Next Generation Sequencing and OLC

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Questions and Answers

Which concept is primarily assessed in the short answer or multiple-choice questions of the test?

Historical developments in genetics

Detailed mechanisms of DNA replication

General principles discussed in lectures (correct)

Specific case studies of disease treatment

What genetic factor is directly linked to drug responsiveness as mentioned in the content?

Chromosomal duplications

Gene rearrangements in tumors

Single Nucleotide Polymorphisms (SNPs) (correct)

Epigenetic modifications

What is the result of a G-to-A mutation in exon 4 of the CYP2D6 gene?

No protein produced due to splicing issues (correct)

Activation of opioid analgesics

Enhanced drug metabolism

Increased enzyme activity for opioid analgesics

Which of the following sequencing techniques is compared to second-generation sequencing?

Third-generation sequencing Signup and view all the answers

What type of graphs are used for sequence assembly as mentioned in the document?

de Bruijn graphs Signup and view all the answers

What type of mutation is indicated in the CYP2C9 gene regarding poor metabolizers of warfarin?

Arg-to-Cys mutation Signup and view all the answers

Which pharmacogenomic test is mentioned as the world's first microarray-based test cleared for clinical use?

AmpliChip CYP450 test Signup and view all the answers

What role do CYP2D6 and CYP2C19 genes play in the body?

Metabolism of a significant portion of prescription drugs Signup and view all the answers

Which of the following correctly describes the consequence of genetic polymorphisms in CYP?

Altered drug clearance from circulation Signup and view all the answers

What effect does a C-to-T single nucleotide polymorphism (SNP) have in the CYP2C9 gene?

Causes a poor metabolizer status Signup and view all the answers

What is the function of a contig in DNA sequencing?

It is a consensus region made up of overlapping DNA segments. Signup and view all the answers

How do repetitive sequences impact DNA assembly?

They can create significant problems in accurately assembling reads. Signup and view all the answers

Which algorithm is primarily used in the assembly problem for overlapping reads?

Greedy algorithm Signup and view all the answers

What is represented by the edges in an overlap layout consensus graph?

The overlap between different sequences. Signup and view all the answers

What happens to fragments that cannot be integrated into a contig?

They are discarded. Signup and view all the answers

What is the main advantage of using an overlap layout consensus graph?

It allows for assembly generation even with repetitive sequence data. Signup and view all the answers

What issue arises when large repeated sequences are present in DNA assembly?

Assembly of the full sequence becomes impossible. Signup and view all the answers

Which of the following statements about the greedy algorithm in sequence assembly is true?

It may lead to suboptimal arrangements if not applied carefully. Signup and view all the answers

What is the primary purpose of amplification in DNA library preparation?

To ensure each library member is detected and quantified Signup and view all the answers

Which method is used in Sanger sequencing to amplify DNA?

Cloning in E.coli Signup and view all the answers

What is a key challenge in next-generation sequencing (NGS) regarding library members?

Isolation and separation of library members Signup and view all the answers

What kind of amplification does Ion semiconductor sequencing use?

Emulsion PCR on a bead Signup and view all the answers

In emulsion PCR, what is the required ratio of DNA strands to beads in each droplet?

At least 1 DNA strand and 1 bead Signup and view all the answers

What is the consequence of having unamplified beads during sequencing?

Reduces sequencing efficiency Signup and view all the answers

What is the role of glycerol gradient centrifugation in the amplification process?

To separate templated beads from DNA-less beads Signup and view all the answers

What is the typical proportion of beads that do not contain amplified DNA after the emPCR process?

60-80% Signup and view all the answers

What advantage does paired-end sequencing offer in contig construction?

It helps resolve ambiguities by reading both ends of the fragment. Signup and view all the answers

Which of the following statements about the Bruijn graph is true?

The master sequence can be easily deduced from it. Signup and view all the answers

Which limitation of 2nd generation sequencing is addressed by 3rd generation sequencing?

The introduction of bias during library generation. Signup and view all the answers

How does the number of passes affect the accuracy of PacBio sequencing?

Increasing passes leads to enhanced prediction of read quality. Signup and view all the answers

Which of the following is NOT a characteristic of 2nd generation sequencing?

It often requires multiple passes for accuracy. Signup and view all the answers

What impact do repeated k-mers have on the Bruijn graph?

They are removed to reduce computational costs. Signup and view all the answers

What is the predicted accuracy of a single pass in PacBio sequencing?

Approximately 90% Signup and view all the answers

In what way does paired-end sequencing differ from single read sequencing?

It reads both ends of the DNA fragment. Signup and view all the answers

What is one of the key benefits associated with multiple passes in PacBio sequencing?

It averages out errors to improve read accuracy. Signup and view all the answers

Study Notes

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

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Description

This quiz covers key concepts related to Next Generation Sequencing (NGS) and the challenges presented by repetitive sequences during the assembly process. Focus is given to the Overlap Layout Consensus Graph (OLC) and its role in creating contigs and scaffolds. Test your understanding of how these techniques work together to produce accurate sequence assemblies.