A MinION™‐based pipeline for fast and cost‐effective DNA barcoding

DNA barcodes are useful for species discovery and species identification, but obtaining barcodes currently requires a well‐equipped molecular laboratory and is time‐consuming, and/or expensive. We here address these issues by developing a barcoding pipeline for Oxford Nanopore MinION? and demonstrating that one flow cell can generate barcodes for ~500 specimens despite the high basecall error rates of MinION? reads. The pipeline overcomes these errors by first summarizing all reads for the same tagged amplicon as a consensus barcode. Consensus barcodes are overall mismatch‐free but retain indel errors that are concentrated in homopolymeric regions. They are addressed with an optional error correction pipeline that is based on conserved amino acid motifs from publicly available barcodes. The effectiveness of this pipeline is documented by analysing reads from three MinION? runs that represent three different stages of MinION? development. They generated data for (i) 511 specimens of a mixed Diptera sample, (ii) 575 specimens of ants and (iii) 50 specimens of Chironomidae. The run based on the latest chemistry yielded MinION? barcodes for 490 of the 511 specimens which were assessed against reference Sanger barcodes (N = 471). Overall, the MinION? barcodes have an accuracy of 99.3%–100% with the number of ambiguous bases after correction ranging from <0.01% to 1.5% depending on which correction pipeline is used. We demonstrate that it requires ~2 hr of sequencing to gather all information needed for obtaining reliable barcodes for most specimens (>90%). We estimate that up to 1,000 barcodes can be generated in one flow cell and that the cost per barcode can be 相似文献   

Gel‐aided sample preparation (GASP)—A simplified method for gel‐assisted proteomic sample generation from protein extracts and intact cells

We describe a “gel‐assisted” proteomic sample preparation method for MS analysis. Solubilized protein extracts or intact cells are copolymerized with acrylamide, facilitating denaturation, reduction, quantitative cysteine alkylation, and matrix formation. Gel‐aided sample preparation has been optimized to be highly flexible, scalable, and to allow reproducible sample generation from 50 cells to milligrams of protein extracts. This methodology is fast, sensitive, easy‐to‐use on a wide range of sample types, and accessible to nonspecialists.     

DNA barcodes from century‐old type specimens using next‐generation sequencing

Type specimens have high scientific importance because they provide the only certain connection between the application of a Linnean name and a physical specimen. Many other individuals may have been identified as a particular species, but their linkage to the taxon concept is inferential. Because type specimens are often more than a century old and have experienced conditions unfavourable for DNA preservation, success in sequence recovery has been uncertain. This study addresses this challenge by employing next‐generation sequencing (NGS) to recover sequences for the barcode region of the cytochrome c oxidase 1 gene from small amounts of template DNA. DNA quality was first screened in more than 1800 century‐old type specimens of Lepidoptera by attempting to recover 164‐bp and 94‐bp reads via Sanger sequencing. This analysis permitted the assignment of each specimen to one of three DNA quality categories – high (164‐bp sequence), medium (94‐bp sequence) or low (no sequence). Ten specimens from each category were subsequently analysed via a PCR‐based NGS protocol requiring very little template DNA. It recovered sequence information from all specimens with average read lengths ranging from 458 bp to 610 bp for the three DNA categories. By sequencing ten specimens in each NGS run, costs were similar to Sanger analysis. Future increases in the number of specimens processed in each run promise substantial reductions in cost, making it possible to anticipate a future where barcode sequences are available from most type specimens.     

A high‐throughput sample preparation method for cellular proteomics using 96‐well filter plates

A high‐throughput sample preparation protocol based on the use of 96‐well molecular weight cutoff (MWCO) filter plates was developed for shotgun proteomics of cell lysates. All sample preparation steps, including cell lysis, buffer exchange, protein denaturation, reduction, alkylation and proteolytic digestion are performed in a 96‐well plate format, making the platform extremely well suited for processing large numbers of samples and directly compatible with functional assays for cellular proteomics. In addition, the usage of a single plate for all sample preparation steps following cell lysis reduces potential samples losses and allows for automation. The MWCO filter also enables sample concentration, thereby increasing the overall sensitivity, and implementation of washing steps involving organic solvents, for example, to remove cell membranes constituents. The optimized protocol allowed for higher throughput with improved sensitivity in terms of the number of identified cellular proteins when compared to an established protocol employing gel‐filtration columns.     

