Terms/Communication in the field Flashcards
(30 cards)
Explain “Plexing” in NGS
In Next-Generation Sequencing (NGS), plexing refers to the practice of pooling multiple samples together in a single sequencing run. This allows for the simultaneous sequencing of many different samples, which can increase throughput and reduce costs.
Can you explain in more detail ?
The term “plex” typically indicates how many samples are being pooled:
2-plex: Two samples pooled together
10-plex: Ten samples pooled together
100-plex: One hundred samples pooled together
More info:
Each sample is usually tagged with a unique barcode or index sequence, allowing them to be identified and separated during data analysis. The more samples that can be multiplexed, the more efficient and cost-effective the sequencing process becomes. However, as the number of samples increases, the amount of sequencing data per sample may decrease, which can impact the sensitivity and accuracy of detecting low-abundance variants.
How does multiplexing work in NGS?
Barcode or Indexing: Each sample in a multiplexed sequencing run is assigned a unique short DNA sequence known as a barcode or index. These barcodes are attached to the samples during the preparation stage, often through PCR amplification.
Pooling Samples: Once all samples are barcoded, they are pooled into a single sequencing reaction. Despite being combined into one pool, each sample maintains its unique barcode, allowing the sequencing platform to distinguish between them during the sequencing process.
Sequencing: The sequencing machine reads the pooled samples in parallel. As it generates sequence data, it also reads the barcode that is associated with each sequence. This means that the platform can assign each sequence read to the correct sample based on its barcode.
Data Analysis: After sequencing, bioinformatics tools are used to sort the reads based on the barcodes. This allows for the reconstruction of each individual sample’s sequence data, even though they were pooled together. The data for each sample is then analyzed separately, as though they were sequenced individually.
what are the benefits?
Benefits of Multiplexing in NGS
Cost Efficiency: Multiplexing enables the sequencing of many samples in one run, reducing the cost per sample. This is particularly important when dealing with large numbers of samples, like in population studies or large-scale clinical testing.
Higher Throughput: By pooling samples together, sequencing platforms can achieve higher throughput, meaning more data can be generated in less time.
Minimized Reagent Consumption: Sequencing reagents and other materials are often a significant part of the cost. Multiplexing helps minimize the amount of reagents needed per sample.
Example of this in NGS
Let’s say a researcher is conducting a genomic study to analyze the mutations in a specific gene across 100 patient samples. If they sequenced each sample individually, the cost could be prohibitively expensive, as each sequencing run requires a certain amount of reagents and sequencing time.
To optimize resources, they decide to use 10-plexing, meaning they will pool 10 samples together in one sequencing run. Here’s how the process works:
Barcode Addition: Each of the 100 patient samples is tagged with a unique barcode. This could be done using a method like PCR, where short sequences (e.g., 8-12 bases long) are added to the ends of the DNA fragments from each sample.
Pooling: The 100 samples, each with its unique barcode, are pooled together in one sequencing library.
Sequencing: The pooled library is then run through the NGS machine. The sequencing platform reads the DNA sequences of all the samples at once and also detects the associated barcode with each read.
Data Sorting and Analysis: After sequencing, bioinformatic software looks at the barcodes attached to each read and sorts the data back into the correct samples. So, although all the samples were sequenced together, the software “untangles” the data and assigns each read to the correct patient sample.
Results: Finally, the mutations in the gene of interest are identified for each of the 100 patients, and the results are reported.
Probes:
In NGS, probes are short DNA or RNA sequences that are designed to bind specifically to a target region of the genome. They are typically used to capture or enrich the sequences of interest from a larger, complex sample, allowing for focused sequencing of certain regions (e.g., genes, exons, or specific genomic loci).
Different Probes:
Capture Probes (Hybridization Probes):
PCR Probes:
Sequencing Probes:
Reactions in NGS
In NGS, a reaction refers to a specific chemical process or series of steps that facilitate the generation of sequence data from the biological sample. Several types of reactions take place throughout the sequencing process. Here’s an overview of the key ones:
Different reactions
- Library Preparation Reactions
- Amplification Reactions (PCR)
- Sequencing Reaction (SBS)
- Cluster Generation Reaction (Illumina)
How probes and reactions work together in NGS
Let’s look at an example where NGS is used for targeted sequencing of a cancer panel to identify mutations in a group of genes associated with cancer:
Probe Design: Specific capture probes (e.g., oligonucleotides complementary to certain regions of the cancer-related genes) are designed to bind to those genes.
