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.     

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.     

Scalable transcriptome preparation for massive parallel sequencing

Background

The tremendous output of massive parallel sequencing technologies requires automated robust and scalable sample preparation methods to fully exploit the new sequence capacity.

Methodology

In this study, a method for automated library preparation of RNA prior to massively parallel sequencing is presented. The automated protocol uses precipitation onto carboxylic acid paramagnetic beads for purification and size selection of both RNA and DNA. The automated sample preparation was compared to the standard manual sample preparation.

Conclusion/Significance

The automated procedure was used to generate libraries for gene expression profiling on the Illumina HiSeq 2000 platform with the capacity of 12 samples per preparation with a significantly improved throughput compared to the standard manual preparation. The data analysis shows consistent gene expression profiles in terms of sensitivity and quantification of gene expression between the two library preparation methods.     

A Microfluidic Device for Preparing Next Generation DNA Sequencing Libraries and for Automating Other Laboratory Protocols That Require One or More Column Chromatography Steps

Library preparation for next-generation DNA sequencing (NGS) remains a key bottleneck in the sequencing process which can be relieved through improved automation and miniaturization. We describe a microfluidic device for automating laboratory protocols that require one or more column chromatography steps and demonstrate its utility for preparing Next Generation sequencing libraries for the Illumina and Ion Torrent platforms. Sixteen different libraries can be generated simultaneously with significantly reduced reagent cost and hands-on time compared to manual library preparation. Using an appropriate column matrix and buffers, size selection can be performed on-chip following end-repair, dA tailing, and linker ligation, so that the libraries eluted from the chip are ready for sequencing. The core architecture of the device ensures uniform, reproducible column packing without user supervision and accommodates multiple routine protocol steps in any sequence, such as reagent mixing and incubation; column packing, loading, washing, elution, and regeneration; capture of eluted material for use as a substrate in a later step of the protocol; and removal of one column matrix so that two or more column matrices with different functional properties can be used in the same protocol. The microfluidic device is mounted on a plastic carrier so that reagents and products can be aliquoted and recovered using standard pipettors and liquid handling robots. The carrier-mounted device is operated using a benchtop controller that seals and operates the device with programmable temperature control, eliminating any requirement for the user to manually attach tubing or connectors. In addition to NGS library preparation, the device and controller are suitable for automating other time-consuming and error-prone laboratory protocols requiring column chromatography steps, such as chromatin immunoprecipitation.     

A novel procedure for efficient genotyping of single nucleotide polymorphisms

Due to the surge in interest in using single nucleotide polymorphisms (SNPs) for genotyping a facile and affordable method for this is an absolute necessity. Here we introduce a procedure that combines an easily automatable single tube sample preparation with an efficient high throughput mass spectrometric analysis technique. Known point mutations or single nucleotide polymorphisms are easily analysed by this procedure. It starts with PCR amplification of a short stretch of genomic DNA, for example an exon of a gene containing a SNP. By shrimp alkaline phosphatase digest residual dNTPs are destroyed. Allele-specific products are generated using a special primer, a conditioned set of α-S-dNTPs and α-S-ddNTPs and a fresh DNA polymerase in a primer extension reaction. Unmodified DNA is removed by 5′-phosphodiesterase digestion and the modified products are alkylated to increase the detection sensitivity in the mass spectrometric analysis. All steps of the preparation are simple additions of solutions and incubations. The procedure operates at the lowest practical sample volumes and in contrast to other genotyping protocols with mass spectrometric detection requires no purification. This reduces the cost and makes it easy to implement. Here it is demonstrated in a version using positive ion detection on described mutations in exon 17 of the amyloid precursor protein gene and in a version using negative ion detection on three SNPs of the granulocyte-macrophage colony stimulating factor gene. Preparation and analysis of SNPs is shown separately and simultaneously, thus demonstrating the multiplexibility of this genotyping procedure. The preparation protocol for genotyping is adapted to the conditions used for the SNP discovery method by denaturing HPLC, thus demonstrating a facile link between protocols for SNP discovery and SNP genotyping. Results corresponded unanimously with the control sequencing. The procedure is useful for high throughput genotyping as it is required for gene identification and pharmacogenomics where large numbers of DNA samples have to be analysed. We have named this procedure the ‘GOOD Assay’ for SNP analysis.     

Protocol of single cells preparation for time of flight secondary ion mass spectrometry

There are several techniques like time of flight secondary ion mass spectrometry (ToF SIMS) that require a special protocol for preparation of biological samples, in particular, those containing single cells due to high vacuum conditions that must be kept during the experiment. Frequently, preparation methodology involves liquid nitrogen freezing what is not always convenient. In our studies, we propose and validate a protocol for preparation of single cells. It consists of four steps: (i) paraformaldehyde fixation, (ii) salt removal, (iii) dehydrating, and (iv) sample drying under ambient conditions. The protocol was applied to samples with single melanoma cells i.e. WM115 and WM266-4 characterized by similar morphology. The surface and internal structures of cells were monitored using atomic force, scanning electron and fluorescent microscopes, used to follow any potential protocol-induced alterations. To validate the proposed methodology for sample preparation, ToF SIMS experiments were carried out using C₆₀⁺ cluster ion beam. The applied principal component analysis (PCA) revealed that chemical changes on cell surface of melanoma cells were large enough to differentiate between primary and secondary tumor sites.     

Solid-phase methods for sequencing of nucleic acids I. Simultaneous sequencing of different oligodeoxyribonucleotides using a new, mechanically stable anion-exchange paper.

