Beginning in the 1980s, automation of DNA sequencing has greatly increased throughput, reduced costs, and enabled large projects to be completed more easily. The development of automation technology paralleled the development of other aspects of DNA sequencing: better enzymes and chemistry, separation and imaging technology, sequencing protocols, robotics, and computational advancements (including base-calling algorithms with quality scores, database developments, and sequence analysis programs). Despite the emergence of high-throughput sequencing platforms, automated Sanger sequencing technology remains useful for many applications. This unit provides background and a description of the "First-Generation" automated DNA sequencing technology. It also includes protocols for using the current Applied Biosystems (ABI) automated DNA sequencing machines.
[Show abstract][Hide abstract] ABSTRACT: Chain termination cycle sequencing, or “first-generation” DNA sequencing, was developed 3 decades ago but remains one of the most commonly used procedures for diagnostic analyses. Automated capillary-gel electrophoresis genetic analyzers greatly improved the efficiency of sequencing DNA templates between 100 and approximately 1,300 nucleotides long. Cycle sequencing may be completed the same day by using fast protocols for the initial amplification and cycle-sequencing reactions and by utilization of commercial sequence interpretation and analysis software. These changes allowed sequencing to become a routine tool for pathogen identification, discovery, and genotyping.
[Show abstract][Hide abstract] ABSTRACT: Individuals who inherit mutations in BRCA1 or BRCA2 are predisposed to breast and ovarian cancers. However, identifying mutations in these large genes by conventional dideoxy sequencing in a clinical testing laboratory is both time consuming and costly, and similar challenges exist for other large genes, or sets of genes, with relevance in the clinical setting. Second-generation sequencing technologies have the potential to improve the efficiency and throughput of clinical diagnostic sequencing, once clinically validated methods become available. We have developed a method for detection of variants based on automated small-amplicon PCR followed by sample pooling and sequencing with a second-generation instrument. To demonstrate the suitability of this method for clinical diagnostic sequencing, we analyzed the coding exons and the intron-exon boundaries of BRCA1 and BRCA2 in 91 hereditary breast cancer patient samples. Our method generated high-quality sequence coverage across all targeted regions, with median coverage greater than 4000-fold for each sample in pools of 24. Sensitive and specific automated variant detection, without false-positive or false-negative results, was accomplished with a standard software pipeline using bwa for sequence alignment and samtools for variant detection. We experimentally derived a minimum threshold of 100-fold sequence depth for confident variant detection. The results demonstrate that this method is suitable for sensitive, automatable, high-throughput sequence variant detection in the clinical laboratory.
The Journal of molecular diagnostics: JMD 09/2013; 15(6). DOI:10.1016/j.jmoldx.2013.07.004 · 4.85 Impact Factor
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