A rapid column-based ancient DNA extraction method for increased sample throughput

Genetic analyses using museum specimens and ancient DNA from fossil samples are becoming increasingly important in phylogenetic and especially population genetic studies. Recent progress in ancient DNA sequencing technologies has substantially increased DNA sequence yields and, in combination with barcoding methods, has enabled large-scale studies using any type of DNA. Moreover, more and more studies now use nuclear DNA sequences in addition to mitochondrial ones. Unfortunately, nuclear DNA is, due to its much lower copy number in living cells compared to mitochondrial DNA, much more difficult to obtain from low-quality samples. Therefore, a DNA extraction method that optimizes DNA yields from low-quality samples and at the same time allows processing many samples within a short time frame is immediately required. In fact, the major bottleneck in the analysis process using samples containing low amounts of degraded DNA now lies in the extraction of samples, as column-based methods using commercial kits are fast but have proven to give very low yields, while more efficient methods are generally very time-consuming. Here, we present a method that combines the high DNA yield of batch-based silica extraction with the time-efficiency of column-based methods. Our results on Pleistocene cave bear samples show that DNA yields are quantitatively comparable, and in fact even slightly better than with silica batch extraction, while at the same time the number of samples that can conveniently be processed in parallel increases and both bench time and costs decrease using this method. Thus, this method is suited for harvesting the power of high-throughput sequencing using the DNA preserved in the millions of paleontological and museums specimens.     

Chelex without boiling, a rapid and easy technique to obtain stable amplifiable DNA from small amounts of ethanol-stored spiders

DNA barcoding projects require high-throughput generation of sequence data to assemble the comprehensive reference databases that are required to perform large-scale biodiversity inventories and molecular ecology studies. With the advent of new sequencing technologies, the extraction step, which often requires a considerable amount of time and money, represents a significant bottleneck in many studies. Here, we present a one-step Chelex double-stranded DNA extraction protocol that is quick, cheap, easy and works with a small quantity of ethanol-stored tissue. We developed this protocol by removing the denaturation step appearing in classic methods. This modification reduces the number of handling steps to one, thus simplifying the extraction procedure and reducing the risk of sample contamination, and yields double-stranded DNA instead of the single-stranded form that classical Chelex extraction protocols usually release. DNA obtained through our method is then suitable for long-term conservation (over 1.5 years). We tested our protocol on a highly diverse genus of spiders comprised of mainly very small species. We also apply the method to two other genera of spiders, one with average size species, the other one with giant species, to test the efficacy of the method with varying amounts of input tissue. We also discuss the advantages and limitations of this DNA extraction technique when working with arthropods.     

Comparative Study of Seven Commercial Kits for Human DNA Extraction from Urine Samples Suitable for DNA Biomarker-Based Public Health Studies

Human genomic DNA extracted from urine could be an interesting tool for large-scale public health studies involving characterization of genetic variations or DNA biomarkers as a result of the simple and noninvasive collection method. These studies, involving many samples, require a rapid, easy, and standardized extraction protocol. Moreover, for practicability, there is a necessity to collect urine at a moment different from the first void and to store it appropriately until analysis. The present study compared seven commercial kits to select the most appropriate urinary human DNA extraction procedure for epidemiological studies. DNA yield has been determined using different quantification methods: two classical, i.e., NanoDrop and PicoGreen, and two species-specific real-time quantitative (q)PCR assays, as DNA extracted from urine contains, besides human, microbial DNA also, which largely contributes to the total DNA yield. In addition, the kits giving a good yield were also tested for the presence of PCR inhibitors. Further comparisons were performed regarding the sampling time and the storage conditions. Finally, as a proof-of-concept, an important gene related to smoking has been genotyped using the developed tools. We could select one well-performing kit for the human DNA extraction from urine suitable for molecular diagnostic real-time qPCR-based assays targeting genetic variations, applicable to large-scale studies. In addition, successful genotyping was possible using DNA extracted from urine stored at −20°C for several months, and an acceptable yield could also be obtained from urine collected at different moments during the day, which is particularly important for public health studies.     

