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9 February 2022

Demystifying the Next-Generation Sequencing Workflow: Part 1

Toward the end of 2021, we highlighted the rising importance of next-generation sequencing (NGS) in diagnostic laboratories. In this two-part series, we will explore the NGS workflow to gain a better understanding of how this testing technology can be implemented in your laboratory. We start with an overview of the NGS workflow to orient clinical diagnostics laboratories that may be considering implementing NGS. In our next blog, we will discuss the challenges associated with NGS in the diagnostic laboratory and ways in which your laboratory can overcome these challenges efficiently.

NGS Workflow Overview

The NGS workflow is a multi-step process that creates some challenges for the diagnostic testing laboratory. The chemical processes generate many daughter samples that must be traced back to the original parent specimen. Laboratories need effective naming conventions and a robust LIMS that can maintain chain of custody across the workflow and the daughter samples. The workflow includes amplification steps, which requires segregation of the workflow to contain amplicon within a single area in the facility. The sequencing portion of the workflow generates a large dataset, which requires the laboratory to have a robust analytic pipeline and data storage capacity.

These topics will be discussed in-depth in this NGS series.

Nucleic Acid Extraction

Nucleic acid extraction utilizes the primary specimen (e.g., dried blood spot, serum, cheek swab) in a series of steps that capture the nucleic acid on a solid matrix, followed by washes to remove debris and contaminants, and finally a release step where the concentrated DNA and RNA are eluted into a small amount of buffer. The nucleic acid extraction process is typically performed using commercially available purification kits that are based on the Boom method (1), using a silica matrix packed into columns or on paramagnetic beads. The nucleic acid extraction process for NGS is the same process used for qPCR-based methods, such as the ones used for molecular COVID-19 testing.

Library Preparation

The library preparation process creates appropriately sized libraries with sequencing adapters in sufficient quantity for the sequencing process through a series of chemical steps. In the first step, the nucleic acids are cleaved into fragments that are the appropriate size for sequencing. Next, molecular adapters that are compatible with the sequencing method are ligated to the ends of the nucleic acid fragments. Alternatively, if the DNA is in low quantity, the adapters may be added using PCR in order to enrich for the sequence regions of interest while also attaching the sequencing adapters in a single step. The resulting libraries can be pooled and run within a single sequencing reaction.

Sequencing

During the sequencing portion of the workflow, the pooled sequencing libraries are loaded onto the sequencer, where individual nucleotide bases are “read”. Several different chemistries are available to perform the sequencing reaction. The Ion Torrent platform uses semiconductor technology to detect changes as nucleotides are added during the sequencing reaction. The Illumina method of sequencing relies on clonal array formation and terminator technology to detect a different fluorescent signal for each type of nucleotide as bases are added.

Data Analysis

After the sequencing completes, the large data set must be analyzed with specialized software that performs a series of quality control steps to assess overall data quality, amplicon size, and agreement between paired ends. Next, the reads are assembled and aligned against a reference genome in order to identify variants that may have clinical significance.

Report Generation

The variants that may have a clinical significance are assembled, along with annotations about those variants, into a report that will be delivered to the clinician. This report must be presented in a way that is clear and meaningful to the clinician. Genetic test result reports that are too long or are unclear will be frustrating to the physician and have no meaningful impact on patient care. Laboratories must work closely with ordering physicians and their LIMS vendor to ensure that the test result reports contain clinically actionable information that is presented in a way that is accessible to the physicians.

Summary

The NGS workflow is a multi-step process that generates a massive amount of data that must be traceable across the workflow and able to be stored and retrieved by the laboratory. In our next blog, we will discuss the software and infrastructure requirements within a diagnostic testing laboratory and how your laboratory can plan for success with NGS testing.

If you have questions about implementing NGS into your laboratory and your LIMS, contact our team at ApolloLIMS. We can help you develop and implement your NGS workflows.

References

  1. Boom R., Sol C.J., Salimans M.M., Jansen C.L., Wertheim-van Dillen P.M., Van Der Noordaa J. Rapid and simple method for purification of nucleic acids. J. Clin. Microbiol. 1990;28(3):495–503.