Illumina Sequencing: Process & Advantages Explained

6 November 2023by Manoj Dadlani

In the world of genomics, and particularly genomics sequencing, few technologies have had greater impact than that of Illumina Sequencing (also known as a technique called Sequencing by Synthesis (SBS)).

By successfully implementing massively parallel sequencing, Illumina Sequencing increased the throughput of genomics sequencing exponentially, propelling the field of genomics into an era of increased discovery in various applications, including human health (cancer, etc.) and microbiology (microbiome, etc.)

This has led to what is known today as the Illumina self-proclaimed “Genome Era”, in which genetic information can be generated and utilized at a scale far greater than ever before.

Illumina sequencing: an overview

Illumina Sequencing succeeded an earlier method of DNA sequencing developed by Fred Sanger (Sanger Sequencing). Instead of sequencing individual DNA fragments, Illumina Sequencing sequences millions to billions of DNA fragments simultaneously. 

This increased the amount of data capable of being generated from a sample while drastically reducing time and cost, paving the way for even greater scientific discoveries in the field of genomics. Where Sanger Sequencing walked, Illumina Sequencing ran. 

Prior to Illumina, a company known as Solexa developed the core function within this approach, known as Sequencing by Synthesis (SBS). In short, SBS is a method in which DNA sequences are determined by adding individual base-pairs to single stranded DNA, resulting in the construction of a complementary DNA strand.

As each individual base-pair is added to the template DNA strand, a fluorescence is emitted, with the color of the fluorescence indicating the base-pair that had attached to the template DNA strand. By stringing together the various colors that are emitted during this process, a full genomic DNA sequence can be distinguished.

This process proved to be highly accurate, as well as highly scalable, thus exponentially increasing the amount of genomic data capable of being generated relative to previous methods (Sanger).

The applications of this technology are wide-ranging, from cancer diagnostics to microbiome research. Due to this technique’s robust data generation capabilities, greater questions and applications could be addressed, such as increasing the number of samples/subjects performed in a research study.

Illumina sequencing vs Nanopore sequencing

One interesting development in the world of genomics has been the continued evolution of how DNA sequencing can be performed, with Nanopore sequencing more recently being discovered and developed.

When it comes to Illumina vs Nanopore sequencing, contrary to how Illumina Sequencing is performed, Nanopore sequencing utilizes a process that pulls DNA strands individually through a carefully manufactured nanopore while quantifying the electric current variation induced by the various nucleotides present within the DNA strand.

In using this technique, nanopore sequencing offers unique advantages, including the ability to generate sequencing data in real-time.Thus, while Illumina Sequencing’s fastest turnaround time for sequencing is at minimum multiple hours, nanopore sequencing data is ready immediately after beginning sequencing.

This offers unique advantages in areas such as infectious disease testing as well as point of care testing. 

However, while the ability to have real time sequencing data is an attractive application, a drawback to nanopore sequencing is the accuracy of sequencing data. Compared to Illumina Sequencing, nanopore sequencing is significantly higher in errors when detecting individual base-pairs within DNA sequences.

Thus, Illumina Sequencing is much more advantageous when it comes to applications in which accuracy down to the base-pair level is important, such as cancer diagnostics, microbial genome assembly, and other genome research applications.

Which sequencing applications utilize Illumina sequencing?

Common microbiome sequencing applications that utilize Illumina Sequencing platforms include:

With its unsurpassed accuracy, Illumina sequencing has enabled a variety of research applications such as de novo assembly projects, large-scale complete genome sequencing, and targeted resequencing. It has also proved to be invaluable in clinical diagnostics where it is used to detect genomic variations associated with diseases. 

The process of Illumina sequencing

Sample preparation

When preparing samples, DNA first needs to be extracted. Once DNA has been extracted, the DNA needs to be high enough in concentration, as well as quality, for Illumina Sequencing. This includes measuring the DNA with instruments such as Qubit and Nanodrop to generate metrics such as ng/uL, 260/280 ratio, and 260/230 ratio.

Once it has been confirmed that the DNA is of suitable concentration and quality, the DNA must be templated appropriately in a process commonly referred to as “library preparation”. In this process, DNA is sheared and adapters are ligated on the ends of each DNA fragment which allows the DNA to bind to the flow cell for Illumina Sequencing.