First record of Teleogryllus (Brachyteleogryllus) marini Otte & Alexander, 1983 (Orthoptera: Gryllidae) in korea and discussion of its continued misidentification using DNA barcoding

DNA barcoding is useful for identifying species that are difficult to distinguish via morphological analysis. However, if the public DNA barcode database includes misidentified DNA samples, subsequent molecular studies could generate incorrect results. Here, we report a misidentified DNA barcode for the North Coastal Black Field Cricket Teleogryllus (Brachyteleogryllus) marini. T. (B.) marini is routinely misidentified using DNA barcoding; it has been reported as Teleogryllus (Brachyteleogryllus) commodus since its misidentification in various studies of Asian samples. Here, we report the first occurrence of T. (B.) marini in Korea, along with its morphological diagnosis, bioacoustic signals, and DNA barcoding findings. T. (B.) marini is transferred to the subgenus Brachyteleogryllus from the subgenus Teleogryllus, based on the male genitalia morphology and phylogenetic analysis. Genetic distance analyses have shown that cytochrome c oxidase I barcoding is useful for species identification of the genus Teleogryllus, but detailed morphological investigations are essential for DNA barcoding before any molecular study.     

Which specimens from a museum collection will yield DNA barcodes? A time series study of spiders in alcohol

We report initial results from an ongoing effort to build a library of DNA barcode sequences for Dutch spiders and investigate the utility of museum collections as a source of specimens for barcoding spiders. Source material for the library comes from a combination of specimens freshly collected in the field specifically for this project and museum specimens collected in the past. For the museum specimens, we focus on 31 species that have been frequently collected over the past several decades. A series of progressively older specimens representing these 31 species were selected for DNA barcoding. Based on the pattern of sequencing successes and failures, we find that smaller-bodied species expire before larger-bodied species as tissue sources for single-PCR standard DNA barcoding. Body size and age of oldest successful DNA barcode are significantly correlated after factoring out phylogenetic effects using independent contrasts analysis. We found some evidence that extracted DNA concentration is correlated with body size and inversely correlated with time since collection, but these relationships are neither strong nor consistent. DNA was extracted from all specimens using standard destructive techniques involving the removal and grinding of tissue. A subset of specimens was selected to evaluate nondestructive extraction. Nondestructive extractions significantly extended the DNA barcoding shelf life of museum specimens, especially small-bodied species, and yielded higher DNA concentrations compared to destructive extractions. All primary data are publically available through a Dryad archive and the Barcode of Life database.     

Influence of killing method on Lepidoptera DNA barcode recovery

The global DNA barcoding initiative has revolutionized the field of biodiversity research. Such large‐scale sequencing projects require the collection of large numbers of specimens, which need to be killed and preserved in a way that is both DNA‐friendly and which will keep voucher specimens in good condition for later study. Factors such as time since collection, correct storage (exposure to free water and heat) and DNA extraction protocol are known to play a role in the success of downstream molecular applications. Limited data are available on the most efficient, DNA‐friendly protocol for killing. In this study, we evaluate the quality of DNA barcode (cytochrome oxidase I) sequences amplified from DNA extracted from specimens collected using three different killing methods (ethyl acetate, cyanide and freezing). Previous studies have suggested that chemicals, such as ethyl acetate and formaldehyde, degraded DNA and as such may not be appropriate for the collection of insects for DNA‐based research. All Lepidoptera collected produced DNA barcodes of good quality, and our study found no clear difference in nucleotide signal strength, probability of incorrect base calling and phylogenetic utility among the three different treatment groups. Our findings suggest that ethyl acetate, cyanide and freezing can all be used to collect specimens for DNA analysis.     

植物DNA条形码与生物多样性数据共享平台构建

DNA条形码基于较短的DNA序列实现物种的快速、准确鉴定, 不仅加快了全球生物物种的鉴定和分类步伐, 也为生物多样性的管理、保护和可持续利用提供了新思路和研究方法。植物DNA条形码标准数据库的不断完善, 将使植物多样性信息的快速获取成为可能; 将不同类型数据资源整合、共享和利用, 构建植物DNA条形码数据共享平台, 是满足公众对物种准确鉴定和快速认知的重要支撑。本文介绍了近年来植物DNA条形码的研究进展; 植物DNA条形码参考数据库的研发现状和存在的问题。结合上述问题, 围绕“大数据”时代背景, 对如何管理和使用好海量的植物信息, 如何构建数据共享平台提出了一些设想: (1)数据共享平台的元数据应尽可能翔实、丰富、准确和多关联; (2)数据标准应统一规范; (3)查询入口方便、迅速、多样, 易于管理, 便于实现更大程度的数据共享和全球化的合作交流。     

