Next Generation Sequencing and OLC

Repetitive Sequence Gaps

• Repetitive sequences can cause issues with assembly, specifically where overlapping read assembly is used.
• The assembly problem is conceptually simple but relies on greedy algorithms where sequences with the most overlaps get combined first.
• Overlap Layout Consensus Graph (OLC) helps overcome repetitive sequences issues.
• OLC creates a directed graph where nodes are specific sequences and edges represent overlaps between them.

Assembly of Reads

• A contig represents a consensus region of DNA constructed from overlapping DNA segments.
• A scaffold is a sequence of contigs, separated by gaps of known length.

Overlap Layout Consensus Graph

• OLC enables assemblies to be generated even with some repetitive sequence data.
• OLC can lead to rearrangements in the sequence if not carefully scrutinized.

Next Generation Sequencing (NGS) - Amplification Issues

• NGS relies on amplification steps to obtain enough of each library member for detection.
• Library members must be individually isolated and amplified without cross-contamination, unlike Sanger sequencing which uses cloning to amplify.

Ion Semiconductor Sequencing

• Relies on emulsion PCR based amplification step on a bead.
• Beads are tethered to emPCR primers.

Sequencing Library Amplification

• The DNA library fragments are clonally attached and amplified on beads using emulsion PCR.
• Beads with DNA are sequenced.
• The emulsion is broken to release beads, and then the beads with DNA are purified.
• The DNA containing beads are sequenced.

emPCR

• emPCR is used for DNA amplification in ion semiconductor sequencing.
• Each droplet must have 1 DNA strand and 1 bead.
• Roughly 1/3 of the droplets will meet this requirement.

Enrichment of Beads with Amplified DNA

• Most beads will not have amplified DNA (60-80%).
• Non-templated beads must be removed before sequencing.

De Bruijn Graph

• De Bruijn graph is a simplified approach to sequencing assembly.
• Used in 3rd generation sequencing because of longer reads.
• Solves assembly problem by visiting each edge only once, creating a simple linear graph.

Paired‑End Sequencing for Contig Assembly

• Single read sequencing reads only one end of a DNA sequence.
• Paired-end sequencing reads both ends of a sequence.
• Paired-end reads can help resolve ambiguities in contig construction.

Limitations of 2nd Generation Sequencing

• PCR introduces bias in library generation and amplification steps.
• Short reads are produced (99% less than 1000bp) and are prone to errors.

PacBio Workflow

• PacBio sequencing provides a circular library, enabling multiple passes to improve accuracy (-5 log10(error) quality scale).
• The accuracy improves with each pass.
• 1 pass = ~90% accuracy, 2 passes = ~96% accuracy, and 3 passes = ~99% accuracy.

Examples of Drug Responsiveness Linked to Genetic Variation

• Cytochrome P450 metabolizing enzymes activate or inactivate drugs.
• Known SNPs affect these enzyme activities.
• CYP2D6 gene has a G-to-A mutation in exon 4 that affects splicing and prevents protein production. This leads to an inability to activate opioid analgesics.
• CYP2C9 gene has a C-to-T SNP causing R144C, resulting in "poor metabolizers" of warfarin.

AmpliChip CYP450 Test (Roche)

• The first microarray-based pharmacogenomic test for clinical use, powered by Affymetrix technology.
• Comprehensive detection of gene variations for CYP2D6 and CYP2C19, genes that metabolize roughly 25% of all prescription drugs.

Critical Notions for Revision - Test 2

• SNPs & Pharmacogenomics
• Sanger Sequencing
• Gaps & Assembly
• Comparison of 2nd and 3rd Generation Sequencing Techniques
• OLC and de Bruijn Graphs for Sequencing Assembly
• Gene Annotation
• Protein and Gene Sequence Similarity Search