GEL, a DNA sequencing project management system.

We have developed an automated system for management of DNA sequencing projects. The system, named GEL, can handle data from both random sequences and from fragments whose relative positions are known. The system is highly interactive, self-documenting, and forgiving; it is designed for use by computer-naive molecular biologists. An editor designed specifically for sequences allows simple entry of data. Special functions allow direct checking and immediate editing of paired readings of the same gel. Merging of new random fragment sequences into the project as a whole is semi-automated. The user is shown probable overlaps if they exist, and can edit either the sequences or the consensus. Heuristic approaches to limiting the kinds of searches made in the merging process reduces the problem of combinatoric data overload as sequencing projects grow large. Complete histories of all entries, editing changes, and generation of consensus sequences are automatically prepared.     

纳米孔测序信号处理及其在DNA数据存储的应用

随着高通量测序技术的不断更新,可以在单个分子水平读取核苷酸序列的第三代测序技术迅速发展,纳米孔测序技术是其具有代表性的单分子测序技术,该技术通过检测DNA单链分子穿过纳米孔时引起的跨膜电流信号的变化,实现碱基识别.纳米孔测序仪在便携性、碱基读取速度、测序读段长度等方面较传统的第一代与第二代测序技术都有明显优势.随着纳米...     

A new DNA sequence assembly program.

We describe the Genome Assembly Program (GAP), a new program for DNA sequence assembly. The program is suitable for large and small projects, a variety of strategies and can handle data from a range of sequencing instruments. It retains the useful components of our previous work, but includes many novel ideas and methods. Many of these methods have been made possible by the program's completely new, and highly interactive, graphical user interface. The program provides many visual clues to the current state of a sequencing project and allows users to interact in intuitive and graphical ways with their data. The program has tools to display and manipulate the various types of data that help to solve and check difficult assemblies, particularly those in repetitive genomes. We have introduced the following new displays: the Contig Selector, the Contig Comparator, the Template Display, the Restriction Enzyme Map and the Stop Codon Map. We have also made it possible to have any number of Contig Editors and Contig Joining Editors running simultaneously even on the same contig. The program also includes a new 'Directed Assembly' algorithm and routines for automatically detecting unfinished segments of sequence, to which it suggests experimental solutions.     

Deletion of GEL2 encoding for a beta(1-3)glucanosyltransferase affects morphogenesis and virulence in Aspergillus fumigatus

The first fungal glycosylphosphatidylinositol anchored beta(1-3)glucanosyltranferase (Gel1p) has been described in Aspergillus fumigatus and its encoding gene GEL1 identified. Glycosylphosphatidylinositol-anchored glucanosyltransferases play an active role in the biosynthesis of the fungal cell wall. We characterize here GEL2, a homologue of GEL1. Both homologues share common characteristics: (i) GEL1 and GEL2 are constitutively expressed during over a range of growth conditions; (ii) Gel2p is also a putative GPI-anchored protein and shares the same beta(1-3)glucanosyltransferase activity as Gel1p and (iii) GEL2, like GEL1, is able to complement the Deltagas1 deletion in Saccharomyces cerevisiae confirming that Gelp and Gasp have the same enzymatic activity. However, disruption of GEL1 did not result in a phenotype whereas a Deltagel2 mutant and the double mutant Deltagel1Deltagel2 exhibit slower growth, abnormal conidiogenesis, and an altered cell wall composition. In addition, the Deltagel2 and the Deltagel1Deltagel2 mutant have reduced virulence in a murine model of invasive aspergillosis. These data suggest for the first time that beta(1-3)glucanosyltransferase activity is required for both morphogenesis and virulence in A. fumigatus.     

Efficient identification of point mutations by automated DNA sequencing of artificial heterozygote samples

DNA sequencing templates of individual point mutants of thelacI target gene were amplified by polymerase chain reaction (PCR). By mixing the PCR fragments from two individual mutants in a defined ratio, samples of artificial heterozygous composition were prepared. These samples were then submitted to automated DNA sequencing. The simultaneous, visual comparison of the mixed mutant traces using a graphics program efficiently revealed all heterozygous positions. Based on the individual intensities of the heterozygous base signals the identified point mutations could be assigned to the corresponding mutants. This efficient approach doubles the sample throughput for both the sequencing reactions and the gel electrophoresis using an automated DNA sequencing system.     

