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Questions and Answers
What role do adapter sequences play in the preparation of sequencing libraries?
What role do adapter sequences play in the preparation of sequencing libraries?
What is the significance of amplification during the sequencing process?
What is the significance of amplification during the sequencing process?
Which of the following describes a characteristic of Sequencing by Synthesis (SBS)?
Which of the following describes a characteristic of Sequencing by Synthesis (SBS)?
What potential issue does Sequencing by Synthesis (SBS) face due to its methodology?
What potential issue does Sequencing by Synthesis (SBS) face due to its methodology?
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Which of the following is true regarding the output from a sequencing machine after a sequencing run?
Which of the following is true regarding the output from a sequencing machine after a sequencing run?
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In the context of NGS, what is the purpose of covalently attached DNA linkers during amplification?
In the context of NGS, what is the purpose of covalently attached DNA linkers during amplification?
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Why is short read sequencing often preferred for high-throughput sequencing applications?
Why is short read sequencing often preferred for high-throughput sequencing applications?
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What is one of the modifications made to nucleotides used in Sequencing by Synthesis?
What is one of the modifications made to nucleotides used in Sequencing by Synthesis?
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What is the purpose of reversible termination in nucleotide incorporation during sequencing?
What is the purpose of reversible termination in nucleotide incorporation during sequencing?
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Which of the following steps is not part of the library preparation in Illumina Sequencing?
Which of the following steps is not part of the library preparation in Illumina Sequencing?
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What is the primary role of bridge PCR in Illumina Sequencing?
What is the primary role of bridge PCR in Illumina Sequencing?
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During the sequencing process, what happens to the fluorescent molecule after the nucleotide is incorporated?
During the sequencing process, what happens to the fluorescent molecule after the nucleotide is incorporated?
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What characteristic of Illumina sequencing allows for the amplification of DNA fragments to remain near their point of origin?
What characteristic of Illumina sequencing allows for the amplification of DNA fragments to remain near their point of origin?
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What is the typical size range of DNA fragments after they have undergone fragmentation in library preparation?
What is the typical size range of DNA fragments after they have undergone fragmentation in library preparation?
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Which sequencing platform is associated with the technology known as 'pyrosequencing'?
Which sequencing platform is associated with the technology known as 'pyrosequencing'?
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What occurs to DNA fragments during the bridge amplification stage of Illumina Sequencing?
What occurs to DNA fragments during the bridge amplification stage of Illumina Sequencing?
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What is a significant role of the labeled dNTPs in the sequencing reaction of Illumina sequencing?
What is a significant role of the labeled dNTPs in the sequencing reaction of Illumina sequencing?
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Why are reverse strands removed after the denaturation of double-stranded clonal bridges?
Why are reverse strands removed after the denaturation of double-stranded clonal bridges?
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In bridge amplification during Illumina sequencing, what is the main purpose of cyclic reversible termination?
In bridge amplification during Illumina sequencing, what is the main purpose of cyclic reversible termination?
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What are the main advantages of using NGS over Sanger sequencing?
What are the main advantages of using NGS over Sanger sequencing?
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What is the role of primers in the sequencing reaction of Illumina sequencing?
What is the role of primers in the sequencing reaction of Illumina sequencing?
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What was the primary method used in the Human Genome Project?
What was the primary method used in the Human Genome Project?
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What does the term 'massively parallel sequencing technology' refer to in the context of NGS?
What does the term 'massively parallel sequencing technology' refer to in the context of NGS?
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Which of the following is an application of NGS?
Which of the following is an application of NGS?
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Which factor is NOT a benefit associated with NGS?
Which factor is NOT a benefit associated with NGS?
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What does the category 'sequencing by hybridization' primarily use?
What does the category 'sequencing by hybridization' primarily use?
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What does NGS allow researchers to analyze besides genomic sequences?
What does NGS allow researchers to analyze besides genomic sequences?
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Which of the following is a defining characteristic of Next Generation Sequencing over first-generation techniques?
Which of the following is a defining characteristic of Next Generation Sequencing over first-generation techniques?
