Glass bead purification of plasmid template DNA for high throughput sequencing of mammalian genomes

To meet the new challenge of generating the draft sequences of mammalian genomes, we describe the development of a novel high throughput 96-well method for the purification of plasmid DNA template using size-fractionated, acid-washed glass beads. Unlike most previously described approaches, the current method has been designed and optimized to facilitate the direct binding of alcohol-precipitated plasmid DNA to glass beads from alkaline lysed bacterial cells containing the insoluble cellular aggregate material. Eliminating the tedious step of separating the cleared lysate significantly simplifies the method and improves throughput and reliability. During a 4 month period of 96-capillary DNA sequencing of the Rattus norvegicus genome at the Baylor College of Medicine Human Genome Sequencing Center, the average success rate and read length derived from >1 800 000 plasmid DNA templates prepared by the direct lysis/glass bead method were 82.2% and 516 bases, respectively. The cost of this direct lysis/glass bead method in September 2001 was ~10 cents per clone, which is a significant cost saving in high throughput genomic sequencing efforts.     

PCR template preparation for capillary DNA sequencing

Fluorescence-based capillary DNA sequencing has facilitated the early completion of several complex sequencing projects. While capillary systems offer great benefits in terms of ease of use and automation, we find that they are sufficiently different from slab gel separation methodologies, demanding re-examination of the protocols used to generate and use DNA sequencing templates. We have recently initiated a large-scale Human Open Reading Frame EST project involving 30 laboratories feeding 11 MegaBace 1000 capillary sequencers. The group has already produced more than 300,000 valid sequences. The most successful template preparation protocol we have found is described here. We have found that a crucial step is the standardization of the quantity and quality of the templates, which have been achieved by overnight bacterial culture followed by PCR using limiting amounts of primers. Using this protocol, there is no need for post-PCR purification, and the final preparation cost is US $0.09/template. After sequencing 10,848 templates using this protocol, 78% of the reads were accepted (after discarding vectors without inserts and inserts smaller than 100 nucleotides), and 85% of the total number of bases had Phred scores of 15 or above.     

An efficient,simple and high throughput protocol for cotton genomic DNA isolation

Cotton is a stubborn plant for genomic DNA isolation due to its high-level of polyphenolics, polysaccharides and secondary metabolites. Genomics and molecular biology studies require high quality and large quantity of DNA. We have standardized an efficient miniprep protocol for cotton genomic DNA isolation, which not only provides higher yield from 800 to 1400 µg of DNA from 200 to 300 mg of fresh leaf tissue but also provide excellent purity. The DNA is amenable to all elementary enzymatic preparations, PCR techniques, Southern blotting and to high end genomic studies. The technique does not require liquid nitrogen, needs small amount of sample, less time, fewer chemicals and one can process up to 100 samples per day. The genomic DNA extracted was good for transgenic event characterization and marker assisted selection.     

Large scale library generation for high throughput sequencing

Background

Large efforts have recently been made to automate the sample preparation protocols for massively parallel sequencing in order to match the increasing instrument throughput. Still, the size selection through agarose gel electrophoresis separation is a labor-intensive bottleneck of these protocols.

Methodology/Principal Findings

In this study a method for automatic library preparation and size selection on a liquid handling robot is presented. The method utilizes selective precipitation of certain sizes of DNA molecules on to paramagnetic beads for cleanup and selection after standard enzymatic reactions.

Conclusions/Significance

The method is used to generate libraries for de novo and re-sequencing on the Illumina HiSeq 2000 instrument with a throughput of 12 samples per instrument in approximately 4 hours. The resulting output data show quality scores and pass filter rates comparable to manually prepared samples. The sample size distribution can be adjusted for each application, and are suitable for all high throughput DNA processing protocols seeking to control size intervals.     

An efficient strategy for high throughput screening of recombinant integral membrane protein expression and stability

Membrane proteins account for about 30% of the genomes sequenced to date and play important roles in a variety of cellular functions. However, determining the three-dimensional structures of membrane proteins continues to pose a major challenge for structural biologists due to difficulties in recombinant expression and purification. We describe here a high throughput pipeline for Escherichia coli based membrane protein expression and purification. A ligation-independent cloning (LIC)-based vector encoding a C-terminal green fluorescence protein (GFP) tag was used for cloning in a high throughput mode. The GFP tag facilitated expression screening in E. coli through both cell culture fluorescence measurements and in-gel fluorescence imaging. Positive candidates from the GFP screening were subsequently sub-cloned into a LIC-based, GFP free vector for further expression and purification. The expressed, C-terminal His-tagged membrane proteins were purified via membrane enrichment and Ni-affinity chromatography. Thermofluor technique was applied to screen optimal buffers and detergents for the purified membrane proteins. This pipeline has been successfully tested for membrane proteins from E. coli and can be potentially expanded to other prokaryotes.     

Improved soybean transformation for efficient and high throughput transgenic production

Transgenic Research - Although genetic transformation of soybean dates back to over two decades, the process remains inefficient. Here, we report the development of an organogenesis-based...     

