NGS Library Validation Using Automated Capillary Gel Electrophoresis

Next-generation sequencing (NGS) is widely used in genomics, epigenetics and transcriptomics, etc.,¹ and also in such clinical diagnostics applications as cancer screening and drug development.² There are multiple commercial NGS platforms available, each incorporating unique operating principles (e.g., Roche 454FLX [454 Life Sciences, Branford, Conn.], Illumina Genome Sequencer³ [Illumina Inc., San Diego, Calif.] and Applied Biosystems/ABI SOLiD Sequencer [Foster City,Calif.]). No matter the platform, library preparation is the most critical step in the NGS workflow.

Figure 1 – Qsep100 capillary gel electrophoresis instrument with disposable cartridge.

The main goal in sequencing library preparation is producing adaptor included DNA fragments with specific size ranges. Therefore, monitoring sample size and quality from sample preparation to sequencing library validation becomes quite important. Here, the authors describe use of the Qsep100 DNA analyzer (Figure 1) (BiOptic, Inc., New Taipei City, Taiwan) to test minimal detectable concentration of NGS library sample. The data demonstrates that the specified detection limit of 50 pg library sample can easily be reached using the instrument.

Qsep100 is simple-to-use, integrating microcapillary electrophoresis technology into a plug-and-play, disposable, pen-shaped capillary gel cartridge with automated liquid handling, real-time fluorescence detection and analysis. Optimized for high resolution and high throughput at ambient temperatures, the system provides improved peak resolution, good linear dynamic range, reproducible migration times and reduced instrument/sample analysis costs. Qsep100 was utilized to test minimal detectable concentration of NGS library samples. The fully automated system completely covers the QC protocol from upstream genomic DNA, to total RNA to the final step of DNA library (Figures 2-5).

Figure 2 – Sequencing library construction workflow.

Figure 3 – a) Quality control of fragmented DNA. DNA was sheared into small fragments with an average size of 895 bp. b) High-sensitivity result of serially diluted library sample. Average size of DNA library was 515 bp. Library concentrations were from 0.33 ng/μL to 0.07 ng/μL.

Figure 4 – Monitoring integrity of total RNA is a typical QC step for downstream experiments such as RNA sequencing. Samples ranged from intact (RQN 8.9) to degraded (RQN 2.3). RQN (RNA quality number) value determines RNA integrity.

Figure 5 – Representative cell-free DNA sample result using the Qsep100 analyzer with N1 high-sensitivity cartridge kit. Low amounts cfDNA (yields of less than 5 pg/μL) were detected.

The instrument was used to analyze the degradation level of smeared genomic DNA samples to assess sample quality (see Figure 3). The ability to rapidly distinguish between intact and degraded genomic DNA samples was crucial for successful sequencing.

Conclusion

The fully automated Qsep100 system provides a simple, effective solution for monitoring each stage of library preparation. It covers the entire QC protocol, from upstream of genomic DNA, to total RNA, to the final DNA library step. The instrument can be set up in 1 minute and produce results in 3–5 minutes. Detailed data can be released in batches or individually.

References

1. Metzker, M.H. Sequencing technologies—the next generation. Nat. Rev. Genetics 2010, 11, 31–46.
2. Desai, A.N. and Jere, A. Next-generation sequencing: ready for the clinics? Clin. Genetics  2012, 81, 503–10.
3. Quail, M.A.; Kozarewa, I. et al. A large genome center’s improvements to the Illumina sequencing system. Nat. Methods  2008, 5, 1005–10.

The authors are with BiOptic, Inc., New Taipei City, Taiwan (ROC), and BiOptic, Inc., 1409½ Foothill Blvd., La Canada Flintridge, Calif. 91011, U.S.A.; tel.: 818- 679-4413; e-mail: [email protected]; www.bioptic.com.tw