The availability of massive amounts of DNA sequence information has begun to revolutionize the practice of biology. As a result, current large-scale sequencing output, while impressive, is not adequate to keep pace with growing demand and, in particular, is far short of what will be required to obtain the 3-billion-base human genome sequence by the target date of 2005. To reach this goal, improved automation will be essential, and it is particularly important that human involvement in sequence data processing be significantly reduced or eliminated. Progress in this respect will require both improved accuracy of the data processing software and reliable accuracy measures to reduce the need for human involvement in error correction and make human review more efficient. Here, we describe one step toward that goal: a base-calling program for automated sequencer traces, phred, with improved accuracy. phred appears to be the first base-calling program to achieve a lower error rate than the ABI software, averaging 40%-50% fewer errors in the data sets examined independent of position in read, machine running conditions, or sequencing chemistry.

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Base-calling of automated sequencer traces using Phred. I. accuracy assessment

Elimination of the data processing bottleneck in high-throughput sequencing will require both improved accuracy of data processing software and reliable measures of that accuracy. We have developed and implemented in our base-calling program phred the ability to estimate a probability of error for each base-call, as a function of certain parameters computed from the trace data. These error probabilities are shown here to be valid (correspond to actual error rates) and to have high power to discriminate correct base-calls from incorrect ones, for read data collected under several different chemistries and electrophoretic conditions. They play a critical role in our assembly program phrap and our finishing program consed.

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Base-Calling of Automated Sequencer Traces Using Phred. II. Error Probabilities

I read this book the same weekend that the Packers took on the Rams, and the experience of the latter event, obviously, colored my judgment. Although I abhor anything that smacks of being a handbook (like, \"How to Earn a Merit Badge in Neurosurgery\") because too many volumes in biomedical science already evince a boyscout-like approach, I must confess that parts of this volume are fast, scholarly, and significant, with certain reservations. I like parts of this well-illustrated book because Dr. Sj6strand, without so stating, develops certain subjects on technique in relation to the acquisition of judgment and sophistication. And this is important! So, given that the author (like all of us) is somewhat deficient in some areas, and biased in others, the book is still valuable if the uninitiated reader swallows it in a general fashion, realizing full well that what will be required from the reader is a modulation to fit his vision, propreception, adaptation and response, and the kind of problem he is undertaking. A major deficiency of this book is revealed by comparison of its use of physics and of chemistry to provide understanding and background for the application of high resolution electron microscopy to problems in biology. Since the volume is keyed to high resolution electron microscopy, which is a sophisticated form of structural analysis, but really morphology in a modern guise, the physical and mechanical background of The instrument and its ancillary tools are simply and well presented. The potential use of chemical or cytochemical information as it relates to biological fine structure , however, is quite deficient. I wonder when even sophisticated morphol-ogists will consider fixation a reaction and not a technique; only then will the fundamentals become self-evident and predictable and this sine qua flon will become less mystical. Staining reactions (the most inadequate chapter) ought to be something more than a technique to selectively enhance contrast of morphological elements; it ought to give the structural addresses of some of the chemical residents of cell components. Is it pertinent that auto-radiography gets singled out for more complete coverage than other significant aspects of cytochemistry by a high resolution microscopist, when it has a built-in minimal error of 1,000 A in standard practice? I don't mean to blind-side (in strict football terminology) Dr. Sj6strand's efforts for what is \"routinely used in our laboratory\"; what is done is usually well done. It's just that …

Methods in Enzymology.

As part of our effort to sequence the 100-megabase (Mb) genome of the nematode Caenorhabditis elegans, we have completed the nucleotide sequence of a contiguous 2,181,032 base pairs in the central gene cluster of chromosome III. Analysis of the finished sequence has indicated an average density of about one gene per five kilobases; comparison with the public sequence databases reveals similarities to previously known genes for about one gene in three. In addition, the genomic sequence contains several intriguing features, including putative gene duplications and a variety of other repeats with potential evolutionary implications.

2.2 Mb of contiguous nucleotide sequence from chromosome III of C. elegans

Fluorescent labeling of biomolecules with organic probes.

The incorporation of fluorescently labeled dideoxynucleotides by T7 DNA polymerase is optimized by the use of Mn2+, fluorescein analogs and four 2'-deoxyribonucleoside 5'-O-(1-thiotriphosphates) (dNTP alpha S's). The one-tube extension protocol was tested on single-stranded templates, as well as PCR fragments which were made single-stranded by digestion with T7 gene 6 exonuclease. Dye primer sequencing using four dNTP alpha S's was shown to give uniform termination patterns which were comparable to four dNTPs. Efficiency of the polymerase also appeared to improve with the dNTP alpha S's. A mathematical model was developed to predict the pattern of termination based on enzyme activity and ratios of ddNTP/dNTPs. This method can be used to optimize sequencing reactions and to estimate enzyme discrimination constants of chain terminators.

DNA sequencing with dye-labeled terminators and T7 DNA polymerase: effect of dyes and dNTPs on incorporation of dye-terminators and probability analysis of termination fragments.

A system is provided for carrying out real time fluorescence-based measurements of nucleic acid amplification products. In a preferred embodiment of the invention, an excitation beam is focused into a reaction mixture through a surface, the reaction mixture containing: (i) a first fluorescent indicator capable of generating a first fluorescent signal whose intensity is proportional to the amount of an amplification product in the volume of the reaction mixture illuminated by the excitation beam and (ii) a second fluorescent indicator homogeneously distributed throughout the reaction mixture capable of generating a second fluorescent signal proportional to the volume of reaction mixture illuminated by the excitation beam. Preferably, the excitation beam is focused into the reaction mixture by a lens through a portion of a wall of a closed reaction chamber containing the reaction mixture. The same lens is used to collect the first and second fluorescent signals generated by the first and second fluorescent indicators, respectively, in response to the excitation beam. The ratio of the fluorescent intensities of the first and second fluorescent signals provides a stable quantitative indicator of the amount of amplification product synthesized in the course of the amplification reaction.

System for real time detection of nucleic acid amplification products

The present invention generally relates to the detection of analytes, particularly biomolecules in samples. The invention also relates to compositions, methods, and kits for detecting the presence of analytes, typically in multiplex detection formats. The invention also relates to methods for determining the presence of at least one analyte in a sample, the methods employing employ single molecule detection techniques to individually detect at least one molecular complex or at least part of a molecular complex.

Multiplex detection compositions, methods, and kits

An apparatus is provided which includes a sample holder for holding a reaction chamber which includes an optical interface, a fiber optic cable for delivering an excitation beam to a sample housed within the reaction chamber and for receiving light emitted by the sample, and a lens co-axially disposed with the fiber optic cable and positioned outside the reaction chamber for focusing the excitation beam through the optical interface and within a volume of the sample and for collecting and transmitting to the fiber optic cable light emitted within the volume of the sample.

Apparatus and method for detecting nucleic acid amplification products

The present teachings generally relate to probes and probes sets for detecting analytes. The teachings also relates to compositions, methods, and kits for assembling probes comprising at least one coded molecular tag.

Charles R. Connell

Papers

DNA sequencing with dye-labeled terminators and T7 DNA polymerase: effect of dyes and dNTPs on incorporation of dye-terminators and probability analysis of termination fragments.

System for real time detection of nucleic acid amplification products

Multiplex detection compositions, methods, and kits

Apparatus and method for detecting nucleic acid amplification products

Compositions, methods, and kits for assembling probes