VIP Barcoding: composition vector‐based software for rapid species identification based on DNA barcoding

Species identification based on short sequences of DNA markers, that is, DNA barcoding, has emerged as an integral part of modern taxonomy. However, software for the analysis of large and multilocus barcoding data sets is scarce. The Basic Local Alignment Search Tool (BLAST) is currently the fastest tool capable of handling large databases (e.g. >5000 sequences), but its accuracy is a concern and has been criticized for its local optimization. However, current more accurate software requires sequence alignment or complex calculations, which are time‐consuming when dealing with large data sets during data preprocessing or during the search stage. Therefore, it is imperative to develop a practical program for both accurate and scalable species identification for DNA barcoding. In this context, we present VIP Barcoding: a user‐friendly software in graphical user interface for rapid DNA barcoding. It adopts a hybrid, two‐stage algorithm. First, an alignment‐free composition vector (CV) method is utilized to reduce searching space by screening a reference database. The alignment‐based K2P distance nearest‐neighbour method is then employed to analyse the smaller data set generated in the first stage. In comparison with other software, we demonstrate that VIP Barcoding has (i) higher accuracy than Blastn and several alignment‐free methods and (ii) higher scalability than alignment‐based distance methods and character‐based methods. These results suggest that this platform is able to deal with both large‐scale and multilocus barcoding data with accuracy and can contribute to DNA barcoding for modern taxonomy. VIP Barcoding is free and available at http://msl.sls.cuhk.edu.hk/vipbarcoding/ .     

Beyond DNA barcoding: The unrealized potential of genome skim data in sample identification

Genetic tools are increasingly used to identify and discriminate between species. One key transition in this process was the recognition of the potential of the ca 658bp fragment of the organelle cytochrome c oxidase I (COI) as a barcode region, which revolutionized animal bioidentification and lead, among others, to the instigation of the Barcode of Life Database (BOLD), containing currently barcodes from >7.9 million specimens. Following this discovery, suggestions for other organellar regions and markers, and the primers with which to amplify them, have been continuously proposed. Most recently, the field has taken the leap from PCR‐based generation of DNA references into shotgun sequencing‐based “genome skimming” alternatives, with the ultimate goal of assembling organellar reference genomes. Unfortunately, in genome skimming approaches, much of the nuclear genome (as much as 99% of the sequence data) is discarded, which is not only wasteful, but can also limit the power of discrimination at, or below, the species level. Here, we advocate that the full shotgun sequence data can be used to assign an identity (that we term for convenience its “DNA‐mark”) for both voucher and query samples, without requiring any computationally intensive pretreatment (e.g. assembly) of reads. We argue that if reference databases are populated with such “DNA‐marks,” it will enable future DNA‐based taxonomic identification to complement, or even replace PCR of barcodes with genome skimming, and we discuss how such methodology ultimately could enable identification to population, or even individual, level.     

Comparison of sample preparation techniques for large‐scale proteomics

Numerous workflows exist for large‐scale bottom‐up proteomics, many of which achieve exceptional proteome depth. Herein, we evaluated the performance of several commonly used sample preparation techniques for proteomic characterization of HeLa lysates [unfractionated in‐solution digests, SDS‐PAGE coupled with in‐gel digestion, gel‐eluted liquid fraction entrapment electrophoresis (GELFrEE) technology, SCX StageTips and high‐/low‐pH reversed phase fractionation (HpH)]. HpH fractionation was found to be superior in terms of proteome depth (>8400 proteins detected) and fractionation efficiency compared to other techniques. SCX StageTip fractionation required minimal sample handling and was also a substantial improvement over SDS‐PAGE separation and GELFrEE technology. Sequence coverage of the HeLa proteome increased to 38% when combining all workflows, however, total proteins detected improved only slightly to 8710. In summary, HpH fractionation and SCX StageTips are robust techniques and highly suited for complex proteome analysis.     