A robust universal method for extraction of genomic DNA from bacterial species

The intactness of DNA is the keystone of genome-based clinical investigations, where rapid molecular detection of life-threatening bacteria is largely dependent on the isolation of high-quality DNA. Various protocols have been so far developed for genomic DNA isolation from bacteria, most of which have been claimed to be reproducible with relatively good yields of high-quality DNA. Nonetheless, they are not fully applicable to various types of bacteria, their processing cost is relatively high, and some toxic reagents are used. The routine protocols for DNA extraction appear to be sensitive to species diversity, and may fail to produce high-quality DNA from different species. Such protocols remain time-consuming and tedious, thus to resolve some of these impediments, we report development of a very simple, rapid, and high-throughput protocol for extracting of high-quality DNA from different bacterial species. Based upon our protocol, interfering phenolic compounds were removed from extraction using polyvinylpyrrolidone (PVP) and RNA contamination was precipitated using LiCl. The UV spectrophotometry and gel electrophoresis analysis resulted in high A ₂₆₀/A ₂₈₀ ratio (>1.8) with high intactness of DNA. Subsequent evaluations were performed using some quality-dependent techniques (e.g., RAPD marker and restriction digestions). The isolated DNA from 9 different bacterial species confirmed the accuracy of this protocol which requires no enzymatic processing and accordingly its low-cost making it an appropriate method for large-scale DNA isolation from various bacterial species.     

Choosing and using a plant DNA barcode

The main aim of DNA barcoding is to establish a shared community resource of DNA sequences that can be used for organismal identification and taxonomic clarification. This approach was successfully pioneered in animals using a portion of the cytochrome oxidase 1 (CO1) mitochondrial gene. In plants, establishing a standardized DNA barcoding system has been more challenging. In this paper, we review the process of selecting and refining a plant barcode; evaluate the factors which influence the discriminatory power of the approach; describe some early applications of plant barcoding and summarise major emerging projects; and outline tool development that will be necessary for plant DNA barcoding to advance.     

Molecular-based rapid inventories of sympatric diversity: A comparison of DNA barcode clustering methods applied to geography-based vs clade-based sampling of amphibians

Molecular markers offer a universal source of data for quantifying biodiversity. DNA barcoding uses a standardized genetic marker and a curated reference database to identify known species and to reveal cryptic diversity within well-sampled clades. Rapid biological inventories, e.g. rapid assessment programs (RAPs), unlike most barcoding campaigns, are focused on particular geographic localities rather than on clades. Because of the potentially sparse phylogenetic sampling, the addition of DNA barcoding to RAPs may present a greater challenge for the identification of named species or for revealing cryptic diversity. In this article we evaluate the use of DNA barcoding for quantifying lineage diversity within a single sampling site as compared to clade-based sampling, and present examples from amphibians. We compared algorithms for identifying DNA barcode clusters (e.g. species, cryptic species or Evolutionary Significant Units) using previously published DNA barcode data obtained from geography-based sampling at a site in Central Panama, and from clade-based sampling in Madagascar. We found that clustering algorithms based on genetic distance performed similarly on sympatric as well as clade-based barcode data, while a promising coalescent-based method performed poorly on sympatric data. The various clustering algorithms were also compared in terms of speed and software implementation. Although each method has its shortcomings in certain contexts, we recommend the use of the ABGD method, which not only performs fairly well under either sampling method, but does so in a few seconds and with a user-friendly Web interface.     

Streamlining DNA Barcoding Protocols: Automated DNA Extraction and a New cox1 Primer in Arachnid Systematics

Background

DNA barcoding is a popular tool in taxonomic and phylogenetic studies, but for most animal lineages protocols for obtaining the barcoding sequences—mitochondrial cytochrome C oxidase subunit I (cox1 AKA CO1)—are not standardized. Our aim was to explore an optimal strategy for arachnids, focusing on the species-richest lineage, spiders by (1) improving an automated DNA extraction protocol, (2) testing the performance of commonly used primer combinations, and (3) developing a new cox1 primer suitable for more efficient alignment and phylogenetic analyses.

Methodology

We used exemplars of 15 species from all major spider clades, processed a range of spider tissues of varying size and quality, optimized genomic DNA extraction using the MagMAX Express magnetic particle processor—an automated high throughput DNA extraction system—and tested cox1 amplification protocols emphasizing the standard barcoding region using ten routinely employed primer pairs.

