Use of FFPE-derived DNA in next-generation sequencing: DNA extraction methods. (2023)

FAQs

How do you extract DNA from FFPE tissue? ›

To extract DNA from paraffin-embedded samples, tissue cores or microdissected tissue are subjected to xylene treatment, which dissolves the paraffin from the tissue, and then rehydrated using a series of ethanol washes.

Next-generation sequencing: DNA extraction protocol

Extraction entails breaking down the extracellular matrix and opening the cell membranes using enzymes, solvents, or surfactants. The DNA in the resulting mixture must then be isolated.

Formalin-Fixed Paraffin-Embedded (FFPE) tissue specimens have been a staple of research and therapeutic applications for decades. FFPE is a form of preservation and preparation for biopsy specimens that aids in examination, experimental research, and diagnostic/drug development.

Regardless of the device used, a tissue surface area of ≥ 1 mm² is adequate for samples to be tested with NGS. Keywords: Oncomine Dx Target Test; bronchoscopy; next-generation sequencing; non-small-cell lung cancer.

The critical difference between Sanger sequencing and NGS is sequencing volume. While the Sanger method only sequences a single DNA fragment at a time, NGS is massively parallel, sequencing millions of fragments simultaneously per run. This process translates into sequencing hundreds to thousands of genes at one time.

There are three main types of DNA sequencing done by NGS: Whole Genome Sequencing, Whole Exome, and Targeted Panels.

Formalin-Fixed Paraffin-Embedded (FFPE) scrolls can be used to extract DNA and can be used in whole-genome sequencing and other genomic applications for pipeline validation and assay development.

Formalin-fixedparaffin-embedded (FFPE) tissues are a very common source of archived tissue samples that have been used by pathologists for decades. It allows for a long-term, room temperature storage solution for tissue samples, making it very convenient.

What does FFPE mean in pathology? ›

Abstract. Paraffin embedding is a standard technique used in clinical and research laboratories to create a formalin-fixed, paraffin-embedded (FFPE) block of tissue. Formalin-fixed tissue undergoes tissue processing and then is embedded in paraffin (wax) to create a FFPE block or paraffin block.

Unlike DNA sequencing, this is called RNA sequencing. Specially designed mRNA sequencing is also often used to detect fusion genes. The most commonly used NGS assay for cancer patients is targeted panel sequencing which usually interrogates dozens or hundreds of targeted genes.

The cost of a human genome sequence decreased from an estimated $1 million in 2007, to $1000 in 2014, and today it is approximately $600.

Since the completion of the Human Genome Project, the cost of next-generation sequencing (NGS) has decreased at a dramatic rate, outpacing Moore's Law. Through continuous innovation, Illumina has helped reduce the cost of NGS, enabling the $1000 human genome.

Question: How should I store my FFPE tissue blocks? Answer: FFPE blocks are typically stored at room temperature but storage at 4°C and protected from light is optimal for 10x Assays as this will slow the degradation of RNA.

Tissue processing is the technique by which fixed tissues are made suitable for embedding within a supportive medium such as paraffin, and consists of three sequential steps: dehydration, clearing, and infiltration.

All tissue specimens should be fixed in 10% neutral buffered formalin for a minimum of 3 days (72 hours) and up to 2 weeks (14 days) for biopsy or autopsy tissue, or up to 4 weeks (28 days) for brain autopsy tissue.

The absorbance quotient (OD₂₆₀/OD₂₈₀) provides an estimate of DNA purity. An absorbance quotient value of 1.8 < ratio (R) < 2.0 was considered to be good, purified DNA. A ratio of <1.8 is indicative of protein contamination, where as a ratio of >2.0 indicates RNA contamination.

Manual methods as well as commercially available kits are used for DNA extraction. Various tissues including blood, body fluids, direct Fine needle aspiration cytology (FNAC) aspirate, formalin-fixed paraffin-embedded tissues, frozen tissue section, etc., can be used for DNA extraction.

DNA extraction is the process where DNA is separated from proteins, membranes, and other cellular material (Butler, 2012). According to Rice (2018), the method involves three necessary steps, namely, lysed, precipitation, and purification.

What are the disadvantages of NGS sequencing? ›

The limitations of NGS in clinical microbiology include unclear antibiotic resistance, inability to detect which pathogen is causing disease, costs, and unknown clinical utility.