Microwave‐assisted 18O labeling of Fmoc‐protected amino acids

Recently, there has been an increased interest in isotopical labeling of peptides. Although there are several techniques allowing for a complete labeling of all carboxyl groups in peptides, regioselective labeling would be beneficial in many situations. Such labeling requires the use of ¹⁸O‐labeled Fmoc amino acids. We have designed a method for such labeling that is an improvement on a technique proposed earlier. The new procedure is suitable for microscale synthesis and could be used in peptide and proteomics laboratories. Although for the majority of tested amino acids our method gives good labeling efficiency, it is time consuming. Therefore, we have decided to use microwave‐assisted procedure. This approach resulted in reduction of reaction time to 15 min and increased reaction efficiency. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

A cost‐effective high‐throughput metabarcoding approach powerful enough to genotype ~44 000 year‐old rodent remains from Northern Africa

We present a cost‐effective metabarcoding approach, aMPlex Torrent, which relies on an improved multiplex PCR adapted to highly degraded DNA, combining barcoding and next‐generation sequencing to simultaneously analyse many heterogeneous samples. We demonstrate the strength of these improvements by generating a phylochronology through the genotyping of ancient rodent remains from a Moroccan cave whose stratigraphy covers the last 120 000 years. Rodents are important for epidemiology, agronomy and ecological investigations and can act as bioindicators for human‐ and/or climate‐induced environmental changes. Efficient and reliable genotyping of ancient rodent remains has the potential to deliver valuable phylogenetic and paleoecological information. The analysis of multiple ancient skeletal remains of very small size with poor DNA preservation, however, requires a sensitive high‐throughput method to generate sufficient data. We show this approach to be particularly adapted at accessing this otherwise difficult taxonomic and genetic resource. As a highly scalable, lower cost and less labour‐intensive alternative to targeted sequence capture approaches, we propose the aMPlex Torrent strategy to be a useful tool for the genetic analysis of multiple degraded samples in studies involving ecology, archaeology, conservation and evolutionary biology.     

Identification of centromeric and telomeric DNA‐binding proteins in rice

Centromeres and telomeres are DNA/protein complexes and essential functional components of eukaryotic chromosomes. Previous studies have shown that rice centromeres and telomeres are occupied by CentO (rice centromere satellite DNA) satellite and G‐rich telomere repeats, respectively. However, the protein components are not fully understood. DNA‐binding proteins associated with centromeric or telomeric DNAs will most likely be important for the understanding of centromere and telomere structure and functions. To capture DNA‐specific binding proteins, affinity pull‐down technique was applied in this study to isolate rice centromeric and telomeric DNA‐binding proteins. Fifty‐five proteins were identified for their binding affinity to rice CentO repeat, and 80 proteins were identified for their binding to telomere repeat. One CentO‐binding protein, Os02g0288200, was demonstrated to bind to CentO specifically by in vitro assay. A conserved domain, DUF573 with unknown functions was identified in this protein, and proven to be responsible for the specific binding to CentO in vitro. Four proteins identified as telomere DNA‐binding proteins in this study were reported by different groups previously. These results demonstrate that DNA affinity pull‐down technique is effective in the isolation of sequence‐specific binding proteins and will be applicable in future studies of centromere and telomere proteins.     

Diversity of DNA‐hydrolyzing antibodies from the sera of autoimmune‐prone MRL/MpJ‐lpr mice