New technologies for large-genome sequencing

Interest in DNA sequencing has increased in recent years and research into new instrument systems to optimize and automate the process has been intensified. A number of commercial devices, from Europe, Japan, and the United States, which instrument aspects of (mainly) the Sanger method, are now available and several new DNA sequencing techniques have been reported. This review briefly describes current methods of DNA sequencing and provides an account of systems presently available or under development for instrumenting the technology. The problems of automation for large-scale DNA sequencing are discussed.     

Rapid quantification of DNA libraries for next-generation sequencing

The next-generation DNA sequencing workflows require an accurate quantification of the DNA molecules to be sequenced which assures optimal performance of the instrument. Here, we demonstrate the use of qPCR for quantification of DNA libraries used in next-generation sequencing. In addition, we find that qPCR quantification may allow improvements to current NGS workflows, including reducing the amount of library DNA required, increasing the accuracy in quantifying amplifiable DNA, and avoiding amplification bias by reducing or eliminating the need to amplify DNA before sequencing.     

Editor's choice: Highly sensitive targeted methylome sequencing by post-bisulfite adaptor tagging

The current gold standard method for methylome analysis is whole-genome bisulfite sequencing (WGBS), but its cost is substantial, especially for the purpose of multi-sample comparison of large methylomes. Shotgun bisulfite sequencing of target-enriched DNA, or targeted methylome sequencing (TMS), can be a flexible, cost-effective alternative to WGBS. However, the current TMS protocol requires a considerable amount of input DNA and hence is hardly applicable to samples of limited quantity. Here we report a method to overcome this limitation by using post-bisulfite adaptor tagging (PBAT), in which adaptor tagging is conducted after bisulfite treatment to circumvent bisulfite-induced loss of intact sequencing templates, thereby enabling TMS of a 100-fold smaller amount of input DNA with far fewer cycles of polymerase chain reaction than in the current protocol. We thus expect that the PBAT-mediated TMS will serve as an invaluable method in epigenomics.     

Computer program for the IBM personal computer which searches for approximate matches to short oligonucleotide sequences in long target DNA sequences.

We describe a program which may be used to find approximate matches to a short predefined DNA sequence in a larger target DNA sequence. The program predicts the usefulness of specific DNA probes and sequencing primers and finds nearly identical sequences that might represent the same regulatory signal. The program is written in the C programming language and will run on virtually any computer system with a C compiler, such as the IBM/PC and other computers running under the MS/DOS and UNIX operating systems. The program has been integrated into an existing software package for the IBM personal computer (see article by Mount and Conrad, this volume). Some examples of its use are given.     

Flow cytometry for enrichment and titration in massively parallel DNA sequencing

Massively parallel DNA sequencing is revolutionizing genomics research throughout the life sciences. However, the reagent costs and labor requirements in current sequencing protocols are still substantial, although improvements are continuously being made. Here, we demonstrate an effective alternative to existing sample titration protocols for the Roche/454 system using Fluorescence Activated Cell Sorting (FACS) technology to determine the optimal DNA-to-bead ratio prior to large-scale sequencing. Our method, which eliminates the need for the costly pilot sequencing of samples during titration is capable of rapidly providing accurate DNA-to-bead ratios that are not biased by the quantification and sedimentation steps included in current protocols. Moreover, we demonstrate that FACS sorting can be readily used to highly enrich fractions of beads carrying template DNA, with near total elimination of empty beads and no downstream sacrifice of DNA sequencing quality. Automated enrichment by FACS is a simple approach to obtain pure samples for bead-based sequencing systems, and offers an efficient, low-cost alternative to current enrichment protocols.     