Among NGS platforms, the accuracy of the original base data obtained by SOLiD platform is greater than 99.94%, though accuracy can reach 99.999% with the sequencing depth of 15×, which is the highest accuracy in NGS platforms (Ronchi et al., 2012).

With over 99% accuracy, the Sanger sequencing method remains the “gold standard” for basic and clinical research applications. In fact, most clinical laboratories rely on Sanger sequencing to validate gene variants (e.g., single-nucleotide variants and insertion/deletions) identified first through NGS.

Due to its sensitivity and relative simplicity in terms of both workflow and technique, Sanger sequencing remains the gold standard in sequencing technology today and is used in a variety of applications from targeted seqencing to confirming variants identified using orthogonal methods.

Illumina, Nanopore, and PacBio are among the most commonly used high-throughput sequencing platforms (Ye et al., 2015) . ...

Key Benefits of NGS

Using capillary electrophoresis-based Sanger sequencing, the Human Genome Project took over 10 years and cost nearly $3 billion. Next-generation sequencing, in contrast, makes large-scale whole-genome sequencing (WGS) accessible and practical for the average researcher.

There are 3 basic steps involved in DNA extraction, that is, lysis, precipitation and purification. In lysis, the nucleus and the cell are broken open, thus releasing DNA. This process involves mechanical disruption and uses enzymes and detergents like Proteinase K to dissolve the cellular proteins and free DNA.

The ability to extract DNA is of primary importance to studying the genetic causes of disease and for the development of diagnostics and drugs. It is also essential for carrying out forensic science, sequencing genomes, detecting bacteria and viruses in the environment and for determining paternity.

Wear personal protective equipment, such as a lab coat, gloves and eye protection. Do not get into eyes, on skin or clothing. Do not breathe in vapours or dust from dried-up solution. Do not ingest.

Therefore, formalin fixation is a major cause of reduced quality of DNA from FFPE tissues, and heat treatment after lysis can dramatically improve the quality.

How long can FFPE sections be stored? ›

Schlumpberger: Generally, we recommend storing FFPE sections or blocks cold at 4° or -20°. At -20°, blocks can be used after ten years. Eventually, there will be a decline in integrity.

Fixation is the foundation step behind the study of pathology and essentially exists to prevent the autolysis and degradation of the tissue and tissue components such that they can be observed both anatomically and microscopically following sectioning.

Based on the results from the assessment of nucleic acid extraction kits, nucleic acids were extracted using the QIAamp® DNA FFPE Tissue kit and RNeasy® FFPE kit. DNA was successfully extracted from all 84 biopsies with a mean yield of 3.7 ng/μl and a purity of 1.7.

A standard method to isolate DNA from tissues is by tissue digestion with proteinase K (PK), followed by heating to 100°C to inactivate the enzyme. After treatment with RNAse, genomic DNA can then be recovered by salt precipitation or with a resin column for downstream applications (Green and Sambrook, 2012‬).

For molecular analysis, the duration of fixation is the major factor affecting the preservation of DNA in formalin fixed tissue. Unbuffered formalin oxidises to formic acid and an acidic environment causes degradation of nucleic acids because the β glycosidic bonds in the purine bases are hydrolysed at pH 4.

The tissue processor finds applications in histopathology laboratories to automatically prepare tissue samples for laboratory testing, by fixing, dehydrating, clearing, and infiltrating them with paraffin.

Solution. The fixative that is used by most laboratories is neutral-buffered, 10% formalin. Formalin is a liquid that is 37-40% formaldehyde and 10% methanol (by weight) in water; thus a 10% formalin solution contains about 3.7% – 4% formaldehyde and 1% methanol (Hewitt, et al., 2008; Kiernan, 2000).

The average tumor cell content was 70% in fresh frozen and 68% in FFPE samples.

In next-generation sequencing workflows, samples of low or variable quality can corrupt downstream processes such as library preparation and ultimately confound analysis. Samples should be assessed for crosslinks, breaks, the accumulation of single-stranded DNA, and other forms of damage.

NGS is a cost-effective strategy compared with single-gene testing (SgT) in the molecular diagnosis of patients with metastatic NSCLC. More targetable genomic alterations could be detected by NGS, and therefore, more patients could potentially be treated with targeted therapies or enrolled in specific clinical trials.

What is next-generation sequencing in simple words? ›

Next generation sequencing (NGS), massively parallel or deep sequencing are related terms that describe a DNA sequencing technology which has revolutionised genomic research. Using NGS an entire human genome can be sequenced within a single day.

