Using next-generation sequencing for high resolution multiplex analysis of copy number variation from nanogram quantities of DNA from formalin-fixed paraffin-embedded specimens

The use of next-generation sequencing technologies to produce genomic copy number data has recently been described. Most approaches, however, reply on optimal starting DNA, and are therefore unsuitable for the analysis of formalin-fixed paraffin-embedded (FFPE) samples, which largely precludes the analysis of many tumour series. We have sought to challenge the limits of this technique with regards to quality and quantity of starting material and the depth of sequencing required. We confirm that the technique can be used to interrogate DNA from cell lines, fresh frozen material and FFPE samples to assess copy number variation. We show that as little as 5 ng of DNA is needed to generate a copy number karyogram, and follow this up with data from a series of FFPE biopsies and surgical samples. We have used various levels of sample multiplexing to demonstrate the adjustable resolution of the methodology, depending on the number of samples and available resources. We also demonstrate reproducibility by use of replicate samples and comparison with microarray-based comparative genomic hybridization (aCGH) and digital PCR. This technique can be valuable in both the analysis of routine diagnostic samples and in examining large repositories of fixed archival material.     

A system for shotgun DNA sequencing.

A multipurpose cloning site has been introduced into the gene for beta-galactosidase (beta-D-galactosidegalactohydrolase, EC 3.21.23) on the single-stranded DNA phage M13mp2 (Gronenborn, B. and Messing, J., (1978) Nature 272, 375-377) with the use of synthetic DNA. The site contributes 14 additional codons and does not affect the ability of the lac gene product to undergo intracistronic complementation. Two restriction endonuclease cleavage sites in the viral gene II were removed by single base-pair mutations. Using the new phage M13mp7, DNA fragments generated by cleavage with a variety of different restriction endonucleases can be cloned directly. The nucleotide sequences of the cloned DNAs can be determined rapidly by DNA synthesis using chain terminators and a synthetic oligonucleotide primer complementary to 15 bases preceeding the new array of restriction sites.     

Assay for nanogram quantities of DNA in cellular homogenates.

The DNA concentration of a crude cellular homogenate can be measured accurately in the nanogram range using the fluorescence enhancement of 4′,6-diamidino-2-phenylindole (DAPI) or bisbenzimidazole (Hoechst H 33258) complexed with DNA. A simple assay has been devised including an internal standard, which allows reliable measurement and compensates for any quenching due to cellular components or buffer. The fluorescence enhancement is highly specific for DNA; no other cell component produces significant fluorescence. The response is linear over a broad dynamic range making the measurement of unknown DNA concentrations convenient.     

Minilibraries constructed from cDNA generated by arbitrarily primed RT-PCR: an alternative to normalized libraries for the generation of ESTs from nanogram quantities of mRNA

The generation of expressed sequenced tags (ESTs) depends on the arbitrary selection of individual cDNA clones from libraries. The efficiency of this process reflects the clonal structure of the library used and can be significantly increased using size selected, directional, normalized cDNA libraries. This strategy, however, is not readily applicable when mRNA is limiting, as is the case in the study of complex microorganisms such as parasites, fetal tissues or tumor biopsies. We show here that the construction and systematic sequencing of minilibraries of cDNAs produced by arbitrarily primed PCR provides an alternative means of efficiently generating ESTs in situations where only nanogram quantities of RNA are available. This methodology greatly compensates for unequal message abundance, avoids the need for complex library construction, is equally applicable to the analysis of abundant or rare biological material and is ideally suited to multicenter programmes.     

A protocol for high-throughput extraction of DNA from rice leaves

Current DNA isolation methods have limitations between speed and purity in high-throughput molecular genetic analysis such as gene mapping and marker-assisted selection programs. We have optimized a simple and rapid method for isolating high-quality genomic DNA from rice that significantly minimizes time and the use of laboratory materials. One person can process as many as 384 samples in 2 h. The isolated DNA is suitable for polymerase chain reaction-based techniques and is stable for no less than 6 mo of storage at 4°C.     

A general coverage theory for shotgun DNA sequencing.

The classical theory of shotgun DNA sequencing accounts for neither the placement dependencies that are a fundamental consequence of the forward-reverse sequencing strategy, nor the edge effect that arises for small to moderate-sized genomic targets. These phenomena are relevant to a number of sequencing scenarios, including large-insert BAC and fosmid clones, filtered genomic libraries, and macro-nuclear chromosomes. Here, we report a model that considers these two effects and provides both the expected value of coverage and its variance. Comparison to methyl-filtered maize data shows significant improvement over classical theory. The model is used to analyze coverage performance over a range of small to moderately-sized genomic targets. We find that the read pairing effect and the edge effect interact in a non-trivial fashion. Shorter reads give superior coverage per unit sequence depth relative to longer ones. In principle, end-sequences can be optimized with respect to template insert length; however, optimal performance is unlikely to be realized in most cases because of inherent size variation in any set of targets. Conversely, single-stranded reads exhibit roughly the same coverage attributes as optimized end-reads. Although linking information is lost, single-stranded data should not pose a significant assembly liability if the target represents predominantly low-copy sequence. We also find that random sequencing should be halted at substantially lower redundancies than those now associated with larger projects. Given the enormous amount of data generated per cycle on pyro-sequencing instruments, this observation suggests devising schemes to split each run cycle between twoor more projects. This would prevent over-sequencing and would further leverage the pyrosequencing method.     