VSQual: a visual system to assist DNA sequencing quality control

A lack of pliant software tools that support small- to medium-scale DNA sequencing efforts is a major hindrance for recording and using laboratory workflow information to monitor the overall quality of data production. Here we describe VSQual, a set of Perl programs intended to provide simple and powerful tools to check several quality features of the sequencing data generated by automated DNA sequencing machines. The core program of VSQual is a flexible Perl-based pipeline, designed to be accessible and useful for both programmers and non-programmers. This pipeline directs the processing steps and can be easily customized for laboratory needs. Basically, the raw DNA sequencing trace files are processed by Phred and Cross_match, then the outputs are parsed, reformatted into Web-based graphical reports, and added to a Web site structure. The result is a set of real time sequencing reports easily accessible and understood by common laboratory people. These reports facilitate the monitoring of DNA sequencing as well as the management of laboratory workflow, significantly reducing operational costs and ensuring high quality and scientifically reliable results.     

Osprey: a comprehensive tool employing novel methods for the design of oligonucleotides for DNA sequencing and microarrays

We have developed a software package called Osprey for the calculation of optimal oligonucleotides for DNA sequencing and the creation of microarrays based on either PCR-products or directly spotted oligomers. It incorporates a novel use of position-specific scoring matrices, for the sensitive and specific identification of secondary binding sites anywhere in the target sequence. Using accelerated hardware is faster and more efficient than the traditional pairwise alignments used in most oligo-design software. Osprey consists of a module for target site selection based on user input, novel utilities for dealing with problematic sequences such as repeats, and a common code base for the identification of optimal oligonucleotides from the target list. Overall, these improvements provide a program that, without major increases in run time, reflects current DNA thermodynamics models, improves specificity and reduces the user's data preprocessing and parameterization requirements. Using a TimeLogic™ hardware accelerator, we report up to 50-fold reduction in search time versus a linear search strategy. Target sites may be derived from computer analysis of DNA sequence assemblies in the case of sequencing efforts, or genome or EST analysis in the case of microarray development in both prokaryotes and eukaryotes.     

纳米孔测序技术应用于食品微生物检测的可行性分析

近些年来DNA测序技术发展迅速,已经从第一代生化测序发展到第三代单分子测序。作为第三代测序技术中的一种不同于当前流行的其他测序技术,纳米孔测序技术是基于电信号的一种物理方法测序。许多研究者通常将高通量测序技术应用于食品微生物的研究,但是将纳米孔测序技术应用于食品中微生物的检测却鲜有报道。Oxford Nanopore Technologies(牛津纳米孔科技公司)研发的DNA测序仪MinION,是世界首例用于商业测序的纳米孔测序仪,经过不断完善,近年来MinION在DNA测序中被广泛应用。MinION 测序一次需要的DNA量约1μg,其标准识别速度为一秒钟识别250个碱基,平均读长可至13kb~20kb,测序准确率可以达到98%。纳米孔测序的高识别速度和高准确率,完全满足快速检测的要求,将其应用于食品中微生物检测是完全可行的。     

SeSaMe PS Function:Functional Analysis of the Whole Metagenome Sequencing Data of the Arbuscular Mycorrhizal Fungi

In this study, we introduce a novel bioinformatics program, Spore-associated Symbiotic Microbes Position-specific Function (SeSaMe PS Function), for position-specific functional analysis of short sequences derived from metagenome sequencing data of the arbuscular mycorrhizal fungi. The unique advantage of the program lies in databases created based on genus-specific sequence properties derived from protein secondary structure, namely amino acid usages, codon usages, and codon contexts of 3-codon DNA 9-mers. SeSaMe PS Function searches a query sequence against reference sequence database, identifies 3-codon DNA 9-mers with structural roles, and creates a comparative dataset containing the codon usage biases of the 3-codon DNA 9-mers from 54 bacterial and fungal genera. The program applies correlation principal component analysis in conjunction with K-means clustering method to the comparative dataset. 3-codon DNA 9-mers clustered as a sole member or with only a few members are often structurally and functionally distinctive sites that provide useful insights into important molecular interactions. The program provides a versatile means for studying functions of short sequences from metagenome sequencing and has a wide spectrum of applications. SeSaMe PS Function is freely accessible at www.fungalsesame.org.     

