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Streamlining DNA Library Preparation for NGS

Whether fragments are long (>1,000bp) or short as in conventional NGS work (<500bp), NGS library preparations benefit from quantitative and qualitative analysis

by Advanced Analytical Technologies
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Problem: Modern high-throughput sequencing technology has led to a boom in sequencing projects spanning a wide range of research. High-throughput sequencing methods like next generation sequencing (NGS) are now a critical tool for both basic and clinical research, as evidenced by the leap from 236 NGS PubMed citations in 2009 to 1,878 in 2013. However, in many cases genomic DNA can’t be accurately sequenced by NGS, a frustrating challenge for researchers. NGS requires input DNA to first be chopped into short fragments, and then sequences the fragments to determine the order of the bases. Finally, these fragments are reassembled into an approximation of the actual genome. Unfortunately, not all genomes are created equal. Genomes with long stretches of repeating sequence, with regions rich in guanine and cytosine, or with missing base pairs can all lead to gaps in the sequencing data. For researchers trying to identify and make sense of sequence variations, this has been a major hurdle. Another obstacle is the chopped DNA fragments—whether they’re short or long—must pass the Goldilocks test: the size and concentration of the input DNA sample must be “just right” to get high quality NGS results.

Scientists have been combining new and old approaches to refine sequencing, but challenges remain. While some researchers turn to traditional Sanger sequencing to validate their NGS results, this method is more labor-intensive and cannot be scaled up to match the high-throughput nature of NGS. Some companies are developing sequencing technologies that deliver longer reads and potentially eliminate some of the data gaps caused by repeating sequence. However, longer reads can be more difficult to resolve. With a new wave of sequencers that have varying requirements for fragment length and concentration, researchers are faced with the challenge of accurately quantifying and qualifying their DNA fragments prior to sequencing.

Advanced Analytical Technologies' fragment analyzer tests DNA quality, concentration and fragment length before sequencing.
Advanced Analytical Technologies, Inc.

Solution: Whether fragments are long (>1,000bp) or short as in conventional NGS work (<500bp), NGS library preparations benefit from quantitative and qualitative analysis. Prioritizing these quality control steps can save researchers thousands of dollars, and days of wasted research time.

A number of companies have focused on developing instruments that help researchers prepare DNA fragments for sequencing. For example, Advanced Analytical Technologies, in Ames, Iowa, has developed the Fragment Analyzer™ Automated Capillary Electrophoresis (CE) System, which analyzes and verifies the integrity, fragment length and concentration of researchers’ DNA samples.

The instrument applies an electric field to a tiny amount of DNA sample, pulling DNA fragments into long, thin capillary tubes. The capillaries contain a gel with a fluorescent dye that binds to the DNA molecules. During electrophoresis, voltage is applied to the capillaries and the DNA fragments move through the gel, separating according to size with longer length fragments moving more slowly. As molecules pass by a window in the capillary, a continuous light source excites the dye bound to the DNA molecules. A camera detects emitted light from the dye, visualizing the DNA fragments. The time required to pass the window relates to DNA size, while the emitted light intensity indicates DNA concentration. Analytical software shows the output as an electropherogram or digital gel image.

The instrument tells researchers whether the size distribution of their DNA fragments is within the appropriate range for their sequencing platform and whether the DNA is at the right concentration. The instrument can resolve DNA fragments of up to 20 kilobases in length, making it an ideal solution for long fragment reads. Furthermore, instruments such as the Fragment Analyzer™ feature highly automated workflows. With an explosion of NGS related studies surfacing in labs everywhere, a streamlined workflow for quantification and qualification of DNA fragments both short and long is critical.

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