ConDeTri--a content dependent read trimmer for Illumina data

During the last few years, DNA and RNA sequencing have started to play an increasingly important role in biological and medical applications, especially due to the greater amount of sequencing data yielded from the new sequencing machines and the enormous decrease in sequencing costs. Particularly, Illumina/Solexa sequencing has had an increasing impact on gathering data from model and non-model organisms. However, accurate and easy to use tools for quality filtering have not yet been established. We present ConDeTri, a method for content dependent read trimming for next generation sequencing data using quality scores of each individual base. The main focus of the method is to remove sequencing errors from reads so that sequencing reads can be standardized. Another aspect of the method is to incorporate read trimming in next-generation sequencing data processing and analysis pipelines. It can process single-end and paired-end sequence data of arbitrary length and it is independent from sequencing coverage and user interaction. ConDeTri is able to trim and remove reads with low quality scores to save computational time and memory usage during de novo assemblies. Low coverage or large genome sequencing projects will especially gain from trimming reads. The method can easily be incorporated into preprocessing and analysis pipelines for Illumina data. AVAILABILITY AND IMPLEMENTATION: Freely available on the web at http://code.google.com/p/condetri.     

微生物全基因组鸟枪法测序

全基因组测序主要有二种策略,一种是分级鸟枪法测序,另一种是全基因组鸟枪法测序。微生物是一种十分重要的遗传资源,运用全基因组鸟枪法可以方便、快捷地完成其基因组的测序任务。本文对微生物全基因组鸟枪法测序中文库构建、插入片段的长短比例、反应投入量、拼接以及补洞等问题作了较细致的描述,有些步骤作了举例说明。 Abstract:Two strategies introduced for whole genome sequencing,one is clone by clone method,the other is whole genome shotgun sequencing,for microbes which are very important to us,whole genome shotgun sequencing method is very convenient.In this article we discussed the library construction、long-to-short-ratio of insert,、total number of reads should be sequenced、assembly and gap filling technologies of the whole microbial genome shotgun sequencing method while some examples presented.     

Method for phosphorothioate antisense DNA sequencing by capillary electrophoresis with UV detection.

The progress of antisense DNA therapy demands development of reliable and convenient methods for sequencing short single-stranded oligonucleotides. A method of phosphorothioate antisense DNA sequencing analysis using UV detection coupled to capillary electrophoresis (CE) has been developed based on a modified chain termination sequencing method. The proposed method reduces the sequencing cost since it uses affordable CE-UV instrumentation and requires no labeling with minimal sample processing before analysis. Cycle sequencing with ThermoSequenase generates quantities of sequencing products that are readily detectable by UV. Discrimination of undesired components from sequencing products in the reaction mixture, previously accomplished by fluorescent or radioactive labeling, is now achieved by bringing concentrations of undesired components below the UV detection range which yields a 'clean', well defined sequence. UV detection coupled with CE offers additional conveniences for sequencing since it can be accomplished with commercially available CE-UV equipment and is readily amenable to automation.     

快速可靠的双链PCR产物直接测序法

利用T7DNA聚合酶在低温下仍具较高活性的特点,在热变性后低温下进行测序反应,使用该方法对多种PCR产物进行序列分析均取得较好的结果．     

A rapid and cost-effective method for sequencing pooled cDNA clones by using a combination of transposon insertion and Gateway technology

Large-scale cDNA-sequencing projects require an efficient strategy for mass sequencing. Here we describe a method for sequencing pooled cDNA clones using a combination of transposon insertion and Gateway technology. Our method reduces the number of shotgun clones that are unsuitable for reconstruction of cDNA sequences, and has the advantage of reducing the total costs of the sequencing project.     

Pyrosequencing™

Nucleotide sequencing is an established method for gaining information relating to partial gene, whole gene, or whole genome sequence. Here we describe some of the background leading to the advent of modern nucleotide sequencing and how it has led to the development of Pyrosequencing™, a relatively new method for real-time nucleotide sequencing. In particular, we describe how this method can be used for typing bacterial pathogens.     

Rapid removal of unincorporated label and proteins from DNA sequencing reactions

This article presents a simple and rapid method for removal of unincorporated label and proteins from DNA sequencing reactions by using Wizard purification resin. This method can be successfully applied for preparation of end-labeled oligonucleotides free of unincorporated label, which is important in experiments (including DNA sequencing) when the level of background should be as low as possible. Also, this method is effective in removal of proteins from DNA sequencing reactions.     