History of chloroplast genomics

The presence of chloroplast DNA was established in 1963. With the development of recombinant DNA technologies, chloroplast DNA was selected as one of the first candidates for genome sequencing. The first physical map was reported for maize chloroplasts in 1976. As tobacco has been popular as an experimental system, tobacco chloroplast DNA has been extensively analyzed and the complete nucleotide sequence was established in 1986. This sequencing and the availability of tobacco chloroplast transformation techniques and of in vitro expression systems have formed the basis of an ongoing functional genomics program. This revised version was published online in August 2006 with corrections to the Cover Date.     

NGSpeciesID: DNA barcode and amplicon consensus generation from long‐read sequencing data

Third‐generation sequencing technologies, such as Oxford Nanopore Technologies (ONT) and Pacific Biosciences (PacBio), have gained popularity over the last years. These platforms can generate millions of long‐read sequences. This is not only advantageous for genome sequencing projects, but also advantageous for amplicon‐based high‐throughput sequencing experiments, such as DNA barcoding. However, the relatively high error rates associated with these technologies still pose challenges for generating high‐quality consensus sequences. Here, we present NGSpeciesID, a program which can generate highly accurate consensus sequences from long‐read amplicon sequencing technologies, including ONT and PacBio. The tool includes clustering of the reads to help filter out contaminants or reads with high error rates and employs polishing strategies specific to the appropriate sequencing platform. We show that NGSpeciesID produces consensus sequences with improved usability by minimizing preprocessing and software installation and scalability by enabling rapid processing of hundreds to thousands of samples, while maintaining similar consensus accuracy as current pipelines.     

Next-generation sequencing technologies for environmental DNA research

Since 2005, advances in next-generation sequencing technologies have revolutionized biological science. The analysis of environmental DNA through the use of specific gene markers such as species-specific DNA barcodes has been a key application of next-generation sequencing technologies in ecological and environmental research. Access to parallel, massive amounts of sequencing data, as well as subsequent improvements in read length and throughput of different sequencing platforms, is leading to a better representation of sample diversity at a reasonable cost. New technologies are being developed rapidly and have the potential to dramatically accelerate ecological and environmental research. The fast pace of development and improvements in next-generation sequencing technologies can reflect on broader and more robust applications in environmental DNA research. Here, we review the advantages and limitations of current next-generation sequencing technologies in regard to their application for environmental DNA analysis.     

Alternative dideoxy sequencing of double-stranded DNA by cyclic reactions using Taq polymerase.

The polymerase chain reaction (PCR) is a technique to amplify a specific DNA sequence millions of times. The thermostable enzyme Taq polymerase allows this procedure to take place under conditions of high specificity and automatization. By combining the techniques of PCR and dideoxy sequencing, it is possible to perform DNA sequencing independently of their structures. The cyclic sequencing reaction is carried out in the presence of an excess amount of sequencing primer and a radioactive nucleotide ([alpha-35S]dATP) using a DNA thermal cycler. Different reaction conditions were investigated and optimized including nucleotide ratios in each termination mix, primer/template ratios, amount of a radioactive nucleotide, and the program of the reaction. This method allows the detection of single base substitutions in heterozygous alleles, and the detection of homozygous deletions. A new RFLP of the human porphobilinogen deaminase (PBGD) gene was identified using this technique. This RFLP is created by one base difference (cytosine or adenine) that changes the restriction site for Apa LI. The alternative sequencing method described in this study is a simple and time-saving procedure that can also be used for large sequencing projects.     

Nanopore sequencing technology: research trends and applications

Nanopore sequencing is one of the most promising technologies being developed as a cheap and fast alternative to the conventional Sanger sequencing method. Protein or synthetic nanopores have been used to detect DNA or RNA molecules. Although none of the technologies to date has shown single-base resolution for de novo DNA sequencing, there have been several reports of alpha-hemolysin protein nanopores being used for basic DNA analyses, and various synthetic nanopores have been fabricated. This review will examine current nanopore sequencing technologies, including recent developments of new applications.