Massively Parallel: Next Generation Sequencing Explained

If a machine can process many different reactions in parallel rather than one by one, then it is capable of high-throughput analysis. If this "massive parallelism" is the core of NGS, then it allows for the sequencing of entire genomes in a very short time.

If Sanger sequencing reads one DNA fragment per capillary, then it is a low-throughput method. If NGS reads millions of fragments simultaneously, then it is a high-throughput method that provides more data in a single run.

If the DNA fragments need to stick to a surface and be recognized by the machine's primers, then they must have known sequences added to their ends. These synthetic sequences are called adapters.

If the goal is to sequence genomic DNA, then the DNA must be prepared (library), copied (amplification), and read (synthesis). While transcription is a biological process, it is not a standard step in the NGS chemical procedure.

If the DNA needs to be spread out so that a camera can take pictures of the glowing bases, then it needs a flat surface. If this specialized glass slide contains billions of tiny wells or spots, then it is a flow cell.

If the machine needs to pause after adding a base to take a photo, then the nucleotide must act as a stop signal. If that stop signal can be chemically removed to allow the next base to join, then it is a reversible terminator.

If the output of a machine is millions of random, short data points, then a computer is needed to manage the complexity. If biology and computer science are combined for this task, then the field is bioinformatics.

If the original Human Genome Project took 13 years using Sanger sequencing, but NGS can process millions of bases per second, then the timeframe is drastically reduced. In a modern lab, the process now takes roughly 1 to 3 days.

If NGS provides a detailed look at DNA sequences, then it can find mutations or identify pathogens. While it provides the data for cloning, the sequencing process itself does not create a living clone.

If the "Exome" represents the 1-2% of the genome that actually codes for proteins, and if most disease-causing mutations occur there, then sequencing just that part is a more efficient diagnostic strategy.

If the sequence is determined by building a new, complementary strand and watching it grow in real-time, then the technique is defined as sequencing by synthesis.

If the chemical process of adding reversible terminators becomes less accurate as the strand gets longer, then the fragments are usually limited to 50-300 bases. If they are short, then piecing them back together (assembly) is computationally difficult.

If millions of single DNA strands are spread on a slide, then they must be copied into "clusters" to be bright enough for a camera to see. If bridge or emulsion PCR creates these identical clusters, then they provide the necessary signal boost.

If a machine reads a base 30 different times from 30 different fragments, then the "coverage" is 30x. If higher coverage leads to more confidence that the sequence is correct, then it is a vital metric for clinical accuracy.

If a hydrogen ion is released every time a nucleotide is added, then the environment becomes more acidic. If the machine uses a semiconductor chip to detect this shift in pH, then it doesn't need a camera.

If a standard reaction tube can only do one test, but an NGS chip has millions of tiny reaction sites, then the system is doing many things at once. If this "simultaneous" action occurs, then it is defined as massive parallelism.

If the DNA needs to attach and the reagents need to flow over them, then the cell needs anchors (oligos) and tubes (channels). It also requires a buffer for the chemistry to work; gold and large proteins are not standard structural parts.

If Sanger sequencing costs about $500 per million bases and NGS costs about $0.01 per million bases, then NGS is significantly cheaper for large projects. Therefore, the statement is false.

If the machine can only read short segments, then the long strands of DNA must be broken down. If they are physically or chemically shattered into smaller pieces, then they have undergone fragmentation.

If an environment contains thousands of different microbes that cannot be grown in a lab, then we can extract all their DNA together. If NGS sequences all those fragments at once, then we can map the entire microbial community.