Alexandre M. Izmailov

A general approach to the analysis of errors and failure modes in the base-calling function in automated fluorescent DNA sequencing.

We describe the analysis of errors and failure modes in the base‐calling function in automated DNA sequencing, on instruments in which fluorescently‐labeled Sanger dideoxy‐sequencing ladders are detected via their times of migration past a fixed detector. A general approach entails the joint use of: (i) well‐defined control samples such as M13mp18, and (ii) mathematical simulation of sequencing electropherograms, with the deliberate introduction of different types of distortion and noise. An algorithm, the electrophoretic trace simulator (ETS), is used to calculate electrophoresis traces corresponding to the output data stream of an automated fluorescent DNA sequencer. The ETS accepts a user‐defined sequence of nucleotide bases (A, C, G, T) as input, and employs user‐adjustable functions to compute the following critical parameters of an electropherogram: peak intensity, peak spacing, peak shape as a function of base number; background, noise, and spectral cross‐talk correction (for a sequencer using multiple dyes). We use a combination of M13mp18 controls and simulated electropherograms to analyze two problems of considerable practical importance: (i) variation in electrophoretic migration rates between different lanes of a gel, and (ii) variation in signal intensity due to user‐dependent loading artifacts. The issue of base‐calling errors and failure modes, for electropherograms that contain noise and distortion, is addressed.

A High-Sensitivity, Medium-Density, and Target Amplification–Free Planar Waveguide Microarray System for Gene Expression Analysis of Formalin-Fixed and Paraffin-Embedded Tissue

BACKGROUND Many microarray platforms and their associated assay chemistries do not work properly with RNA extracted from formalin-fixed, paraffin-embedded (FFPE) tissue samples, a feature that severely hampers the use of microarrays in oncology applications, for which FFPE tissue is the routine specimen. Furthermore, the limited sensitivity of most microarray platforms requires time-consuming and costly amplification reactions of the target RNA, which negatively affects clinical laboratory work flow. METHODS We developed an approach for sensitively and reliably measuring mRNA abundances in FFPE tissue samples. This approach involves automated RNA extractions, direct hybridization of extracted RNA to immobilized capture probes, antibody-mediated labeling, and readout with an instrument applying the principle of planar waveguides (PWG). A 14-gene multiplex assay conducted with RNA isolated from 20 FFPE blocks was correlated to an analysis of the same with reverse-transcription quantitative real-time PCR (RT-qPCR). RESULTS The assay sensitivity for gene expression analysis obtained for the PWG microarray platform was <10 fmol/L, eliminating the need for target preamplification. We observed a correlation coefficient of 0.87 to state-of-the-art RT-qPCR technology with RNA isolated from FFPE tissue, despite a compressed dynamic range for the PWG system (a 2.9-log dynamic range for PWG in our test system vs 5.0 logs for RT-qPCR). The precision of the PWG platform was comparable to RT-qPCR (Pearson correlation coefficient of 0.9851 for PWG vs 0.9896 for RT-qPCR) for technical replicates. CONCLUSIONS The presented PWG platform demonstrated excellent sensitivity and precision and is especially well suited for any application for which fast, simple, and robust multiplex assays of RNA in FFPE tissue are required.

Improvement of base-calling in multilane automated DNA sequencing by use of electrophoretic calibration standards, data linearization, and trace alignment.

We present a new method for the linearization and alignment of data traces generated by multilane automated DNA sequencing instruments. Application of this method to data generated with the Visible Genetics Open Gene DNA sequencing system (using Micro Cel 700 gel cassettes, with a 25 cm separation distance) allows read lengths of >1000 nucleotides to be routinely obtained with high confidence and >97% accuracy. This represents an increase of 10–15% in average read length, relative to data from this system that have not been processed in the fashion described herein. Most importantly, the linearization and alignment method allows usable sequence to be obtained from a fraction of 10–15% of data sets which, because of original trace misalignment problems, would otherwise have to be discarded. Our method involves adding electrophoretic calibration standards to the DNA sequencing fragments. The calibration standards are labeled with a dye that differs spectrally from the dye attached to the sequencing fragments. The calibration standards are identical in all the lanes. Analysis of the mobilities of the calibration standards allows correction for both systematic and random variation of electrophoretic properties between gel lanes. We have successfully used this method with two‐dye and three‐dye DNA sequencing instruments.