Cluster generation

For cluster generation, the library is loaded into a flow cell where the templated DNA fragments are captured to surface-bound oligos that are complementary to the adapters added during sequencing library preparation. 

Once bound, the fragments undergo “bridge amplification”, in which the DNA fragment binds to neighboring oligos, resulting in a replication and amplification process. DNA polymerase is also used to extend the bridge amplicons, allowing clusters of DNA fragments to form.


Once clusters have been generated, the Illumina Sequencing performs SBS chemistry reactions, in which individual nucleotides are added into the flow cell. 

Once the nucleotide attaches to the DNA strand from cluster generation, the color-specific fluorescence emitted from the attachment signifies the nucleotide that had attached to the DNA strand. Over time, the sequential color pattern from nucleotides attaching to the DNA fragment provides the DNA fragment’s sequence. 

Data Analysis

During the sequencing process, the Illumina sequencing instrument translates the fluorescent imaging to files known as Binary Base Call files (BCL). BCL files are primary data, and need to be further converted to files known as FASTQ files which are then interpreted for additional data analysis. 

This process is performed via bcl2fastq. Once bcl2fastq is performed, FASTQ data is utilized for subsequent downstream analysis pipelines.

Case studies of Illumina sequencing

In the beginning of Illumina Sequencing (SBS), Solexa performed sequencing of the bacteriophage phiX-174 in 2005, the same genome Sanger used to validate Sanger sequencing, however the Illumina Sequencing technology produced significantly more data with over 3 million reads. This resulted in significantly increased coverage, which can in turn enable even greater genomic based initiatives.

Some of these initiatives and accomplishments include:

  • 2008: Sequenced the first ever African human genome
  • 2018: Pan-Cancer assays established for Cancer Dx
  • 2020: Illumina sequence data used for COVID-19 vaccine development

Closing Thoughts

With all this in mind, Illumina Sequencing (SBS) has enabled the field of genomics to accelerate over the past two decades at rates previously unimaginable with traditional sequencing methods.

The scalability, paired with accuracy, has enabled population-level genome sequencing which has resulted in numerous discoveries, including new diagnostic markers, therapeutic targets, etc.

As genomics and sequencing continues to evolve, look for Illumina Sequencing (SBS) to become even more affordable, and scalable, to enable even larger research and diagnostic initiatives to further advance science into the “Genome Era”. 

Unlock the Power of the Microbiome With CosmosID

For any microbiome applications in which you need Illumina Sequencing, CosmosID has the experts you need to properly plan and execute. Our expertise in bioinformatics analysis and microbiome sequencing applications utilizing Illumina Sequencing platforms include:

Contact our microbiome experts today to get started.

Illumina sequencing process FAQs

What is the Illumina method of gene sequencing?

Illumina sequences using the process of Sequencing by Synthesis (SBS). Sequencing by DNA synthesis is a sequencing method in which a complementary strand of DNA is built, base-pair by base-pair, with each base-pair addition resulting in fluorescence corresponding to the particular DNA sequence.

What are the 4 steps of Next Generation Sequencing (NGS)?

The 4 steps of next generation sequencing (NGS) include:

  • Sample Preparation
  • Cluster Generation
  • Sequencing
  • Data Analysis.

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Manoj Dadlani

Mr. Manoj Dadlani serves as Chief Executive Officer at CosmosID, Inc., the Maryland based provider of industry-leading solutions for unlocking the microbiome. Previously, Mr. Dadlani served as a partner at Applied Value Group, a management consulting and investment firm, and was co-founder and CEO at Rasa Industries, Ltd., a leading beverage manufacturing company. Mr. Dadlani has substantial experience in strategy, M&A, supply chain management, product development, marketing and business development. Mr. Dadlani received his bachelor’s and master’s degrees in Biological Engineering from Cornell University. Services offered by CosmosID’s CLIA certified and GLP laboratory cover the entire workflow from study design to sample collection, extraction, library preparation, sequencing, data analysis and publication support. CosmosID’s cloud-based metagenomics application offers user-friendly access to the largest curated databases for microbial genomics, antimicrobial resistance and virulence data and has been independently validated to return metagenomic analyses at strain level resolution with industry-leading sensitivity and precision.