and medical importance and cause substantial socioeconomic losses worldwide. The genetic characterization of parasitic nematodes using advanced molecular tools is central to the diagnosis of infections and the control of parasitism. The accurate analysis of genetic variation also underpins studies of their taxonomy, epidemiology and evolutionary history. Although the nuclear genome contains suitable genetic markers (e.g., in ribosomal DNA) for the identification of many species, the large size and high variability of the mt genome consistently provides a rich source of such markers for informative systematic and epidemiological studies both within and among species. There is significant value in establishing a practical platform for the rapid sequencing, annotation and analysis of mt genomic datasets to underpin such fundamental and applied studies of parasitic worms (= helminths). In the last decade, there have been some important advances in the mt genomics of helminths, but next-generation sequencing (NGS) technologies now provide opportunities for high throughput sequencing, assembly and annotation. In this article, we provide a background on mt genomics, cover technological challenges and recent advances, and provide a perspective on future mt genome research of parasitic helminths and its fundamental scientific and biotechnological implications.     

高通量测序分析多种典型生境中厌氧氨氧化细菌的多样性分布特征

【背景】厌氧氨氧化过程是氮素循环过程的重要途径之一,在氮素循环中发挥重要作用。先前的研究已经证实了厌氧氨氧化细菌存在于多种生境中,但对其多样性分布还没有系统的研究。【目的】对厌氧氨氧化细菌在不同类型生境中的多样性分布规律进行深入分析,充分展示其在不同生境中的群落结构特点,并揭示多样性分布与环境因素之间的关系。【方法】在建立厌氧氨氧化细菌16S rRNA基因序列数据库的基础上,运用高通量测序技术分析其在不同生境中的多样性分布特征。【结果】厌氧氨氧化细菌在红树林、海湾和河口生境中的多样性水平较高,而污泥和红壤的多样性水平明显较低。系统发育分析表明,这些生境中的厌氧氨氧化细菌主要由Candidatus Brocadia、Ca.Scalindua和未明确分类地位的菌属组成;从河流到红树林生态系统,随着盐度的增加,厌氧氨氧化细菌的优势种属由Ca. Brocadia转变到Ca. Scalindua,相关性分析也表明了盐度是导致不同生境中厌氧氨氧化细菌群落结构差异的主要因素。【结论】不同生境中存在不同的厌氧氨氧化细菌种群结构,环境条件的差异影响了厌氧氨氧化细菌的种群分布和系统演化。     

Characterization of 11 novel polymorphic microsatellite loci in the threatened Korean loach,Iksookimia koreensis,isolated using a next-generation sequencing method

To investigate the genetic diversity and population structure of Iksookimia koreensis, we characterized 11 novel polymorphic microsatellite markers developed using next-generation sequencing. The number of alleles per locus ranged from 4 to 10 (average = 6.26). Observed and expected heterozygosities ranged from 0.333 to 0.866 and from 0.375 to 0.866, respectively. No loci showed significant deviation from Hardy–Weinberg equilibrium (HWE). These loci were also used successfully to study the genetic diversity of a closely related species, Iksookimia longicorpa. Four of the 11 loci amplified in the two species showed different allele frequency and distribution, indicating deep genetic divergence between I. koreensis and I. longicorpa. The newly developed microsatellite markers reported here will provide a useful tool for examining gene flow, population genetic structure, and genetic diversity of these species.     

不同生境草鱼肠道微生物组成和群落特征分析

[目的]分析不同生境来源的草鱼前肠、中肠和后肠的微生物组成和群落特征.[方法]利用16S rRNA高通量测序技术比较河流、湖泊、高密度池塘养殖与水库低密度养殖4种不同生境来源的草鱼其前、中、后肠的微生物组成和群落特征.[结果]Venn图、稀释性曲线和Alpha指数分析结果显示,前肠微生物群落多样性以养殖生境草鱼更高,而...