Third-Generation Sequencing: Pacific Biosciences' SMRT
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Questions and Answers

What is a characteristic of Single-Molecular Real-Time Sequencing (SMRT) that allows for accurate sequencing?

  • Use of Sanger sequencing technology
  • Parallelized sequencing and fluorescence detection (correct)
  • Amplification of DNA molecules before sequencing
  • Sequencing of short DNA molecules

    • Which of the following is a limitation of Nanopore Sequencing?

  • Difficulty in extracting and delivering high-molecular weight DNA (correct)
  • High cost of the Oxford Nanopore MinION device
  • Short read lengths
  • Need for complex libraries

    • What is an application of Single-Molecular Real-Time Sequencing (SMRT) in medicine?

  • Studying the microbiome of the human gut
  • Detecting gene mutations in cancer cells
  • Sequencing of entire human genomes
  • Detecting drug resistance in Chronic myeloid leukemia (correct)

    • What was 454 sequencing used for, despite its high error rates and high cost?

    <p>Sequencing microbial genomes and investigating the decline of honey bee populations</p> Signup and view all the answers

    What is an advantage of Nanopore Sequencing over other sequencing technologies?

    <p>Lower cost compared to other sequencing technologies</p> Signup and view all the answers

    What is a limitation of Polony sequencing?

    <p>It generates a lot of raw data</p> Signup and view all the answers

    What is a characteristic of Third-Generation Sequencing technologies?

    <p>Single molecule sequencing</p> Signup and view all the answers

    What is a characteristic of Illumina technology?

    <p>It is highly accurate and cost-effective</p> Signup and view all the answers

    What is an application of Nanopore Sequencing in agriculture?

    <p>Understanding gene structures in rice species</p> Signup and view all the answers

    What is NOT a use of 454 sequencing?

    <p>Sanger sequencing</p> Signup and view all the answers

    What is a strength of Polony sequencing?

    <p>It is cost-effective and accurate</p> Signup and view all the answers

    What is a limitation of Single-Molecular Real-Time Sequencing (SMRT)?

    <p>Polymerase longevity affecting read length</p> Signup and view all the answers

    Why is Illumina technology popular among scientists?

    <p>It is highly accurate and cost-effective</p> Signup and view all the answers

    What is a feature of Nanopore Sequencing that allows for direct detection of nucleotides?

    <p>Measuring electrical current changes as DNA molecules pass through a nanopore</p> Signup and view all the answers

    What is the primary advantage of High-throughput Sequencing (HTS) over traditional Sanger sequencing?

    <p>Significantly reduced cost and time required for sequencing</p> Signup and view all the answers

    Which technique is commonly used in Massively Parallel Sequencing (MPSS) to label genetic material?

    <p>Fluorescent linker</p> Signup and view all the answers

    What is another name for Massively Parallel Sequencing (MPSS)?

    <p>Next-generation sequencing</p> Signup and view all the answers

    What is a key application of Massively Parallel Sequencing (MPSS)?

    <p>Screening entire genomes for new mutations or pathogens that may cause human disease</p> Signup and view all the answers

    What is the main difference between 454 sequencing and traditional Sanger sequencing?

    <p>454 sequencing uses a special enzyme to cut genetic material, while Sanger sequencing uses PCR</p> Signup and view all the answers

    Which of the following is NOT an application of Massively Parallel Sequencing (MPSS)?

    <p>Studying the genetic code of extinct species</p> Signup and view all the answers

    What is the primary goal of High-throughput Sequencing (HTS) in terms of disease diagnosis?

    <p>Diagnosing diseases more quickly and accurately</p> Signup and view all the answers

    Study Notes

    Third-Generation Sequencing

    • Allows for much longer reads (over 10,000 base pairs), single molecule sequencing, de novo assemblies of many microbial genomes, and reconstructions of animal and plant genomes

    Single-Molecular Real-Time Sequencing (Pacific Biosciences)

    • Sequences DNA in real-time, uses long DNA molecules, and combines parallelized single molecule sequencing and fluorescence detection
    • Applications: environment research, medicine, and agriculture
    • Advantages: high sequencing accuracy, long read lengths (up to 30 kb), and enables full gene isoforms and splice variants to be sequenced
    • Limitations: polymerase longevity affects read length

    Nanopore Sequencing (Oxford Nanopore Technologies)

    • Uses a device called Oxford Nanopore MinION, measures electrical current changes as DNA molecules pass through a nanopore, and directly detects nucleotides without DNA synthesis
    • Advantages: long read lengths (up to 2.3 Mb), lower cost (around $1000), and enables de novo genome assembly and determination of genome structural variations
    • Limitations: difficulty in extracting and delivering high-molecular weight DNA, higher error rate and lower accuracy compared to short-read sequencing, and requires correction using short-read sequencing data
    • Useful for: large complex genomes, identifying pathogens in clinical samples, and comparing microbial composition in soil samples

    High-Throughput Sequencing (HTS)

    • Based on pyrosequencing and PCR (polymerase chain reaction)
    • Revolutionized the field of genetics, making it possible to sequence entire genomes in weeks instead of years and reducing the cost of sequencing from billions of dollars to just $600
    • Transformed many fields, including science, medicine, and research

    Massively Parallel Sequencing (MPSS)

    • A powerful technology that allows for high-throughput genetic sequencing at a lower cost
    • Also known as next-generation sequencing and has revolutionized the field of genomics
    • Many applications, including: discovering new genes in plants, screening entire genomes for new mutations or pathogens that may cause human disease

    454 Sequencing

    • First next-generation sequencing (NGS) technology, developed in 2005
    • Important for sequencing microbial genomes, studying metagenomics, and investigating the decline of honey bee populations
    • Limitations: high error rates in certain regions, higher cost compared to other technologies, and discontinued in 2016

    Polony Sequencing

    • A genetic sequencing technique developed in the 1990s and 2000s, cost-effective and accurate
    • Uses a special film to amplify genetic material, making it possible to: detect rare genetic messages, identify changes in gene expression, and sequence genomes of bacteria
    • While it generates a lot of raw data, it's a low-cost and highly accurate technique

    Illumina Technology

    • A powerful tool for genetic sequencing, dominating the field since 2007
    • Highly accurate and cost-effective, making it a popular choice for scientists

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    Description

    This quiz covers the development of third-generation sequencing, specifically Pacific Biosciences' Single-Molecule Real-Time Sequencing technology, allowing for longer reads and de novo assemblies of genomes.

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