Sequencing the DNA of recombinant chromosomes

With contemporary molecular cloning and DNA sequencing techniques, deriving the primary structure of higher cell genes is now routine. This publication reviews the chemical DNA sequencing method, and suggests strategies for sequencing recombinant DNA, whether arising naturally by chromosome rearrangement in vivo or created experimentally by gene splicing in vitro. One such strategy prepares end-labeled restriction fragments from an inserted or rearranged DNA region, for sequencing by the chemical method. Another maps the point at which the sequences of two related DNA regions diverge, and indicates which restriction endonucleases would be useful for sequencing across that point. These techniques can facilitate sequencing of DNA integrations, excisions, translocations, inversions, and introns in cloned chromosomal segments.     

From micrograms to picograms: quantitative PCR reduces the material demands of high-throughput sequencing

Current efforts to recover the Neandertal and mammoth genomes by 454 DNA sequencing demonstrate the sensitivity of this technology. However, routine 454 sequencing applications still require microgram quantities of initial material. This is due to a lack of effective methods for quantifying 454 sequencing libraries, necessitating expensive and labour-intensive procedures when sequencing ancient DNA and other poor DNA samples. Here we report a 454 sequencing library quantification method based on quantitative PCR that effectively eliminates these limitations. We estimated both the molecule numbers and the fragment size distributions in sequencing libraries derived from Neandertal DNA extracts, SAGE ditags and bonobo genomic DNA, obtaining optimal sequencing yields without performing any titration runs. Using this method, 454 sequencing can routinely be performed from as little as 50 pg of initial material without titration runs, thereby drastically reducing costs while increasing the scope of sample throughput and protocol development on the 454 platform. The method should also apply to Illumina/Solexa and ABI/SOLiD sequencing, and should therefore help to widen the accessibility of all three platforms.     

Multiplex random priming of internal restriction fragments for DNA sequencing.

An approach for DNA sequencing is described that circumvents the need for synthetic oligonucleotide primers, which seriously restrict the progress of DNA sequencing in the commonly used protocol. The method is based on the use of short restriction fragments as primers randomly distributed along single-stranded templates. Premapping of target DNA is eliminated and subcloning manipulation is minimized. This method has been used successfully for sequencing genes in the range of 2 kb, for which about 10 restriction fragment primers per kilobase were sufficient to generate a continuous overlapping sequence in alignment. The approach has also been readily applied for an automated sequencing system with the fluorescent chain-terminating dideoxynucleotides, thus implying its potential for sequencing large genomic DNAs.     

HTS-PEG: A Method for High Throughput Sequencing of the Paired-Ends of Genomic Libraries

Second generation sequencing has been widely used to sequence whole genomes. Though various paired-end sequencing methods have been developed to construct the long scaffold from contigs derived from shotgun sequencing, the classical paired-end sequencing of the Bacteria Artificial Chromosome (BAC) or fosmid libraries by the Sanger method still plays an important role in genome assembly. However, sequencing libraries with the Sanger method is expensive and time-consuming. Here we report a new strategy to sequence the paired-ends of genomic libraries with parallel pyrosequencing, using a Chinese amphioxus (Branchiostoma belcheri) BAC library as an example. In total, approximately 12,670 non-redundant paired-end sequences were generated. Mapping them to the primary scaffolds of Chinese amphioxus, we obtained 413 ultra-scaffolds from 1,182 primary scaffolds, and the N50 scaffold length was increased approximately 55 kb, which is about a 10% improvement. We provide a universal and cost-effective method for sequencing the ultra-long paired-ends of genomic libraries. This method can be very easily implemented in other second generation sequencing platforms.     

Strategies for automated sequencing of human mitochondrial DNA directly from PCR products.

A rapid, robust and sensitive method has been developed for the amplification and direct sequencing of human mitochondrial DNA. A 403-bp hypervariable segment was amplified by two successive rounds of nested PCR. This was then sequenced by the dideoxy chain termination method using dye-labeled universal sequencing primers in conjunction with an automated DNA sequencer. This paper describes the assessment of four different strategies for this amplification and sequencing process. Optimal results were obtained by immobilizing the biotinylated PCR product on Dynabeads followed by solid-phase sequencing with Sequenase. Degraded samples and those containing trace amounts of DNA such as extracts from hair shafts can be analyzed by this method.     

Rapid screening of complex DNA samples by single-molecule amplification and sequencing

Microbial cloning makes Sanger sequencing of complex DNA samples possible but is labor intensive. We present a simple, rapid and robust method that enables laboratories without special equipment to perform single-molecule amplicon sequencing, although in a low-throughput manner, from sub-picogram quantities of DNA. The method can also be used for quick quality control of next-generation sequencing libraries, as was demonstrated for a metagenomic sample.     