Results

The best results were obtained with the commonly used Folmer primers (LCO1490/HCO2198) that capture the standard barcode region, and with the C1-J-2183/C1-N-2776 primer pair that amplifies its extension. However, C1-J-2183 is designed too close to HCO2198 for well-interpreted, continuous sequence data, and in practice the resulting sequences from the two primer pairs rarely overlap. We therefore designed a new forward primer C1-J-2123 60 base pairs upstream of the C1-J-2183 binding site. The success rate of this new primer (93%) matched that of C1-J-2183.

Conclusions

The use of C1-J-2123 allows full, indel-free overlap of sequences obtained with the standard Folmer primers and with C1-J-2123 primer pair. Our preliminary tests suggest that in addition to spiders, C1-J-2123 will also perform in other arachnids and several other invertebrates. We provide optimal PCR protocols for these primer sets, and recommend using them for systematic efforts beyond DNA barcoding.     

A new cost‐effective and fast direct PCR protocol for insects based on PBS buffer

Insect DNA barcoding is a species identification technique used in biodiversity assessment and ecological studies. However, DNA extraction can result in the loss of up to 70% of DNA. Recent research has reported that direct PCR can overcome this issue. However, the success rates could still be improved, and tissues used for direct PCR could not be reused for further genetic studies. Here, we developed a direct PCR workflow that incorporates a 2‐min sample preparation in PBS‐buffer step for fast and effective universal insect species identification. The developed protocol achieved 100% success rates for amplification in six orders: Mantodea, Phasmatodea, Neuroptera, Odonata, Blattodea and Orthoptera. High and moderate success rates were obtained for five other species: Lepidoptera (97.3%), Coleoptera (93.8%), Diptera (90.5%), Hemiptera (81.8%) and Hymenoptera (75.0%). High‐quality sequencing data were also obtained from these amplifiable products, allowing confidence in species identification. The method was sensitive down to 1/4th of a 1‐mm fragment of leg or body and its success rates with oven‐dried, ethanol‐preserved, food, bat guano and museum specimens were 100%, 98.6%, 90.0%, 84.0% and 30.0%, respectively. In addition, the pre‐PCR solution (PBS with insect tissues) could be used for further DNA extraction if needed. The workflow will be beneficial in the fields of insect taxonomy and ecological studies due to its low cost, simplicity and applicability to highly degraded specimens.     

Optimized genotyping with microsatellite markers in the fungal banana pathogen Mycosphaerella fijiensis (Mycosphaerellaceae)

? Premise of the study: Large-scale population genetics studies are required to investigate the dispersal processes underlying the emergence of Mycosphaerella fijiensis, a fungal pathogen of banana. To this end, we have developed an optimized genotyping procedure combining novel microsatellite markers and a modified DNA extraction protocol. ? Methods and Results: Primers for tetranucleotide loci were designed directly from the recently published genome sequence of M. fijiensis. A total of 19 new polymorphic and easy-to-score markers were developed. Their use was combined with an adapted protocol for total DNA extraction starting from young lesions collected from banana leaves, thus avoiding a pathogen isolation step. ? Conclusions: The combination of the two technical developments presented here will permit the expansion of genotyping capacity in M. fijiensis, allowing large-scale analysis of samples from various geographic locations.     

Rapid and simple methodology for isolation of high quality genomic DNA from coniferous tissues (<Emphasis Type="Italic">Taxus baccata</Emphasis>)

Various investigations have been so far performed for extraction of genomic DNA from plant tissues, in which the extracted intact DNA can be exploited for a diverse range of biological studies. Extraction of high quality DNA from leathery plant tissues (e.g., coniferous organs) appears to be a critical stage. Moreover, for some species such as Taxus trees, bioprocess engineering and biosynthesis of secondary metabolites (e.g., paclitaxel) is a crucial step due to the restrictions associated with extinction of these species. However, extraction of intact genomic DNA from these plants still demands a rapid, easy and efficient protocol. To pursue such aim, in the current work, we report on the development of a simple and highly efficient method for the extraction of DNA from Taxus baccata. Based upon our protocol, interfering phenolic compounds were removed from extraction using polyvinylpyrrolidone and RNA contamination was resolved using LiCl. By employing this method, high quality genomic DNA was successfully extracted from leaves of T. baccata. The quality of extracted DNA was validated by various techniques such as RAPD marker, restriction digestions and pre-AFLP. Upon our findings, we propose this simple method to be considered for extraction of DNA from leathery plant tissues.     

