DNA Sequencing 2

Overview of DNA Sequencing Techniques

  • Three main types of sequencing:

    • Next Generation Sequencing (NGS)

    • Targeted Sequencing

    • Third Generation Sequencing (TGS)

Next Generation Sequencing (NGS)

  • Massively parallel method of sequencing.

  • Steps involved in NGS:

    • 1. Genomic DNA (gDNA) Isolation

    • 2. NGS Library Construction:

    • gDNA is fragmented.

    • Preparation of fragments to interact with the sequencing platform.

    • 3. Library Partitioning:

    • Segregating fragments of DNA into clusters of identical copies.

    • 4. Cluster Sequencing

    • 5. Data Analysis.

Illumina Sequencing

  • Steps for NGS library construction:

    • DNA is broken into fragments.

    • Adaptors are added to the fragments.

    • Fragmentation Methods:

    • 1. Ultrasonic Disruption

    • 2. Tagmentation

    • PCR Amplification:

    • Optional step that increases the number of copies of each fragment.

    • Can include index sequence or barcode for multiplexing.

Fragmentation by Ultrasonic Disruption

  • Visual aids: Figures 8.08 & 8.09 from Clark et al. (2019).

Fragmentation by Tagmentation

  • Visual aids: Figures 8.10 from Clark et al. (2019).

Adapters in Illumina Sequencing

  • Key features of adapters:

    • A sequence complementary to the oligos bound to flow cell.

    • An index sequence unique to each genomic fragment from one sample.

    • A sequence that is complementary to the sequencing primer.

  • Visual aid: Figure 8.11 from Clark et al. (2019).

Library Partitioning and Bridge Amplification in Illumina Sequencing

  • Visual aids: Figures 8.12 & 8.13 from Clark et al. (2019).

Sequencing by Synthesis in Illumina Sequencing

  • Visual aid: Figure 8.14 from Clark et al. (2019).

Analysis of Illumina Sequencing Data

  • The final sequence from each cluster is referred to as a read.

  • Four reads generated:

    • Read 1: from left to right.

    • Read 2: from right to left.

    • Read 3: from left to right (including index/barcode).

    • Read 4: from right to left (including index/barcode).

  • Data Analysis:

    • With a reference genome: alignment and mapping to identify variants such as SNPs, SNVs, etc.

    • Without a reference genome (de novo sequencing):

    • Align the reads to create a contig.

    • Compare contigs to form a consensus sequence.

  • Read Depth: Number of reads covering an individual site.

  • Visual aid: Figure 8.15 from Clark et al. (2019).

Ion Torrent Sequencing Technology

  • Key steps in library preparation include:

    • DNA isolation, fragmentation, and addition of adaptors.

    • Partitioning each fragment into its own location (using microbeads instead of a flow cell).

    • Emulsion PCR:

    • Separation of beads using oil and water to avoid contamination.

    • Replicates DNA using PCR to produce thousands of copies of each fragment.

    • Sequencing Stage:

    • Oil is removed, and beads are put into microwells.

    • Primer and Polymerase are added.

    • dNTPs are introduced one at a time, with the addition recorded.

  • Advantage: Faster than Illumina sequencing. Limitation: Cannot differentiate multiple dNTPs added consecutively.

  • Visual aid: Figures 8.18 & 8.19 from Clark et al. (2019).

Targeted Sequencing

  • Focus on specific sequences or areas of interest.

  • Advantages: Increased coverage in selected regions.

  • Techniques include:

    • Whole Exome Sequencing:

    • Sequences all of the exons.

    • Using PCR to amplify regions of interest:

    • Add adaptors for sequencing.

    • Use of biotinylated oligonucleotide probes:

    • Fragmented DNA mixed with biotinylated probes to form a panel.

    • Probes have sequences complimentary to specific genes or exons.

  • Effective for improving accessibility to sequencing in health-related fields.

Third Generation Sequencing (TGS)

  • Unique feature: sequences from one strand of DNA; does not require multiple copies.

  • Two primary techniques:

    • Nanopore Detectors:

    • ssDNA passes through a pore (1 nm).

    • Variation in electrical signal is recorded; each base produces a distinct signal.

    • Error-prone with 1X coverage; suitable for quick analyses or re-sequencing.

    • Single Molecule Real Time (SMRT) Sequencing:

    • Developed by Pacific Biosciences.

    • Features:

      • Nanocontainers that hold a single piece of template DNA (diameter of 20 nm).

      • Zero-mode waveguides (ZMW).

      • Fluorescent dNTPs tagged with pyrophosphate.

      • Light flash as pyrophosphate is released.

      • Typical read length approximately 20,000 bases.

      • Effective for repetitive sequences.

  • Visual aid: Figure 8.26 from Clark et al. (2019).

Learning Objectives

  • Post-lecture, students will be able to:

    • Define key terms: adapters, NGS, tagmentation, barcode sequence, consensus sequence, contig, read depth, emulsion PCR, third generation sequencing.

    • Describe two methods for fragmenting genomic DNA for NGS library preparation.

    • Discuss two methods of creating a targeted sequencing library for NGS.

    • Explain the basic steps involved in Illumina, Ion Torrent, Nanopore, and SMRT sequencing methods.