3) Next Generation Sequencing (NGS) - Coverage & Sample Quality Control

Summary

This video covers key considerations for preparing DNA samples for Next Generation Sequencing (NGS), a powerful tool for sequencing genomes, exomes, and methylated cytosines. It explains the importance of proper sample quality and quantity, using methods like qPCR and Qubit for quantification, and the Bioanalyzer for size distribution analysis. The video also highlights the concept of coverage in NGS, emphasizing the need for multiple reads to avoid errors in sequencing. Applied Biological Materials (abm) offers a range of NGS services to ensure reliable results.

Takeaways

• 😀 NGS (Next Generation Sequencing) is a powerful tool for analyzing DNA sequences and advancing our understanding of biological processes.
• 😀 NGS is flexible and can be applied to a wide range of applications, including whole genome sequencing, RNA sequencing, exome sequencing, and methylation analysis.
• 😀 Before starting an NGS experiment, it's important to consider key parameters to ensure the experiment is successful.
• 😀 Applied Biological Materials (abm) offers a variety of affordable NGS services, including sequencing, lane rentals, and expert consultation.
• 😀 NGS platforms are highly accurate but still prone to errors, and sequencing multiple times (coverage) helps mitigate this.
• 😀 Coverage (or depth) refers to the number of times a nucleotide is sequenced, which helps ensure accurate data by reducing errors.
• 😀 To calculate coverage, divide the total output from sequencing by the size of the sample. For example, a human genome typically requires 333X coverage on Illumina's HighSeq 2500 platform.
• 😀 Sample library validation is crucial to ensure a sufficient amount of high-quality DNA, with specific DNA quantity requirements based on the sequencing protocol.
• 😀 Low or excessive DNA quantity can lead to sequencing issues like flow cell saturation, read problems, or reduced coverage.
• 😀 A good sample library should contain a diverse set of DNA fragments with minimal duplication, as duplicates can bias the sequencing results.
• 😀 Library quantification can be performed using qPCR or fluorometric methods like Qubit. qPCR is more sensitive, while Qubit is faster but less accurate for certain sample types.
• 😀 The Bioanalyzer checks the size distribution and quality of DNA libraries, helping ensure that the right fragment sizes are present before sequencing.
• 😀 At abm, all sample libraries undergo quality control to ensure valid sequencing results, and customers can consult experts for advice on coverage and library preparation.

Q & A

What is Next Generation Sequencing (NGS) and why is it important?

Next Generation Sequencing (NGS) is a technology used to determine the DNA sequence of organisms. It is important because it enables researchers to gain detailed insights into biological processes, genetic variations, and diseases by sequencing the genome, exome, and other components like methylated cytosines.

How does NGS compare to other sequencing technologies?

NGS is highly flexible and accurate compared to traditional sequencing methods. It can handle a wide range of applications, such as whole genome sequencing, RNA sequencing, and exome sequencing, and provides higher throughput and more detailed data.

What is meant by 'coverage' in the context of NGS sequencing?

'Coverage' refers to how many times each nucleotide in the sample is sequenced. It is important because higher coverage increases the reliability of the sequencing results by reducing errors, ensuring that each base is accurately represented.

Why is sequencing multiple times important in NGS?

Sequencing multiple times helps to correct errors that may occur in individual reads. Even with a 99% accuracy rate, errors can still accumulate, and repeating the sequencing process increases confidence in the accuracy of the final results.

What is the role of qPCR in NGS sample validation?

qPCR is used to quantify sample libraries before sequencing. It selectively amplifies adapter-ligated fragments, providing sensitive measurements of DNA quantity. This is particularly useful when there is insufficient DNA or no prior PCR amplification.

What is the advantage of using Qubit for sample quantification?

Qubit is faster than qPCR for quantifying DNA and is useful when PCR enrichment is present. However, it is less sensitive than qPCR and requires more sample to obtain accurate results.

How does the Bioanalyzer ensure sample quality?

The Bioanalyzer is used to check the size distribution of DNA fragments in the sample library before sequencing. It helps confirm that the library preparation is accurate and that the fragment sizes are suitable for sequencing.

What happens if there is too much or too little DNA in a sample library?

If there is too much DNA, it can lead to flow cell saturation and reduced sequencing efficiency. Conversely, insufficient DNA results in reduced coverage and poor-quality data, as there may not be enough material for the sequencing reaction to work properly.

What are the consequences of having duplicate DNA fragments in a sample library?

Duplicate fragments can lead to biased sequencing results. During PCR amplification, duplicates are generated, which can overrepresent certain fragments in the final sequencing data and skew the results by limiting the diversity of fragments sequenced.

Why is consulting NGS experts important before starting an experiment?

Consulting NGS experts is important because they can guide you on the optimal coverage and sequencing methods for your specific experiment. This ensures that you get high-quality data and avoid common pitfalls like inadequate DNA quantity or incorrect fragment sizes.