A solid-phase method for simultaneous sequencing of large numbers of oligodeoxyribonucleotides has been developed using a new, mechanically stable anion-exchange paper. The excellent mechanical properties of the polymer allow the processing of several paper segments in one reaction vessel or to carry out all necessary operations on a larger area of the paper. In addition, DNA material can be chemically eluted from the new carrier during the piperidine reaction, thus avoiding salt elution of DNA and subsequent ethanol precipitation steps - a prerequisite for sequencing oligonucleotides. The approach involves 7 operations including: i) immobilization; ii) washing; iii) modification; iv) washing; v) sorting of the papers; vi) piperidine reaction and chemical elution and vii) lyophilization. All steps can be carried out in 4 to 5 hours independently of the number of oligonucleotides to be sequenced. It is also possible to sequence small oligonucleotides with 3 to 4 base pairs. The method can be fully automated.     

Weighted gene co‐expression network analysis can sort cancer‐associated fibroblast‐specific markers promoting bladder cancer progression

The role of cancer‐associated fibroblasts (CAFs) has been thoroughly investigated in tumour microenvironments but not in bladder urothelial carcinoma (BLCA). The cell fraction of CAFs gradually increased with BLCA progression. Weighted gene co‐expression network analysis (WGCNA) revealed a specific gene expression module of CAFs that are relevant to cancer progression and survival status. Fifteen key genes of the module were consistent with a fibroblast signature in single‐cell RNA sequencing, functionally related to the extracellular matrix, and significant in survival analysis and tumour staging. A comparison of the luminal‐infiltrated versus luminal‐papillary subtypes and fibroblast versus urothelial carcinoma cell lines and immunohistochemical data analysis demonstrated that the key genes were specifically expressed in CAFs. Moreover, these genes are highly correlated with previously reported CAF markers. In summary, CAFs play a major role in the progression of BLCA, and the 15 key genes act as BLCA‐specific CAF markers and can predict CAF changes. WGCNA can, therefore, be used to sort CAF‐specific gene set in cancer tissues.     

Asymptotic Conditional Singular Value Decomposition for High‐Dimensional Genomic Data

Summary High‐dimensional data, such as those obtained from a gene expression microarray or second generation sequencing experiment, consist of a large number of dependent features measured on a small number of samples. One of the key problems in genomics is the identification and estimation of factors that associate with many features simultaneously. Identifying the number of factors is also important for unsupervised statistical analyses such as hierarchical clustering. A conditional factor model is the most common model for many types of genomic data, ranging from gene expression, to single nucleotide polymorphisms, to methylation. Here we show that under a conditional factor model for genomic data with a fixed sample size, the right singular vectors are asymptotically consistent for the unobserved latent factors as the number of features diverges. We also propose a consistent estimator of the dimension of the underlying conditional factor model for a finite fixed sample size and an infinite number of features based on a scaled eigen‐decomposition. We propose a practical approach for selection of the number of factors in real data sets, and we illustrate the utility of these results for capturing batch and other unmodeled effects in a microarray experiment using the dependence kernel approach of Leek and Storey (2008, Proceedings of the National Academy of Sciences of the United States of America 105 , 18718–18723) .     

A review of DNA sequencing techniques

The four best known DNA sequencing techniques are reviewed. Important practical issues covered are read-length, speed, accuracy, throughput, cost, as well as the automation of sample handling and preparation. The methods reviewed are: (i) the Sanger method and its most important variants (enzymic methods); (ii) the Maxam & Gilbert method and other chemical methods; (iii) the Pyrosequencing method--DNA sequencing in real time by the detection of released pyrophosphate (PPi); and (iv) single molecule sequencing with exonuclease (exonuclease digestion of a single molecule composed of a single strand of fluorescently labelled deoxynucleotides). Each method is briefly described, the current literature is covered, advantages, disadvantages, and the most suitable applications of each method are discussed.     

A Method for Preparing DNA Sequencing Templates Using a DNA-Binding Microplate

A DNA-binding matrix was immobilized on the surface of a 96-well microplate and used for plasmid DNA preparation for DNA sequencing. The same DNA-binding plate was used for bacterial growth, cell lysis, DNA purification, and storage. In a single step using one buffer, bacterial cells were lysed by enzymes, and released DNA was captured on the plate simultaneously. After two wash steps, DNA was eluted and stored in the same plate. Inclusion of phosphates in the culture medium was found to enhance the yield of plasmid significantly. Purified DNA samples were used successfully in DNA sequencing with high consistency and reproducibility. Eleven vectors and nine libraries were tested using this method. In 10 μl sequencing reactions using 3 μl sample and 0.25 μl BigDye Terminator v3.1, the results from a 3730xl sequencer gave a success rate of 90–95% and read-lengths of 700 bases or more. The method is fully automatable and convenient for manual operation as well. It enables reproducible, high-throughput, rapid production of DNA with purity and yields sufficient for high-quality DNA sequencing at a substantially reduced cost.     

Analysis of p53 mutations in single cells obtained from histological tissue sections

We have previously reported on direct sequence analysis of the p53 gene in laser-dissected single cells from tissue sections, where each allele of two fragments (exons 7 and 8) could be accurately analyzed in only 14% of the cells due to the high frequency of exon and allele dropout. Here in an effort to minimize this problem, we have investigated various approaches for sample preparation and gene amplification. By pinpointing some critical steps in the procedure, we could increase the number of investigated exons and substantially improve the genetic analysis of single cells obtained from histochemically stained frozen tissue sections. The biggest improvement was achieved by minimizing DNA degradation using EDTA as a nuclease inhibitor in all sample preparation steps. Efforts to increase primer annealing, by increasing the concentration of template and primers, in addition to prolonging the annealing and extension times, also improved the amplification efficiency. With these measures we can now amplify six individual exons of the p53 gene (exons 4-9) in 70% of the cells and in 50% of these cells both alleles are amplified. This allows application of the method in various investigations such as within the field of tumor pathology.