Fundamental Next-Generation Sequencing Technologies to Know for Computational Genomics

Next-generation sequencing (NGS) technologies have revolutionized genomics by enabling rapid and cost-effective DNA sequencing. These methods, like Illumina and PacBio, provide diverse applications, from whole genome sequencing to RNA analysis, driving advancements in computational genomics.

  1. Illumina sequencing (sequencing by synthesis)

    • Utilizes reversible dye terminators to identify nucleotides as they are incorporated into growing DNA strands.
    • High throughput capability allows for sequencing millions of fragments simultaneously.
    • Generates short reads (typically 50-300 bp), which are useful for a variety of applications including whole genome sequencing and targeted resequencing.
  2. Ion Torrent semiconductor sequencing

    • Measures changes in pH as nucleotides are added to a growing DNA strand, allowing for real-time sequencing.
    • Offers a faster and more cost-effective alternative to optical methods, with a focus on smaller-scale projects.
    • Produces shorter reads (up to 400 bp) and is particularly useful for targeted sequencing and small genomes.
  3. 454 pyrosequencing

    • Based on the detection of pyrophosphate release during nucleotide incorporation, generating light signals proportional to the number of nucleotides added.
    • Capable of producing longer reads (up to 1,000 bp) compared to other NGS technologies.
    • Primarily used for applications such as metagenomics and de novo sequencing, though it has largely been phased out in favor of other technologies.
  4. SOLiD sequencing

    • Employs ligation-based methods to sequence DNA, using fluorescently labeled oligonucleotides.
    • Provides high accuracy and the ability to generate short reads (50-75 bp), making it suitable for applications requiring precise variant detection.
    • Often used in targeted resequencing and transcriptome analysis.
  5. Pacific Biosciences (PacBio) SMRT sequencing

    • Utilizes single-molecule real-time (SMRT) technology to sequence long DNA fragments (up to 15,000 bp or more).
    • Offers high accuracy with circular consensus sequencing (CCS) and is particularly useful for complex genomes and structural variant detection.
    • Enables comprehensive transcriptome analysis and epigenetic studies due to its long-read capabilities.
  6. Oxford Nanopore sequencing

    • Employs nanopore technology to sequence DNA by measuring changes in ionic current as nucleotides pass through a nanopore.
    • Capable of producing ultra-long reads (up to several megabases), which is advantageous for resolving repetitive regions and structural variants.
    • Portable devices allow for real-time sequencing and field applications, making it versatile for various genomic studies.
  7. Paired-end and mate-pair sequencing

    • Paired-end sequencing involves sequencing both ends of a DNA fragment, providing information about the distance between reads, which aids in genome assembly.
    • Mate-pair sequencing uses longer fragments to link distant regions of the genome, improving assembly and structural variant detection.
    • Both methods enhance the accuracy of genome mapping and facilitate the identification of complex genomic rearrangements.
  8. RNA-Seq

    • A technique for sequencing RNA to analyze gene expression levels, alternative splicing, and non-coding RNA.
    • Provides a comprehensive view of the transcriptome, allowing for the identification of novel transcripts and isoforms.
    • Utilizes various NGS platforms, with data analysis requiring specialized computational tools for quantification and differential expression analysis.
  9. ChIP-Seq

    • Combines chromatin immunoprecipitation (ChIP) with sequencing to identify binding sites of proteins (e.g., transcription factors) on DNA.
    • Provides insights into gene regulation and epigenetic modifications by mapping protein-DNA interactions across the genome.
    • Data analysis involves peak calling and integration with other genomic datasets to understand regulatory networks.
  10. Whole genome sequencing (WGS)

    • Involves sequencing the entire genome of an organism, providing a comprehensive view of genetic variation.
    • Useful for applications such as population genomics, evolutionary studies, and personalized medicine.
    • Requires substantial computational resources for data storage, processing, and analysis, including variant calling and annotation.


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© 2024 Fiveable Inc. All rights reserved.
AP® and SAT® are trademarks registered by the College Board, which is not affiliated with, and does not endorse this website.