Solution-based targeted genomic enrichment for precious DNA samples

ABSTRACT: BACKGROUND: Solution-based targeted genomic enrichment (TGE) protocols permit selective sequencing of genomic regions of interest on a massively parallel scale. These protocols could be improved by: 1) modifying or eliminating time consuming steps; 2) increasing yield to reduce input DNA and excessive PCR cycling; and 3) enhancing reproducible. RESULTS: We developed a solution-based TGE method for downstream Illumina sequencing in a non-automated workflow, adding standard Illumina barcode indexes during the post-hybridization amplification to allow for sample pooling prior to sequencing. The method utilizes Agilent SureSelect baits, primers and hybridization reagents for the capture, off-the-shelf reagents for the library preparation steps, and adaptor oligonucleotides for Illumina paired-end sequencing purchased directly from an oligonucleotide manufacturing company. CONCLUSIONS: This solution-based TGE method for Illumina sequencing is optimized for small- or medium-sized laboratories and addresses the weaknesses of standard protocols by reducing the amount of input DNA required, increasing capture yield, optimizing efficiency, and improving reproducibility.     

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...     

粘虫核型多角体病毒919bp片段的PCR双链直接序列测定

本文发展了ＰＣＲ克隆和亚克隆技术制备ＤＮＡ测序模板。首先,我们用ｐＵＣ／Ｍ１３系列质粒的通用正反向引物ＰＣＲ扩增出质粒ｐＢｌｕｅｓｃｒｉｐｔＫＳＤＮＡ的多克隆位点及其侧翼序列,用ＥｃｏＲＶ和ＸｈｏＩ消化成为左右两个引物多克隆臂,与粘虫核型多角体病毒（ＬｓＮＰＶ）的ＥｃｏＲＶ和ＸｈｏＩ约４００ｂｐ和５００ｂｐ片段分别连接,经ＰＣＲ扩增,得到两端具有上述正反向引物结合位点的测序模板,用ｄｄＮＴＰ链终止法／ＰＣＲ扩增／银染色,从片段两端测定了全部９１９ｂｐ序列,这种ｄｄＮＴＰ／ＰＣＲ／银染测序法简化了操作,大大缩短了测序模板的制备时间,易于实现自动化操作。     

Ultrafast sequencing of oligodeoxyribonucleotides by FAB-mass spectrometry.

A fully instrumental method is described for the bidirectional sequencing of oligodeoxyribonucleotides. The method makes use of the negative ion fragmentation patterns of fast atom bombardment mass spectrometry. It is less time consuming than any other sequencing procedure known to date. Since one sequencing run takes as little as one hour, this new method is anticipated to cut down considerably the time required for the controlled synthesis of oligodeoxyribonucleotides of (currently) up to ten nucleotide units in length.     

Automated sequencing and mapping of cosmid DNA with fluorescently-labeled dideoxynucleotide terminators.

We have established a method for directly sequencing cosmid DNA on an automated DNA sequencer. The major advantage of this method is that only small amounts of cosmid template DNA are needed for the sequencing reactions.     

Exoquence DNA sequencing.

We have developed a strategy for DNA sequencing based on exonuclease III digestion followed by double strand specific endonuclease digestion and direct dideoxynucleotide sequencing reaction. This strategy eliminates the need for subcloning, oligonucleotide primers, and prior knowledge of the DNA to be sequenced. All template and primer duplexes needed for sequencing a complete insert can be prepared in one day from uncharacterized starting DNA. Sequence information can be obtained from different regions of the DNA simultaneously. The method uses double-stranded DNA to generate single-stranded template and primer, and thus produces high quality sequence results. Commercially available dideoxy-sequencing kits are well suited for this method. The strategy should be applicable for both automatic and routine laboratory DNA sequencing.     

A cost-effective plasmid purification protocol suitable for fluorescent automated DNA sequencing

A cost-effective, reliable, and reproducible method has been developed to produce good-quality, double-stranded plasmid DNA for automated sequence analysis. The method incorporates modifications to a previously described plasmid-purification protocol used in manual sequencing. The quality of the DNA produced from the present protocol is suitable for automated fluorescent sequencing. Using a dye-terminator sequencing protocol, most runs using plasmid DNA prepared using this protocol produced over 700 bases with greater than 99% base-calling accuracy.