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Capillary electrophoresis (CE) is not a new technology, but the introduction of automated, high-throughput CE instruments has made it possible to streamline the analysis of next-generation sequencing (NGS) libraries. These instruments help ensure that DNA libraries yield high-quality data. Use of CE is critical to any NGS workflow, as poor-quality DNA libraries can hamper sequencing and, in turn, slow research or disease diagnosis.
Role of CE in NGS data quality
Before performing NGS, a scientist must prepare DNA samples in a process called library preparation. The Fragment Analyzer from Advanced Analytical Technologies, Inc. (AATI, Ankeny, IA) is an automated CE system that can help ensure these libraries are of high quality. Generating high-quality libraries helps optimize flow cells for the next preparatory step in sequencing—cluster generation—and reduces the cost and time it takes to complete a sequencing run.
NGS laboratories typically need their libraries to be of a certain size range and concentration. CE provides both parameters in one run. Like traditional gel electrophoresis, CE separates DNA fragments by their size by exposing them to an electric field. In a CE platform, the DNA fragments flow through a narrow capillary tube that can more accurately determine the size of each fragment as it runs past a fluorescence detector.
Sizing up DNA in high resolution
The narrow capillary tubes used in CE provide higher resolution than traditional electrophoresis. In a CE tube, the buffer flows at a constant rate across its diameter. This means that DNA fragments do not diffuse laterally (i.e., across the width of the tube); rather, the molecules only move in one direction, down the tube, leading to high separation efficiency.1
Quantifying DNA fragments
CE also more accurately and precisely measures the initial extracted DNA fragment concentration. This is particularly useful during library preparation when adapters are added to the sheared genomic DNA. It is important to know the initial DNA fragment concentration to determine the right proportion of adapters to add to the sample. A concentration of adapter that is too high, and subsequently is not removed properly in the cleanup steps, can result in a series of repetitive DNA sequences that can bind to the flow cell and produce poor sequencing data.
Pulsed-field capillary electrophoresis
It can be difficult to separate large DNA molecules effectively using standard electrophoresis; molecules larger than 20 kb tend to migrate together when exposed to a constant voltage, regardless of size.2 The FEMTO Pulse Automated Pulsed-Field CE Instrument (AATI) (Figure 1) is optimal for the separation of genomic DNA over 20 kb. Pulsed-field CE applies a voltage that shifts among multiple parameters. DNA of various lengths react differently to these shifts and, during electrophoresis, these differences translate into their separation, making it easier to analyze larger individual fragments.2 The instrument is able to separate and resolve fragments up to 165 kb.3 It automates quality control of longread sequencing library preps, analysis of bacterial artifi cial chromosomes, and synthetic genomes, decreasing the time to results.4
Figure 1 – FEMTO Pulse
Automated Pulsed-Field CE Instrument.Cell-free DNA applications
CE helps resolve fragments corresponding to nucleosome fragmentation patterns in cellfree DNA (cfDNA) (Figure 2). When cellular DNA is fragmented following cell death, it is cut around nucleosomes, yielding fragments of distinct sizes (e.g., ~165 bp for a mono-nucleosome fragment, ~350 bp for a di-nucleosome fragment, and ~565 bp for a tri-nucleosome fragment). The fragmentation pattern may indicate where the cfDNA originated, suggesting where diseased cells, such as a tumor, might be found.5
Figure 2 – cfDNA extracted with the QIAmp kit (Qiagen, Germantown, MD) using carrier RNA and separated on the Fragment Analyzer using the DNF-477 HS small fragment kit (AATI). LM = lower marker; UM = upper marker.Compared to chip-based systems, CE may also better separate contaminating high-molecular-weight DNA from these common nucleosomal peaks. Researchers can size cfDNA fragments more precisely, enabling them to optimize DNA libraries for sequencing.
Potential of cfDNA sequencing
The ability to detect and quantify cfDNA for sequencing has facilitated the development of noninvasive tests for detecting cancer mutations and fetal abnormalities.6,7 Sequencing-based noninvasive prenatal testing will continue to grow as new research demonstrates how a mother’s cfDNA affects the health of her fetus. In cancer diagnostics, clinical researchers use tumor-derived cfDNA as biomarkers to monitor recurrence, determine the effectiveness of therapy, and ultimately detect cancer in asymptomatic patients. Overall, as cfDNA becomes a more desirable target for liquid biopsies, sequencing workflows involving cfDNA will become more common.
While NGS is considered the gold standard for profiling genetic markers of disease, its cost and complexity have created a demand to optimize the workflow. CE streamlines DNA library quality control by characterizing libraries and screening for poor-quality samples, and is therefore an indispensable component in the NGS workflow.
References
- https://www.chromatographytoday.com/news/electrophoretic-separations/35/breaking-news/ advantages-and-disadvantages-of-capillary-electrophoresis/32342
- https://link.springer.com/chapter/10.1007%2F978-3-642-59811-1_7
- https://www.businesswire.com/news/home/20161128006134/en/World%E2% 80%99sAutomated-Pulsed-Field-Capillary-Electrophoresis-System-AATI
- https://www.aati-us.com/instruments/femto-pulse/
- https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4715266/
- https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5412506/
- http://onlinelibrary.wiley.com/doi/10.1002/uog.14791/abstract
Steve Siembieda is vice president of commercialization, Advanced Analytical Technologies, Inc., 2450 SE Oak Tree Ct., Ankeny, IA 50021, U.S.A.; tel.: 515-964-8500; e-mail: [email protected]; www.aati-us.com