It has been shown for the first time that polyclonal IgG abzymes (Abzs) with DNase activity from the sera of autoimmune‐prone MRL/MpJ‐lpr mice can be separated by isoelectric focusing into many subfractions having the isoelectric points (pI) from 4.5 to 9, with the maximal activity for Abzs with pI = 6.5–9.0. Affinity chromatography on DNA‐cellulose separated DNase IgGs into many subfractions demonstrating a range of affinities for DNA and different levels of the relative DNase activities (RDA) due to intrinsically bound metals and after addition of external Mg²⁺, Mn²⁺, Ca²⁺, and Mg²⁺+Ca²⁺. Some fractions significantly increase RDAs in the presence of external ions (Mg²⁺+Ca²⁺ > Mg²⁺ > Mn²⁺ > Ca²⁺), while each of this cofactor can also inhibit or have no influence on the RDAs of another fractions. It is known that complexes of DNA with histones and other proteins of apoptotic cells are the primary immunogens in systemic lupus erythematosus (SLE). Bovine serum albumin (BSA) and methylated BSA (mBSA) increase the RDAs of only some fractions, while have no effect or inhibit other IgG fractions. The ratio of the RDAs in the presence of all metal ions, BSA, and mBSA was individual for every abzyme fraction. Mn²⁺ and Ca²⁺ stimulated accumulation of only relaxed form of supercoiled DNA (scDNA) in the case of all subfractions, while in the presence of Mg²⁺ antibodies (Abs) of some subfractions (and in the presence of Mn²⁺ +Ca²⁺ all subfractions) produced relaxed DNA (rDNA) and linear DNA (linDNA) in a variable extent. The data obtained show that the polyclonal Abzs of mice may be a cocktail of Abs directly to DNA, RNA, and their complexes with proteins and anti‐idiotypic Abs to active centers of different nucleases. The diversity of the physicochemical and kinetic characteristics of the Abzs seems to be significantly widened when pre‐diseased mice spontaneously develop the disease. Copyright © 2010 John Wiley & Sons, Ltd.     

Integration of conventional quantitative and phospho‐proteomics reveals new elements in activated Jurkat T‐cell receptor pathway maintenance

Recent years have seen a constant development of tools for the global assessment of phosphoproteins. Here, we outline a concept for integrating approaches for quantitative proteomics and phosphoproteomics. The strategy was applied to the analysis of changes in signalling and protein synthesis occurring after activation of the T‐cell receptor (TCR) pathway in a T‐cell line (Jurkat cells). For this purpose, peptides were obtained from four biological replicates of activated and control Jurkat T‐cells and phosphopeptides enriched via a TiO₂‐based chromatographic step. Both phosphopeptide‐enriched and flow‐through fractions were analyzed by LC–MS. We observed 1314 phosphopeptides in the enriched fraction whereas 19 were detected in the flow‐through, enabling the quantification of 414 and eight phosphoproteins in the respective fractions. Pathway analysis revealed the differential regulation of many metabolic pathways. Among the quantified proteins, 11 kinases with known TCR‐related function were detected. A kinase‐substrate database search for the phosphosites identified also confirmed the activity of a further ten kinases. In total, these two approaches provided evidence of 19 unique TCR‐related kinases. The combination of phosphoproteomics and conventional quantitative shotgun analysis leads to a more comprehensive assessment of the signalling networks needed for the maintenance of the activated status of Jurkat T‐cells.     

DNA barcoding of Cymbidium by genome skimming: Call for next-generation nuclear barcodes

Cymbidium is an orchid genus that has undergone rapid radiation and has high ornamental, economic, ecological and cultural importance, but its classification based on morphology is controversial. The plastid genome (plastome), as an extension of plant standard DNA barcodes, has been widely used as a potential molecular marker for identifying recently diverged species or complicated plant groups. In this study, we newly generated 237 plastomes of 50 species (at least two individuals per species) by genome skimming, covering 71.4% of members of the genus Cymbidium. Sequence-based analyses (barcoding gaps and automatic barcode gap discovery) and tree-based analyses (maximum likelihood, Bayesian inference and multirate Poisson tree processes model) were conducted for species identification of Cymbidium. Our work provides a comprehensive DNA barcode reference library for Cymbidium species identification. The results show that compared with standard DNA barcodes (rbcL + matK) as well as the plastid trnH-psbA, the species identification rate of the plastome increased moderately from 58% to 68%. At the same time, we propose an optimized identification strategy for Cymbidium species. The plastome cannot completely resolve the species identification of Cymbidium, the main reasons being incomplete lineage sorting, artificial cultivation, natural hybridization and chloroplast capture. To further explore the potential use of nuclear data in identifying species, the Skmer method was adopted and the identification rate increased to 72%. It appears that nuclear genome data have a vital role in species identification and are expected to be used as next-generation nuclear barcodes.     