A flexible and efficient template format for circular consensus sequencing and SNP detection

A novel template design for single-molecule sequencing is introduced, a structure we refer to as a SMRTbell™ template. This structure consists of a double-stranded portion, containing the insert of interest, and a single-stranded hairpin loop on either end, which provides a site for primer binding. Structurally, this format resembles a linear double-stranded molecule, and yet it is topologically circular. When placed into a single-molecule sequencing reaction, the SMRTbell template format enables a consensus sequence to be obtained from multiple passes on a single molecule. Furthermore, this consensus sequence is obtained from both the sense and antisense strands of the insert region. In this article, we present a universal method for constructing these templates, as well as an application of their use. We demonstrate the generation of high-quality consensus accuracy from single molecules, as well as the use of SMRTbell templates in the identification of rare sequence variants.     

Rapid genotyping of soybean cultivars using high throughput sequencing

Soybean (Glycine max) breeding involves improving commercially grown varieties by introgressing important agronomic traits from poor yielding accessions and/or wild relatives of soybean while minimizing the associated yield drag. Molecular markers associated with these traits are instrumental in increasing the efficiency of producing such crosses and Single Nucleotide Polymorphisms (SNPs) are particularly well suited for this task, owing to high density in the non-genic regions and thus increased likelihood of finding a tightly linked marker to a given trait. A rapid method to develop SNP markers that can differentiate specific loci between any two parents in soybean is thus highly desirable. In this study we investigate such a protocol for developing SNP markers between multiple soybean accessions and the reference Williams 82 genome. To restrict sampling frequency reduced representation libraries (RRLs) of genomic DNA were generated by restriction digestion followed by library construction. We chose to sequence four accessions Dowling (PI 548663), Dwight (PI 597386), Komata (PI200492) and PI 594538A for their agronomic importance as well as Williams 82 as a control.MseI was chosen to digest genomic DNA based on predictions that it will cut sparingly in the mathematically defined high-copy-number regions of the genome. All RRLs were sequenced on the Illumina genome analyzer. Reads were aligned to the Glyma1 reference assembly and SNP calls made from the alignments. We identified from 4294 to 14550 SNPs between the four accessions and the Williams 82 reference. In addition a small number of SNPs (1142) were found by aligning Williams 82 reads to the reference assembly (Glyma1) suggesting limited genetic variation within the Williams 82 line. The SNP data allowed us to estimate genetic diversity between the four lines and Williams 82. Restriction digestion of soybean genomic DNA with MseI followed by high throughput sequencing provides a rapid and reproducible method for generating SNP markers.     

Rethinking microbial diversity analysis in the high throughput sequencing era

The analysis of amplified and sequenced 16S rRNA genes has become the most important single approach for microbial diversity studies. The new sequencing technologies allow for sequencing thousands of reads in a single run and a cost-effective option is split into a single run across many samples. However for this type of investigation the key question that needs to be answered is how many samples can be sequenced without biasing the results due to lack of sequence representativeness? In this work we demonstrated that the level of sequencing effort used for analyzing soil microbial communities biases the results and determines the most effective type of analysis for small and large datasets. Many simulations were performed with four independent pyrosequencing-generated 16S rRNA gene libraries from different environments. The analysis performed here illustrates the lack of resolution of OTU-based approaches for datasets with low sequence coverage. This analysis should be performed with at least 90% of sequence coverage. Diversity index values increase with sample size making normalization of the number of sequences in all samples crucial. An important finding of this study was the advantage of phylogenetic approaches for examining microbial communities with low sequence coverage. However, if the environments being compared were closely related, a deeper sequencing would be necessary to detect the variation in the microbial composition.     

miRTRAP, a computational method for the systematic identification of miRNAs from high throughput sequencing data

MicroRNAs (miRs) have been broadly implicated in animal development and disease. We developed a novel computational strategy for the systematic, whole-genome identification of miRs from high throughput sequencing information. This method, miRTRAP, incorporates the mechanisms of miR biogenesis and includes additional criteria regarding the prevalence and quality of small RNAs arising from the antisense strand and neighboring loci. This program was applied to the simple chordate Ciona intestinalis and identified nearly 400 putative miR loci.     

Large scale purification of linear plasmid DNA for efficient high throughput cloning

In this report we describe a rapid, simple, and efficient method for large-scale purification of linear plasmid DNA to answer demand from high-throughput gene cloning. The process is based on the separation of the linear vector from small DNA fragments by anion exchange chromatography. Gene cloning experiments by restriction/ligation or the In-Fusion(tm) technique confirmed the high quality of the linearized vector as 100% of the genes were successfully cloned.     

高通量测序数据的基因组拷贝数变异检测方法综述

拷贝数变异是指基因组中发生大片段的DNA序列的拷贝数增加或者减少。根据现有的研究可知,拷贝数变异是多种人类疾病的成因,与其发生与发展机制密切相关。高通量测序技术的出现为拷贝数变异检测提供了技术支持,在人类疾病研究、临床诊疗等领域,高通量测序技术已经成为主流的拷贝数变异检测技术。虽然不断有新的基于高通量测序技术的算法和软件被人们开发出来,但是准确率仍然不理想。本文全面地综述基于高通量测序数据的拷贝数变异检测方法,包括基于reads深度的方法、基于双末端映射的方法、基于拆分read的方法、基于从头拼接的方法以及基于上述4种方法的组合方法,深入探讨了每类不同方法的原理,代表性的软件工具以及每类方法适用的数据以及优缺点等,并展望未来的发展方向。