Isolation of cell free / circulating tumor DNA from plasma

tubes

Identification of biomarkers that indicate presence of disease are highly sought after. Non-invasive methods to measure those biomarkers are even more valuable. By extracting and measuring cell-free DNA, scientists have satisfy both.

Cell free DNA are degraded fragments released in plasma. Elevated levels of cfDNA are found in cancer states, making assessment of somatic genomic alterations from tumors possible using sequencing. Cell free fetal DNA (cffDNA) can be found as early as 7 weeks gestation, and analysis of cffDNA is already being used in non-invasive prenatal diagnostics. Cell free DNA (cfDNA) in blood was first described by Mandel and Metais in 1948 [1] but only recently has been identified as having utility for prenatal testing and disease diagnostics and monitoring.

Unlike mutations that are passed from a parent to child and are in every cell of your body, somatic mutations form during a person’s life. These somatic mutations are present in tumor cell DNA and are an excellent biomarker if they can be measured and monitored.

Acquiring tumor DNA often requires a biopsy, a potentially risky and invasive procedure. In many cases presence of a tumor or the ability to biopsy is not even an option for patient. During tumor turnover and progression, apoptotic and necrotic cells release small pieces of their DNA (cfDNA) into the bloodstream. The amount of cfDNA in the blood steam is influenced by clearance and filtering of the blood and lymphatic circulation.

Detecting cfDNA in plasma is called a ‘liquid biopsy’ and is already a popular method for obtaining clinical samples for prenatal testing, disease diagnostics and monitoring. One of the challenges of liquid biopsies, are standardization of the isolation procedure and maintaining  uniform specificity and sensitivity. Extraction of cfDNA can be carried out using magnetic beads or silica matrices along with chaotrophic salts, such as guanidine thiocyanate. While several commercial approaches (Table 1) exist, none have undergone rigorous large patient scale studies. Once more information is known, universal standardization should allow greater clinical utility.

Commercial kits for extraction of cfDNA need to be designed to extract uniform DNA copies from varying biopsy volumes. Scalability and adaptability for cell free fetal and ctDNA are important considerations. Below we highlight current kits available in the market. In a future blog post we’ll discuss isolation and sequencing standardizations required for broader use of cfDNA liquid biopsy.

Table 1.

Kit Company Method Digestion Prep Time (min) Plasma Volume (mL) Elution (uL) DNA sizes

(bp)

NextPrep-Mag

 

Bioo Scientific Mag Beads Proteinase K (optional) 30 1 – >5 12 >50
Chemagic cfNA Chemagen Mag Beads Proteinase K 120 2 – 10 60 >100
MagMAX Cell Free DNA Kit Thermo Fisher Mag Beads Proteinase K

(optional)

40 1 – >5 15 >50
QIAamp

 

Qiagen Column Proteinase K 120 1-5 20 >70
Quick-cfDNA

 

Zymo Research Column Proteinase K 60 3- 10 35 >100

Targeted gene panels vs. whole exome sequencing

gene-panels

One frequent question we hear on Genohub is, ‘should I make a custom panel for this gene set, or not bother and do whole exome sequencing?’. While whole genome sequencing approaches can capture all possible mutations, whole exome or targeted gene panel sequencing are cost-effective approaches for capturing phenotype altering mutations. We go into the advantages of WGS vs. WES in an earlier blog post. A remaining question however is, among targeting approaches, which is best. We attempt to address this here:

Advantages of targeting all exons – whole exome sequencing (WES)

If your study is discovery based, in other words you don’t know what genes you need to target, WES is the obvious choice.

  • Better for discovery based applications where you’re not sure what genes you should be targeting.
  • Exome panels are commercially available, they don’t need to be customized or designed.
  • Exome sequencing services are fairly standard, costs range between $550-800 for 100-150x mean on target coverage.

Advantages of targeted gene panels (amplicon-seq or targeted hybridization methods)

Targeted gene panels are ideal for analyzing specific mutations or genes that have suspected associations with disease.

  • Focusing on individual genes or gene regions allows you to sequence at a much higher depth than exome-seq, e.g. 2,000-10,000x as opposed to 200x which is typical with exome-seq.
  • High depth sequencing enables the identification of rare variants
  • Can be customized for different samples types, e.g. FFPE, cf/ctDNA, degraded samples.
  • Lower input amounts can be used with targeted gene panels (1 ng vs. 100 ng with whole exome sequencing).
  • Gene panels can be customized to only include genomic regions of interest. Why sequence everything when you don’t need that extra information?
  • Panels can be easily designed for non-human species. Designing a non-human exome is much more laborious.
  • Gene panel workflows are a lot simpler and time to results is often as little as 1-2 days.
  • You can process thousands of samples on a single sequencing run. Targeted gene panels can be run at a higher throughput and are often more cost-effective than whole exome sequencing.

By focusing on genes likely to be involved with disease, you can reduce expense and focus sequencing resources on your targeted region. However, if you only have a few samples that you need to sequence at a low depth of coverage, consider whether it’s worth designing a panel vs. performing whole exome sequencing using an existing commercial panel.

If you’re interested in designing a custom gene panel or already have an existing panel you’d like to sequence, submit a request describing your project or view several of the existing commercially available panels here.

Guides to improve your next generation sequencing project

read length, output and instrument recommendations for next generation sequencing

If you’re new to next generation sequencing or if you’re simply looking for tips to improve your next project, we recommend you take some time to look at the guides available on Genohub. As researchers order sequencing services it’s completely normal for there to be numerous questions related to nucleic acid extraction, library prep and best practices for loading a sequencing instrument. Over the years, we’ve curated these questions and published guides to help those embarking on their next NGS project. Topics covered include: library prep applications, batch effects, optimal cluster densities, read lengths and instrument output.

Next generation sequencing is a tool that can be applied to answer any number of questions related to the genome, transcriptome or epigenome. Regardless of the organism being sequenced or the library method used to prepare nucleic acid from that organism, the fundamentals of how a sequencing platform works, is similar across all samples. There are currently four main sequencing platforms that researchers regularly use. These include Illumina, Ion, PacBio and Oxford Nanopore. The guides below tend to be Illumina focused because that’s the platform most people are currently using today. Despite that, we review the read throughput of each available instrument and discuss hybrid methodologies where short and long reads are combined from two different instruments to improve assemblies.

Guides for sequencing

  1. Designing a sequencing project
  2. Recommended coverage by library preparation application
  3. Comparison of instrument read lengths and read outputs

Guides for preparing your samples

  1. Best practices for shipping tissue and nucleic acid 
  2. Library preparation kits and tips

Guides by application

  1. Transcriptome and mRNA-Seq
  2. Genome sequencing and re-sequencing
  3. Exome
  4. Metagenomics 
  5. Small RNA (microRNA)
  6. WGS vs. WES

Tips and considerations for commonly used sequencing instruments

  1. HiSeq X
  2. HiSeq 3000/4000
  3. Nextseq and low diversity

These are evolving guides, meaning our goal is to continuously improve them. If you have any feedback or would like to contribute please send us a message. We hope these guides will be helpful in designing your next NGS run. If you have technical questions related to an upcoming NGS project, feel free to submit them on our consultation page.