Sequencing

Think of these methods as different ways of reading the book of life—each with its own purpose, speed, and level of detail

Overview of Clinical Sequencing Technologies

DNA sequencing is the process of determining the precise order of nucleotides (A, T, C, and G) within a DNA molecule. In the clinical lab, this isn’t just an academic exercise; it’s a powerful diagnostic tool that allows us to find the root cause of genetic diseases, guide cancer therapy, and identify pathogens. Let’s explore the main tools in our sequencing toolbox

Sanger Sequencing: The Gold Standard for Targeted Analysis

Sanger sequencing is the classic, foundational method. If you need to read a single, specific “sentence” or “paragraph” of the genetic code with the highest possible accuracy, Sanger is your go-to technique

• The Principle: Chain Termination The magic of Sanger sequencing lies in dideoxynucleotides (ddNTPs). These are special nucleotide mimics that lack the 3’-OH group needed for DNA polymerase to continue building a DNA strand. In the sequencing reaction, we mix the patient’s DNA template with a primer, DNA polymerase, regular nucleotides (dNTPs), and a small amount of fluorescently labeled ddNTPs (each base, A, T, C, and G, gets a different color). As the polymerase copies the DNA, it will occasionally incorporate a colored ddNTP, which stops the chain from growing further. This process creates a collection of DNA fragments of every possible length, each ending with a specific colored tag indicating the final base

• The Workflow and Role in the Lab

  1. The resulting fragments are separated by size with single-base resolution using capillary electrophoresis
  2. As the fragments pass a detector, a laser excites the colored tags, and the instrument records the sequence of colors
  3. The output is a clean, easy-to-read electropherogram showing the sequence

  Clinical Niche Because it is highly accurate but has low throughput, Sanger is perfect for confirming specific findings. If an NGS panel finds a suspicious variant, Sanger sequencing is used to validate it. It’s also the method of choice for single-gene testing, analyzing PCR products, or investigating known mutation hotspots

Next-Generation Sequencing (NGS): Massively Parallel Discovery

If Sanger is reading a single paragraph, NGS is like reading thousands or millions of sentences from every book in a library, all at the same time. It’s a high-throughput approach designed to analyze huge amounts of DNA simultaneously

• The Principle: Massively Parallel Sequencing by Synthesis NGS starts by taking a patient’s DNA, fragmenting it into millions of small pieces, and attaching synthetic DNA “adapters” to the ends of each piece. This collection of prepared DNA is called a library. The library fragments are then loaded onto a special surface called a flow cell, where they are clonally amplified into dense clusters. The instrument then sequences all clusters at once using a “sequencing by synthesis” approach, where it adds one fluorescently labeled base at a time, takes a picture to see which base was added to each cluster, and then repeats the cycle

• The Workflow and Role in the Lab This technology is the engine behind large-scale clinical tests

  • Targeted Gene Panels: Sequencing hundreds of genes related to a specific condition, like inherited cancers or cardiomyopathies
  • Whole Exome Sequencing (WES): Sequencing all the protein-coding regions (~2% of the genome) to diagnose complex or rare genetic disorders
  • Cancer Genomics: Analyzing tumor DNA to find targetable mutations, assess tumor mutational burden, and guide therapy
  • Non-Invasive Prenatal Testing (NIPT): Analyzing fetal DNA from a mother’s blood sample

Pyrosequencing: The Specialist for Quantitative Analysis

Pyrosequencing is a unique “sequencing by synthesis” method that occupies a niche between Sanger and NGS. It’s not for discovery but is exceptionally good at analyzing short, known DNA sequences with quantitative precision

• The Principle: Detecting Light on Incorporation Instead of using chain terminators, pyrosequencing detects the release of pyrophosphate (PPi) each time DNA polymerase adds a correct nucleotide. This PPi kicks off an enzyme cascade that ultimately generates a flash of light via the luciferase enzyme (the same one in fireflies!). The instrument dispenses one nucleotide at a time (A, then T, then C, then G) and measures whether light is produced

• The Workflow and Role in the Lab The key output is a pyrogram, where the height of the light peak is proportional to the number of identical bases incorporated. For example, if a “G” is added and the sequence is “…GG…”, the light peak will be twice as high as a single “G” incorporation Clinical Niche This quantitative ability is its superpower

  • Allele Frequency Quantification: Determining the percentage of a somatic mutation in a tumor sample (e.g., how much of the KRAS gene is mutated)
  • Methylation Analysis: Quantifying the percentage of methylation at specific CpG sites after bisulfite treatment, which is critical in epigenetics and cancer
  • Antimicrobial Resistance Testing: Quickly checking for known resistance mutations in pathogens

Bioinformatics: Turning Raw Data into a Clinical Report

Bioinformatics is the essential bridge between the massive data generated by sequencers (especially NGS) and a clinically meaningful result. It’s a computational pipeline that processes, analyzes, and interprets the sequence data

• The Principle: A Standardized Analysis Pipeline Without bioinformatics, NGS data is just billions of unorganized letters. The pipeline gives it structure and meaning
  1. Quality Control (QC) The pipeline first assesses the raw data (FASTQ files) to ensure it’s high quality
  2. Alignment It then takes the millions of short sequence “reads” and aligns them to a human reference genome, like assembling a giant jigsaw puzzle. The aligned data is stored in a BAM file
  3. Variant Calling The software systematically compares the patient’s aligned sequence to the reference, creating a list of all the differences (SNPs, indels, etc.). This list is stored in a VCF file
  4. Annotation & Interpretation This is the most critical step. The pipeline cross-references each variant against massive databases (e.g., ClinVar) to add context: What gene is it in? What does it do to the protein? Is it common or rare? Has it been linked to a disease before? This annotated list is then filtered down, and a molecular pathologist or geneticist performs the final review to write the patient report
• The Role of the MLS As an MLS, you are a critical user of this pipeline. Your role includes initiating the analysis, performing the first-line QC checks on the run data, recognizing when a pipeline fails, and managing the large data files produced