Specifically, the Centers for Medicare & Medicaid Services (CMS) established coverage of NGS as a diagnostic laboratory test when performed in a Clinical Laboratory Improvement Amendments (CLIA)-certified laboratory, ordered by a treating physician, and specific criteria are met.

FDA Approves Liquid Biopsy Next-Generation Sequencing Companion Diagnostic Test. On August 26, 2020, the Food and Drug Administration approved the liquid biopsy next-generation sequencing-based FoundationOne Liquid CDx test (Foundation Medicine, Inc.)

The testing typically takes 2 to 3 weeks for results, while individual tests often take a week or less. The cost of testing (typically several thousand dollars) is more than the cost of testing one or a few markers, if no more are needed.

There are three main types of DNA sequencing done by NGS: Whole Genome Sequencing, Whole Exome, and Targeted Panels. Choosing between the type of NGS that may be required for research or clinical diagnosis depends on what you are looking for.

Researchers have created a new DNA sequencing technique called Chem-map, which enables researchers to perform in situ mapping of small molecule-genome interactions with unparalleled precision.

Conventional intrinsic NGS error rates range between 0.1 and 1% (Phred quality score of 20–30) (1, 11) depending on the sequencing platform, the GC content of the target regions (12), and the fragment length, as shown in Illumina paired-end sequencing (13).

Often, DNA elution is facilitated by high temperature, high pH, and low ionic strength conditions. Higher pH conditions likely facilitate DNA elution by increasing the negative charge density on the silica surface, resulting in greater electrostatic repulsion between the DNA and silica surface.

With deceased and decayed bodies, personal identification is performed using hard tissue DNA, commonly extracted from bone. The quantity and quality of DNA used in the polymerase chain reaction (PCR) amplification step is critical for a successful outcome.

The quality of the DNA extracted from the samples was measured. Results No difference was observed between fresh and stored blood samples. Conclusions The quality of DNA extracted from liquid or dried blood is not adversely affected by storage at 4° C for up to 24 h.

How do you extract DNA step by step? ›

There are 3 basic steps involved in DNA extraction, that is, lysis, precipitation and purification. In lysis, the nucleus and the cell are broken open, thus releasing DNA. This process involves mechanical disruption and uses enzymes and detergents like Proteinase K to dissolve the cellular proteins and free DNA.

For DNA sequencing, the amount is dependent upon the type of the sample you have. Soil and tissue samples require at least 0.25g. We recommend sending a few grams extra especially if multiple extraction replicates per sample are requested.

The purpose of genomic DNA extraction is to separate this genetic material from the rest of the cell (proteins, RNA, cell membrane, etc.). Once purified, scientists can study individual genes, sequence the entire genome, modify sections of DNA, and more.

You can expect your results within eight weeks of the date the laboratory receives your sample.

About a month to a million years, theoretically. The decay rate of DNA depends on the conditions of its storage and packaging. Above all, it depends on whether the DNA is exposed to heat, water, sunlight, and oxygen.

We recommend freeze drying of DNA samples, especially when they have to be shipped for longer distances. No special packaging or declaration is needed for freeze dried samples, and the risk of thawing is excluded.

No, it does not matter what tissue is used in an organism, the DNA sequence is the same in all cells of an organism. One must keep in mind that in blood, red blood cells do not have a nucleus, therefore, no DNA. When a DNA extraction process is done on blood, the DNA actually comes from the white blood cells.

It is suggested that the brain tissue preserved at −80°C is the best among soft issues for DNA extraction.

As any enzyme activity is sensitive to temperature, the degradation process is reduced at lower temperatures. Thus, keeping samples cold will slow down the enzymatic degradation of DNA.

Cryopreservation of whole blood is useful for DNA collection, and clinical and basic research. Blood samples in ethylenediaminetetraacetic acid disodium salt (EDTA) tubes stored at - 80 °C are suitable for DNA extraction, but not for high-quality RNA extraction.

What are 5 ways DNA can be extracted? ›

For DNA testing the most popular and reliable way to collect samples is the oral buccal swab method. A buccal swab closely resembles a one ended Q-Tip in appearance. Using swabs as a collection method is quick and painless and is the recommended way to collect DNA samples for testing.

By adding salt, we help neutralize the DNA charge and make the molecule less hydrophilic, meaning it becomes less soluble in water. The salt also helps to remove proteins that are bound to the DNA and to keep the proteins dissolved in the water.