Gap statistics for whole genome shotgun DNA sequencing projects

MOTIVATION: Investigators utilize gap estimates for DNA sequencing projects. Standard theories assume sequences are independently and identically distributed, leading to appreciable under-prediction of gaps. RESULTS: Using a statistical scaling factor and data from 20 representative whole genome shotgun projects, we construct regression equations that relate coverage to a normalized gap measure. Prokaryotic genomes do not correlate to sequence coverage, while eukaryotes show strong correlation if the chaff is ignored. Gaps decrease at an exponential rate of only about one-third of that predicted via theory alone. Case studies suggest that departure from theory can largely be attributed to assembly difficulties for repeat-rich genomes, but bias and coverage anomalies are also important when repeats are sparse. Such factors cannot be readily characterized a priori, suggesting upper limits on the accuracy of gap prediction. We also find that diminishing coverage probability discussed in other studies is a theoretical artifact that does not arise for the typical project.     

A mercury-thiol affinity system for rapid generation of overlapping labeled DNA fragments for DNA sequencing.

We describe an in vitro protocol for quickly generating overlapping terminal-labeled restriction fragments for DNA sequence analysis via the Maxam-Gilbert technique. The protocol involves introducing mercurated nucleotides into one end of a region to be sequenced, partial digestion with several restriction enzymes and terminal-labeling, separation of the mercurated restriction enzymes and terminal-labeling, separation of the mercurated restriction fragments from non-mercurated ones on a thiol column and resolution of the different mercurated fragments on one preparative agarose gel. The protocol was used to determine the nucleotide sequence of a 980 base pair cDNA that contains the coding region for a variable surface glycoprotein of Trypanosoma brucei. It could just as quickly and easily be used to obtain many terminal-labeled overlapping restriction fragments covering a region of several kilobases.     

Apple II software for M13 shotgun DNA sequencing.

A set of programs is presented for the reconstruction of a DNA sequence from data generated by the M13 shotgun sequencing technique. Once the sequence has been established and stored other programs are used for its analysis. The programs have been written for the Apple II microcomputer. A minimum investment is required for the hardware and the software is easily interchangeable between the growing number of interested researchers. Copies are available in ready to use form.     

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.     

Bst DNA polymerase permits rapid sequence analysis from nanogram amounts of template

A simple, rapid method is presented for the enzymatic sequence analysis of nanogram amounts of single-stranded or double-stranded DNA. This approach employs the thermostable DNA polymerase from Bacillus sterothermophilus and exploits its ability to efficiently extend all of the template-primer complex, even at low substrate concentrations. The procedure requires few pipetting steps, no preannealing step and very short reaction time. This method can significantly reduce the cost associated with DNA polymerase and the amount of template and time required to perform the enzymatic sequencing reactions. As little as a 10-ng aliquot of such sequencing reactions can be analyzed on a fluorescence-based capillary gel electrophoresis instrument recently developed in our laboratory. This highly sensitive detection, in conjunction with the ability to efficiently sequence nanogram amounts of template, strongly suggests the feasibility of direct DNA sequencing of single bacteriophage M13 plaques without prior amplification.     

Experience in shotgun sequencing a 134 kilobase pair DNA molecule.

Until now, large DNA sequences have been obtained by cloning fragments of the target molecule into plasmid, cosmid or bacteriophage lambda vectors. The 134 kbp DNA sequence of channel catfish virus was determined with relative ease by shotgun cloning of random fragments of genomic DNA directly into a bacteriophage M13 vector, sequencing by dideoxynucleotide chain termination, and compilation of the data using Staden's database handling programs. Experience gained during this endeavour indicates that sequences substantially larger than 134 kbp may be obtained using this approach.     

A high-throughput protocol for extracting high-purity genomic DNA from plants and animals

DNA extraction techniques that employ the reversible binding of DNA to silica via chaotropic salts can deliver high-quality genomic DNA from plant and animal tissues, while avoiding the use of toxic organic solvents. Existing techniques that use this method are either prohibitively expensive, or are applicable to only a restricted set of taxa. Here we describe a cost-effective DNA extraction technique suitable for a wide range of plant and animal taxa that yields microgram quantities of high-molecular-weight genomic DNA at a throughput of 192 samples per day. Our technique is particularly robust for tissue samples that are insoluble or are rapidly discoloured or oxidized in standard DNA extraction buffers. We demonstrate the quality of DNA extracted using this method by applying the amplified fragment length polymorphism technique to plant species.     

THetA: inferring intra-tumor heterogeneity from high-throughput DNA sequencing data

Tumor samples are typically heterogeneous, containing admixture by normal, non-cancerous cells and one or more subpopulations of cancerous cells. Whole-genome sequencing of a tumor sample yields reads from this mixture, but does not directly reveal the cell of origin for each read. We introduce THetA (Tumor Heterogeneity Analysis), an algorithm that infers the most likely collection of genomes and their proportions in a sample, for the case where copy number aberrations distinguish subpopulations. THetA successfully estimates normal admixture and recovers clonal and subclonal copy number aberrations in real and simulated sequencing data. THetA is available at http://compbio.cs.brown.edu/software/.     

Recovery of nanogram quantities of DNA from plasma and quantitative measurement using labeling by nick translation

A method which allows for the quantitative measurement of DNA in plasma is described. After treatment of plasma with phenol, DNA is precipitated by ethanol using gelatin as a coprecipitating agent. DNA is then measured by nick translation labeling. This assay takes a few hours. It is suitable for the measurement of DNA within a range of 0.02 to 20 ng in 10 microliters of plasma. For example, it is applied to the measurement of DNA in plasma from mice injected with bacterial lipopolysaccharide.     

Extension of Lander-Waterman theory for sequencing filtered DNA libraries

Background  

The degree to which conventional DNA sequencing techniques will be successful for highly repetitive genomes is unclear. Investigators are therefore considering various filtering methods to select against high-copy sequence in DNA clone libraries. The standard model for random sequencing, Lander-Waterman theory, does not account for two important issues in such libraries, discontinuities and position-based sampling biases (the so-called "edge effect"). We report an extension of the theory for analyzing such configurations.