Library Preparation:
DNA from patient samples is fragmented.
The fragmented DNA is ligated to adapters.
The DNA with adapters is hybridized with the capture probes that target the cancer genes.
Target Enrichment: Using the capture probes, the regions of interest (cancer genes) are selectively pulled out and enriched from the rest of the genome. This is done through hybridization and capture on beads, or through solid-phase methods.
PCR Amplification: The enriched target regions are amplified using PCR to make enough copies for sequencing.
Sequencing Reaction: The prepared library is loaded onto the sequencer, and the sequencing reaction begins. Bases are incorporated one by one as complementary strands are synthesized, and the sequencing machine detects the base at each position.
Data Analysis: The sequenced reads are aligned, and the mutations in the cancer genes are identified by comparing the sequence to a reference genome.
Conclusion
Probes are short DNA or RNA sequences designed to specifically bind to regions of interest, such as genes or exons, in NGS. They play a crucial role in enriching the target DNA and making the sequencing process more focused and efficient.
Reactions are the chemical processes involved in preparing, amplifying, and sequencing DNA. These reactions include library preparation, amplification (PCR), and sequencing-by-synthesis.
Together, probes and reactions ensure that NGS is able to sequence the correct regions of DNA accurately and efficiently.
What are “arrays” in NGS?
arrays are specialized platforms used to analyze large amounts of genetic data. These arrays allow researchers to examine specific parts of the genome, typically focusing on predetermined sites of interest, like gene expression, DNA methylation, or single nucleotide polymorphisms (SNPs). Arrays are useful for high-throughput analysis, providing a cost-effective and relatively simple way to process genomic information compared to full NGS sequencing.
Types of arrays in NGS?
Human Arrays (EPIC and 450K)
EPIC Array (Illumina Human MethylationEPIC BeadChip): The EPIC array is a DNA methylation microarray that contains over 850,000 probes. It is used for measuring DNA methylation levels across the human genome at specific CpG sites. Methylation is an epigenetic modification that can affect gene expression and has implications in various diseases, including cancer. The EPIC array is an updated version of the previous 450K array with more coverage of the genome, especially focusing on regulatory regions, promoters, and enhancers.
450K Array (Illumina Human Methylation 450K BeadChip): This array is similar to the EPIC array but covers fewer probes (about 485,000). It focuses on methylation analysis at CpG sites and is often used for studies involving epigenetic regulation and disease-related modifications in DNA.
Methylation Arrays Methylation arrays like the EPIC and 450K are a specific type of array designed to capture and quantify DNA methylation. Methylation refers to the addition of a methyl group to the DNA, often at CpG sites, which can influence gene expression without altering the underlying DNA sequence. Methylation arrays are used to study epigenetic modifications across large numbers of samples to investigate changes in gene regulation, disease mechanisms, and environmental impacts on gene expression.
How do arrays work?
Probes: The arrays are covered with thousands of tiny probes that can specifically bind to DNA regions of interest (like CpG sites). These probes are designed to capture particular sequences or modifications (e.g., methylation) at specific sites.
Fluorescent Tags: After DNA is isolated from samples and hybridized to the probes on the array, a fluorescent marker is often used to measure the level of binding, which is related to the presence or absence of the modification (like methylation).
Data Generation: The signal intensity from the fluorescent tags is then captured by scanning the array, and the data is analyzed to determine the degree of methylation or other genomic alterations.
Why are array’s used?
High Throughput: Arrays allow for the simultaneous analysis of thousands or even millions of genetic sites, making them much more efficient than traditional methods of sequencing or individual gene analysis.
Cost-effective: Arrays are generally less expensive than full genome sequencing techniques, making them ideal for large-scale studies where researchers are focused on specific genes, regions, or modifications.
Focus on Specific Questions: Arrays are designed to target specific genetic questions. For example, methylation arrays focus on understanding epigenetic changes in the genome, which can be critical for studying complex diseases like cancer.
What are CpG’s?
A CpG site is a location in the genome where a cytosine is followed directly by a guanine in the 5’ to 3’ direction. This sequence can be found throughout the genome, though it’s more common in some regions than others, such as in CpG islands (regions rich in CpG sites, often located near gene promoters).