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What type of sequencing technology has been further developed from Sanger sequencing?
What type of sequencing technology has been further developed from Sanger sequencing?
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Study Notes
Human Genome Project
- First major sequencing project
- Used Sanger Sequencing
- Completed in 2003
- Took 13 years to complete
- Cost 3 Billion dollars
Next Generation Sequencing
- Introduced in 2004 and 2006
- Offers high-throughput sequencing
- Massively parallel sequencing technology
- Allows sequencing of entire genomes or regions of interest
- Reduced cost per megabase
- Increased speed and accuracy
- Allows for a wide variety of applications
Next Generation Sequencing Applications
- Rapidly sequence whole genomes
- Deeply sequence target regions
- Utilize RNA sequencing (RNA-seq) to discover novel RNA variants and splice sites
- Quantify mRNAs for gene expression analysis
- Analyze epigenetic factors such as genome-wide DNA methylation and DNA-protein interactions
- Sequence cancer samples to study rare somatic variants and tumour subclones
- Identify novel pathogens
Categories of NGS
- Two categories
- Sequencing by hybridization
- Uses probes to interrogate sequences
- Used in diagnostic applications for identifying:
- Disease-related SNPs
- Gross chromosome abnormalities – rearrangements, deletions, duplications, copy number variants (CNVs)
- Sequencing by synthesis (SBS)
- A development of Sanger sequencing (but without ddNTPs)
- Uses repeated synthesis cycles and methods to incorporate nucleotides into a growing chain
- Sequencing by hybridization
Similarities Between Different NGS Technologies
-
Sample preparation
- Amplification or ligation with custom adapter sequences
- Adapter sequences allow library hybridization to the sequencing chips
- Universal priming site for sequencing primers
-
Sequencing machines
- Library fragments are amplified on a solid surface – beads or flat silicon
- Amplification creates clusters of DNA, each originating from a single library fragment
- Sequence from each cluster is optically read - by light or fluorescence
-
Data output
- Each machine provides raw data at the end of the sequencing run
- This data is analyzed to provide meaningful results
Sequencing by Synthesis Properties
- Relies on shorter reads (300-500bp)
- Intrinsically higher error rate relative to Sanger sequencing
- Requires high sequence coverage – "massively parallel sequencing"
- Utilizes step-by-step incorporation of reversibly fluorescent and terminated nucleotides
Illumina Sequencing - Library Preparation
- DNA is broken into manageable fragments (~200-600bp)
- Adapters are attached to DNA fragments
- DNA fragments attached to adapters are denatured
- Libraries are constructed to give a mixture of adapter-flanked fragments
Illumina Sequencing - Bridge Amplification
- Utilizes bridge PCR to amplify the genomic region being sequenced
- Primers densely coat the surface of a solid substrate
- Amplification products from the template library remain locally attached near the point of origin
- Each clonal cluster contains ~1000 copies of a single member of the template library
- DNA fragments are made single-stranded
- DNA fragments are washed across the flow cell
- Complementary DNA binds to primers on the surface of the flow cell
- DNA that doesn’t attach is washed away
Illumina Sequencing – Bridge Amplification - Clonal Clustering
- Double-stranded clonal bridges are denatured
- Reverse strands are removed
- Forward strands remain as clusters for sequencing
Illumina Sequencing – Sequencing Reaction
- Cyclic reversible termination
- One of the four fluorescent dNTPs is added per cycle
- Images of clusters are captured after incorporation of each nucleotide
- After imaging, the dye and terminator are cleaved and released
When To Use NGS vs Sanger Sequencing
- Sanger Sequencing
- Suitable for small regions of DNA or limited number of samples
- NGS
- Cost-effective for screening multiple samples
- Allows detection of multiple variants across targeted areas of the genome
- More cost-effective and time-saving when Sanger sequencing would be too costly or time-consuming
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Description
Explore the key milestones of the Human Genome Project and the advancements brought by Next Generation Sequencing (NGS). Discover the technologies, applications, and categories of NGS that have revolutionized genomic research since the early 2000s. This quiz will challenge your understanding of these pivotal developments in genetics.