DNA barcoding of microgastrine parasitoid wasps (Hymenoptera: Braconidae) using high‐throughput methods more than doubles the number of species known for Australia

The Microgastrinae are a hugely diverse subfamily of endoparasitoid wasps of lepidopteran caterpillars. They are important in agriculture as biological control agents and play a significant ecological role in the regulation of caterpillar populations. Whilst the group has been the focus of intensive rearing and DNA barcoding studies in the Northern Hemisphere, the Australian fauna has received little attention. In total, 99 species have been described from or have been introduced into Australia, but the real species diversity for the region is clearly much larger than this. In this study, museum ethanol samples and recent field collections were mined for hundreds of specimens of microgastrine wasps, which were then barcoded for the COI region, ITS2 ribosomal spacer and the wingless nuclear genes, using a pooled sequencing approach on an Illumina Miseq system. Full COI sequences were obtained for 525 individuals which, when combined with 162 publicly available sequences, represented 417 haplotypes, and a total of 236 species were delimited using a consensus approach. By more than doubling the number of known microgastrine wasp species in Australia, our study highlights the value of DNA barcoding in the context of employing high‐throughput sequencing methods of bulk ethanol museum collections for biodiversity assessment.     

Drawbacks in the use of unconventional hydrophobic anhydrides for histone derivatization in bottom‐up proteomics PTM analysis

MS‐based proteomics has become the most utilized tool to characterize histone PTMs. Since histones are highly enriched in lysine and arginine residues, lysine derivatization has been developed to prevent the generation of short peptides (<6 residues) during trypsin digestion. One of the most adopted protocols applies propionic anhydride for derivatization. However, the propionyl group is not sufficiently hydrophobic to fully retain the shortest histone peptides in RP LC, and such procedure also hampers the discovery of natural propionylation events. In this work we tested 12 commercially available anhydrides, selected based on their safety and hydrophobicity. Performance was evaluated in terms of yield of the reaction, MS/MS fragmentation efficiency, and drift in retention time using the following samples: (i) a synthetic unmodified histone H3 tail, (ii) synthetic modified histone peptides, and (iii) a histone extract from cell lysate. Results highlighted that seven of the selected anhydrides increased peptide retention time as compared to propionic, and several anhydrides such as benzoic and valeric led to high MS/MS spectra quality. However, propionic anhydride derivatization still resulted, in our opinion, as the best protocol to achieve high MS sensitivity and even ionization efficiency among the analyzed peptides.     

Exploiting formalin-preserved fish specimens for resources of DNA barcoding

With the development of the DNA barcoding project, a large number of specimens are required to establish the library of reference barcode. Formalin-fixed samples from museums provide a potential resource for it. However, recovery of DNA and amplification of the target gene from formalin-fixed samples are challenging. In this study, a hot alkali pre-treatment accompanied by the use of cetyltrimethylammonium bromide (CTAB) method was employed for DNA recovery from formalin-preserved samples, with the purpose of pursuing the optimal condition for high quantity and quality of DNA and minimizing PCR inhibition. Meanwhile, a semi-nested PCR-based method was developed to enhance the efficacy of amplification. This advanced protocol was demonstrated to be reliable and effective. Even for 23-year-old samples, genomic DNA could be extracted, and COI gene was correctly sequenced.     

DNA‐Functionalized Plasmonic Nanomaterials for Optical Biosensing

Plasmonic nanomaterials, especially Au and Ag nanomaterials, have shown attractive physicochemical properties, such as easy functionalization and tunable optical bands. The development of this active subfield paves the way to the fascinating biosensing platforms. In recent years, plasmonic nanomaterials–based sensors have been extensively investigated because they are useful for genetic diseases, biological processes, devices, and cell imaging. In this account, a brief introduction of the development of optical biosensors based on DNA‐functionalized plasmonic nanomaterials is presented. Then the common strategies for the application of the optical sensors are summarized, including colorimetry, fluorescence, localized surface plasmon resonance, and surface‐enhanced resonance scattering detection. The focus is on the fundamental aspect of detection methods, and then a few examples of each method are highlighted. Finally, the opportunities and challenges for the plasmonic nanomaterials–based biosensing are discussed with the development of modern technologies.