Next Generation Sequencing (NGS)

Module 1: Next Generation Sequencing (NGS)

• Genomics are covered in Bioinformatics Spring 2025.

Overview

• Covers DNA and its composition.
• Discusses what sequencing really is.
• Touches on Sanger sequencing.
• Focuses on Next-generation sequencing: Illumina seq and Ion sequencing.
• Talks about Next-generation sequencing: Experimental design.
• Discusses Paired-end reads
• Touches on the appearance of data.
• Covers Third Generation Sequencing.
• Discusses genetic variation

In Class Exercise: Sequencing so far: Concept Map

• Concept maps visually represent information.
• Focus should be on the interrelatedness of objects with each other.
• Should focus on effortful information retrieval, and supplement the map during study.

Solid-Phase Amplification

• Genomic DNA is prepared by fragmenting and ligating adapters to both ends.
• Single-stranded fragments bind randomly to the flow cell channel's inner surface.
• Enzymes incorporate nucleotides to build double-stranded bridges on the solid-phase substrate.
• Denaturation leaves single-stranded templates anchored to the substrate.
• Unlabeled nucleotides and enzymes initiate solid-phase bridge amplification.
• After the P5 region is cleaved, clusters containing only fragments attached by the P7 region remain, ensuring sequencing in the same direction.
• Millions of double-stranded DNA are generated in each flow cell channel, creating unique "clusters."
• Clusters are all the same DNA, clonally amplified via bridge amplification.

Illumina Sequencing

• Labeled nucleotides are sent over the flow cell for sequencing.
• After the sequence index, Paired-end sequencing is performed.
• The bridge amplification process repeats
• P7 is cut instead of P5.
• Resulting clusters contain fragments attached by the P5 region that creates the opposite Second Read.

The Cost and Output of Illumina Machines

• iSeq output per is 1.2 Gb with a instrument price of $19.9K.
• MiniSeq output per run is 7.5 Gb with a instrument price of $49.5K.
• MiSeq output per run is 15 Gb with a instrument price of $99K.
• NextSeq output per run is 120 Gb with a instrument price of $275K.
• HiSeq output per run is 1.5 Tb with a instrument price of $900K.
• HiSeq X output per run is 1.8 Tb with a instrument price of $6M/$10M.
• NovaSeq output per run is 1 Tb - 6 Tb with a instrument price of $985K.

Remember the H+

• The incorporation of a deoxyribonucleotide triphosphate (dNTP) into an extending DNA strand involves DNA Polymerase.
• This process involves the formation of a covalent bond and releases pyrophosphate and a positively charged hydrogen ion.

Thermo Fisher Ion Sequencing

• Genomic DNA fragments are clonally amplified onto acrylamide beads.
• DNA polymerase and primers are added to each bead-bound template strand, and the beads are delivered to individual wells on sequencing chips (emPCR).
• Each well has a sensor sensitive to pH shifts, converting the release of protons into voltage.
• During sequencing, each of the four nucleotides is added in turn.
• When a single proton is released, it converts to an electrical signal.
• The semiconductor gathers and processes information from each well as nucleotides are added.
• Signals are then processed into base calls.
• Multiple bases can be added to the growing complimentary strand.

High-Level Sequencing Jargon

• NGS is a type of assay to measure many analytes at once.
• The type of NGS assay used depends on the desired information.
• Full genomes or "targeted" regions can be sequenced.
• "De novo" sequencing doesn't require prior knowledge of the target organism and sequences everything potentially.
• Whole-Genome Sequencing (WGS) is an example of de novo sequencing.
• "Targeted" sequencing selects for the localized regions of a genome.
• Whole-Exome Sequencing (WES), Targeted regions (amplicon), and Whole-Transcriptome Sequencing (RNA-Seq) are examples of targeted sequencing.
• Different assays need different library preparations to select for the desired pool of templates.
• Templates are fragmented DNA or PCR amplicons selected for size.

Different Types of NGS Assays

• Whole-Genome sequencing includes all genetic information of an organism.
• Whole-Exome sequencing includes the complete exon with no introns.
• Target Regions sequencing researches key genomic regions.
• Whole-Transcriptome sequencing includes the total RNA molecules can differ in the same individual.

Targeted Regions (Again, it's a type of Assay)

• Targeted assays are far less expensive, enabling use on many individuals.
• Targeted assays can be used for screen, like screening for the cause (OC).
• Allows researchers and clinicians to focus on regions of the genome under investigation.
• Much deeper sequencing allows identifies rare variants missed by CE tools.
• The 395-gene and 120-gene panel are examples of targeted assays.
• Primers are designed for PCR for specific genes, such as KRAS, EGFR, and BRAF.

How does NGS work in Practice?

• Process involves library preparation, sequencing, and the mapping of reads to the genome.
• After reads have been mapped to the genome, the variants discovery and annotated.

Different Types of Experimental Design

• Experimental design types: whole genome and exome

Library Preparation

• The library preparation process typically involves extracting nucleic acids from samples like blood, tissue, or saliva.
• Double-stranded DNA is sheared into fragments through enzymatic or mechanical means.
• Adapter ligation is performed for cluster generation and clonal amplification.
• Protocols vary based on the experimental design (WGS, exome, etc.).

Sequencing the Library

• DNA libraries are deposited on a flow cell and amplified to form clusters, processed by a HTS machine.
• Each lane contains samples (indexed with a DNA barcode).
• A Read Group (RG) combines the sample and the flow cell.
• Next, sequencing occurs by synthesis with reversible terminators.
• Generates enormous piles of reads.

What Was Actually Sequenced (Illumina)?

• Its a piece of fragmented DNA, with two adapter sequences at the end.
• With on insert of 220bp, and performing 100bp PE sequencing, the inner distance is 20 bp.

How Are Reads Aligned?

• Single-end reads align for WGS/WES using the same reference for short fragment sequenced

Aligning the Reads to the Reference Genome

• The reads are easy to sort by chromosome and then position.
• Paired-end reads can help with difficult sorting decisions.

Why Use Paired-End Reads?

• Sequences aligned as read pairs enables accurate read alignment.
• Alignment algorithms can map repeats using algorithms with precise distances.

Once Reads Aligned: High-Level Sequencing Data Jargon

• Coverage (20x) indicates that each base is covered by 20 sequencing reads on average.
• Variant Allele Fraction (VAF) estimates the proportion of reads containing a variant allele.
• Quality scores asses base calling accuracy.

What WGS and WES Reads look like in practice?

• Comparing how Whole genome (WGS) and Exome aligned reads differs over base pairs (bp)

Different Exome Kits Produce Different Analyzable Territory

• Exome kits produce different analyzable territory, which leads to decent and bad coverage differences.

Depth of Coverage with NGS

• Can be Biased (e.g. GC bias or experimental)
• Hard to know what's the true reads mapped.

GC Bias

• Can make GC-rich region poorly covered and unusable Uneven Coverage in different WGS Examples
• Reaches an unevenly distribution coverage with piles of unmapped data due to different DNA and leads to changes.

This lesson covers genomics in Bioinformatics Spring 2025. It discusses DNA, sequencing, Sanger sequencing, Illumina seq, Ion sequencing, experimental design, paired-end reads, data appearance, third generation sequencing, and genetic variation.