Ex: Why Are CpGs Important in NGS?
Epigenetic Regulation: CpG methylation plays a key role in regulating gene expression and is central to many biological processes. Understanding methylation at CpG sites can provide insights into disease mechanisms and potential therapeutic targets.
Marker for Disease: Changes in CpG methylation patterns are often associated with diseases such as cancer. For instance, hypermethylation of CpG sites in tumor suppressor genes can lead to gene silencing and contribute to cancer development.
Population Studies: Since DNA methylation is influenced by both genetic and environmental factors, studying CpG methylation at specific loci can reveal information about how environmental exposures, aging, and genetic variation impact human health.
What is Twist Bioscience?
Answer: Twist Bioscience is a biotechnology company that develops and manufactures synthetic DNA using its proprietary silicon-based platform. The company specializes in providing high-quality DNA products, including custom genes, NGS target enrichment panels, and synthetic biology solutions for researchers and industries. Twist’s products are used for a variety of applications, including genomics, drug discovery, diagnostics, and synthetic biology.
What are Twist NGS Target Enrichment Panels?
Answer: Twist NGS Target Enrichment Panels are ready-to-use, customizable panels designed for targeted sequencing of specific regions of interest in the genome. These panels allow researchers to focus on genes or genomic regions relevant to their research, enabling high-throughput sequencing with improved cost efficiency and coverage. Twist offers a range of panels, including oncology panels, infectious disease panels, and custom panels tailored to specific research needs.
How does Twist Target Enrichment work?
Answer: Twist’s NGS Target Enrichment technology works by using probe-based hybridization to selectively capture and enrich the target regions from a whole genome sample. In this method, biotinylated probes complementary to the targeted genomic regions are used to pull down the desired DNA fragments. These enriched fragments are then sequenced using NGS technologies. This approach allows for more focused sequencing, which reduces the amount of sequencing data required and improves depth of coverage for the regions of interest.
What is Twist’s Universal Library Prep?
Answer: Twist’s Universal Library Prep is a high-efficiency, cost-effective method for preparing DNA samples for NGS. It is designed to streamline the sequencing process by reducing time and cost while maintaining high performance. The protocol is compatible with a wide range of input DNA types and works with both low-quantity and degraded samples. This library prep method simplifies NGS workflows and is suitable for various applications, including targeted sequencing, whole-genome sequencing, and exome sequencing.
What is Twist’s Synthetic DNA?
Answer: Twist Bioscience produces synthetic DNA using its proprietary silicon-based platform, which allows for the mass production of high-quality, precise, and scalable synthetic DNA. This synthetic DNA is used in a variety of applications, such as gene synthesis, cloning, protein production, synthetic biology, and diagnostic assay development. Twist’s synthetic DNA is known for its high accuracy and low error rates, making it suitable for a range of high-performance genomic applications.
. What is the Twist DNA Synthesis Platform?
Answer: The Twist DNA Synthesis Platform is an innovative technology that allows for high-throughput, precise, and cost-effective synthesis of DNA. Unlike traditional methods, which rely on liquid-phase oligonucleotide synthesis, Twist’s platform uses a silicon-based approach that dramatically reduces errors and increases production speed. This enables the production of complex DNA sequences at scale, which can be used for applications such as gene synthesis, DNA libraries, and targeted sequencing panels.
How does Twist ensure high coverage and sensitivity in its NGS panels?
Answer: Twist ensures high coverage and sensitivity in its NGS panels through its proprietary probe design and target enrichment methods. By designing highly specific probes and optimizing hybridization conditions, Twist maximizes the capture efficiency and minimizes off-target binding. Additionally, the uniformity of the probes ensures that every targeted region is enriched equally, allowing for deep and consistent sequencing coverage, even for challenging or low-abundance targets. This approach improves the sensitivity of detecting rare variants or low-frequency mutations.
What is Twist’s approach to Custom Panels?
Answer: Twist provides custom NGS panels that can be tailored to the specific needs of a research project. Researchers can design custom panels to focus on specific genes, pathways, or regions of the genome that are relevant to their studies. Twist’s custom panels use the same high-quality technology as their pre-designed panels, ensuring high performance in terms of target enrichment, coverage, and sensitivity. Twist’s team works with researchers to help design panels that fit their exact specifications and ensure reliable results.
