b'Sanger sequencing methods and tools sample, can be identified and confirmed by Sanger sequencing. Sanger sequencing is the trusted standard for obtaining DNAApplied Biosystems Minor Variant Finder Software is easy-to-sequence information. It powered the Human Genome Project,use desktop software designed for the accurate detection and and investigators continue to rely on this method to generatereporting of minor variants in Sanger sequencing traces with highly accurate reliable sequencing results. Sanger sequencinga detection level of minor alleles as low as 5%. On a test set is a specialized form of fragment analysisit relies on chain- of 632,452 base positions, it exhibited a 5% limit of detection terminating fluorescent nucleotides to generate a series ofwith 95.3% sensitivity and 99.83% specificity [15]. Minor Variant fragments that differ by one nucleotide (Figure 4). Thermo FisherFinder Software can also readily align sequences with the offers fast and straightforward Sanger sequencing workflows thathuman reference genome and VCF files from NGS experiments, provide a high degree of accuracy, long-read capabilities, andproviding a smooth workflow for NGS confirmation with simple data analysis. Applied Biosystems BigDye Terminatorannotations in the dbSNP database.v1.1 and Terminator v3.1 cycle-sequencing chemistries are the gold standard for Sanger sequencing by CE. After cyclePCR methods and toolssequencing, there are various options for cleanup beforeThe portfolio of TaqMan Assays is the most comprehensive set electrophoresis, including Applied Biosystems Centri-Sepof real-time PCR (qPCR) products available for analyzing gene purification columns and plates, ExoSAP-IT enzyme mix, andexpression, miRNA levels, protein abundance, copy number BigDye XTerminator kits. An entire sequencing workflow canvariation, SNP genotyping, and rare-allele mutation detection. be completed in a few hours with minimal hands-on time fromThe typical TaqMan Assay consists of forward and reverse sample to answer, providing the flexibility to support a diverseprimers to amplify a target by PCR (Figure 5). TaqMan Assays range of applications in many research areas.also contain a sequence-specific fluorescent probe, designed with a minor groove binder (MGB) moiety at the 3 end that Discovery-based genomic research, such as NGS, oftenincreases the melting temperature (T ) of the probe and stabilizes uncovers novel or unexpected variants or other sequencemprobetarget hybrids. This means that TaqMan MGB probes anomalies. Investigators look for ways to verify these newcan be significantly shorter than traditional probes, providing discoveries using orthogonal methods. Sanger sequencingbetter sequence discrimination and flexibility to accommodate is the method of choice for confirming NGS results becausemore targets. In addition, TaqMan probes have a nonfluorescent of its workflow simplicity and unambiguous results. For thesequencher (NFQ) that binds to the fluorophore, until it is cleaved by confirmatory studies, short amplicons, usually covering only thethe polymerase, and minimizes background. All TaqMan Assays region to be confirmed, need to be sequenced. Moreover, minorcome with a performance guarantee.allelic variants, present in a heterogeneous PCR amplication PCR cleanup Cycle sequencing Sequencing cleanup Capillary electrophoresis Data analysisT C A C G C AA G TCA A A T A C GT T G G A AT CG C G TCT T GFigure 4. Basics of a Sanger sequencing workflow. The target region that will be sequenced is amplified by PCR. The primers and PCR reagents Capillary electrophoresis workoware removed before a second linear amplification is performed. This step generates the fragments that are chain-terminated with a fluorescent dideoxynucleotide. The cycle sequencing reaction is purified, and the resulting fragments are separated by capillary electrophoresis and detected with a laser. The sequence can be read from the lengths of the fragments and the colors of the dideoxynucleotide terminators.Forward primer Probe Figure 5. Basics of a TaqMan Assay. Unlabeled forward and reverse R Q M oligonucleotide primers define the region that will be queried. A third 5 3 oligonucleotide binds to the region between the primers. The probe has 3 an end-label fluorophore reporter, a nonfluorescent quencher, and a minor groove binding (MGB) moiety that stabilizes binding of the short 5 probe sequence. When the probe is hybridized to its target sequence, the quencher greatly reduces the fluorescence of the reporter. When the sequence is copied during the PCR reaction, the nuclease activity Reverse primer of the polymerase cleaves the reporter from the rest of the probe, releasing the reporter be detected by a laser in the instrument. The Reporter dye Nonuorescent quencher MGB amount of fluorescence is therefore directly related to the amount of target amplified.Contents 18'