DNA条形码技术在植物中的研究现状

DNA条形码技术(DNA barcoding)是用短的DNA片段对物种进行识别和鉴定的分子生物学技术。在动物研究中该技术已经成功应用于利用线粒体细胞色素c氧化酶亚基I(COI)进行物种鉴定和发现隐种或新物种。相对于动物, COI基因在高等植物中进化速率较慢, 因此植物条形码研究以叶绿体基因组作为重点, 但目前还处于寻找合适的基因片段阶段。许多学者对此进行了积极的探索, 报道了多种植物条形码的候选片段或组合, 但还没有获得满足所有标准的特征位点片段。该文介绍了DNA条形码的标准、优点、工作流程及数据分析方法, 总结了DNA条形码在植物中的研究现状。     

Use of DNA barcoding to detect invertebrate invasive species from diapausing eggs

The global transhipment of ballast water and associated flora and fauna by cargo vessels has increased dramatically in recent decades. Invertebrate species are frequently carried in ballast water and sediment, although identification of diapausing eggs can be extremely problematic. Here we test the application of DNA barcoding using mitochondrial cytochrome c oxidase subunit I and 16S rDNA to identify species from diapausing eggs collected in ballast sediment of ships. The accuracy of DNA barcoding identification was tested by comparing results from the molecular markers against each other, and by comparing barcoding results to traditional morphological identification of individuals hatched from diapausing eggs. Further, we explored two public genetic databases to determine the broader applicability of DNA barcodes. Of 289 diapausing eggs surveyed, sufficient DNA for barcoding was obtained from 96 individuals (33%). Unsuccessful DNA extractions from 67% of eggs in our study were most likely due to degraded condition of eggs. Of 96 eggs with successful DNA extraction, 61 (64%) were identified to species level, while 36% were identified to possible family/order level. Species level identifications were always consistent between methodologies. DNA barcoding was suitable for a wide range of taxa, including Branchiopoda, Copepoda, Rotifera, Bryozoa and Ascidia. Branchiopoda and Copepoda were respectively the best and worst represented groups in genetic databases. Though genetic databases remain incomplete, DNA barcoding resolved nearly double the number of species identified by traditional taxonomy (19 vs. 10). Notorious invaders are well represented in existing databases, rendering these NIS detectable using molecular methods. DNA barcoding provides a rapid and accurate approach to identification of invertebrate diapausing eggs that otherwise would be very difficult to identify.     

Isolation of DNA from bacterial samples of the human gastrointestinal tract

The human gastrointestinal (GI) tract contains a complex microbial community that develops in time and space. The most widely used approaches to study microbial diversity and activity are all based on the analysis of nucleic acids, DNA, rRNA and mRNA. Here, we present a DNA isolation protocol that is suitable for a wide variety of GI tract samples, including biopsies with minute amounts of material. The protocol is set up in such a way that sampling can be performed outside the laboratory, which offers possibilities for implementation in large intervention studies. The DNA isolation is based on mechanical disruption, followed by isolation of nucleic acids using phenol:chloroform:isoamylalcohol extraction. In addition, it includes an alternative DNA isolation protocol that is based on a commercial kit. These protocols have all been successfully used in our laboratory, resulting in isolation of DNA of sufficient quality for microbial diversity studies. Depending on the number of samples and sample type, the whole procedure will take approximately 2.5-4 hours.     

Rapid and reliable high-throughput methods of DNA extraction for use in barcoding and molecular systematics of mushrooms

We present two methods for DNA extraction from fresh and dried mushrooms that are adaptable to high-throughput sequencing initiatives, such as DNA barcoding. Our results show that these protocols yield ∼85% sequencing success from recently collected materials. Tests with both recent (<2 year) and older (>100 years) specimens reveal that older collections have low success rates and may be an inefficient resource for populating a barcode database. However, our method of extracting DNA from herbarium samples using small amount of tissue is reliable and could be used for important historical specimens. The application of these protocols greatly reduces time, and therefore cost, of generating DNA sequences from mushrooms and other fungi vs. traditional extraction methods. The efficiency of these methods illustrates that standardization and streamlining of sample processing should be shifted from the laboratory to the field.