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Illumina CMOS Chip and One-Channel
SBS Chemistry
The iSeq™ 100 System combines CMOS technology with innovative one-channel SBS
chemistry to deliver high-accuracy data in a compact system.
Introduction
Illumina sequencing platforms leverage proven, highly accuratesequencing by synthesis (SBS) chemistry—the most widely adopted
next-generation sequencing chemistry in the world.1 Illumina SBS
allows massively parallel sequencing using a proprietary reversible
terminator–based method that enables detection of single bases as
they are incorporated into growing DNA strands. Illumina SBS
chemistry employs natural competition among all four nucleotides,
which reduces incorporation bias and allows more robust sequencing
of repetitive regions and homopolymers.2 Compared to capillaryelectrophoresis–based Sanger sequencing, NGS can detect a
broader range of DNA variants, including low-frequency variants andadjacent phased variants, with a faster time to results and fewer
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hands-on steps. ,While SBS chemistry forms the foundation of all Illumina sequencing
platforms, the optical systems have evolved to include four-, two-,and one-channel detection methods. The iSeq 100 System employs
the latest evolution in Illumina SBS technology: one-channel SBS on
a complementary metal-oxide semiconductor (CMOS) chip. The
one-channel SBS method supports lower sequencing costs and theconvenience of an instrument footprint measuring just over one cubic
foot.

Highly Accurate SBS Chemistry
Illumina SBS chemistry uses reversibly labeled nucleotides tosequence hundreds of millions of clusters on a flow cell surface inparallel (Figure 1). During each sequencing cycle, a single labeleddeoxynucleotide triphosphate (dNTP) is added to the nucleic acidchain. The nucleotide label serves as a terminator for polymerization,
so that after each dNTP incorporation, the fluorescent dye is imaged
and then chemically cleaved to allow incorporation of the next
nucleotide. Base calls are made directly from signal intensity
measurements during each cycle. Because all four reversible
terminator-bound dNTPs (A, C, T, G) are present as single, separate
molecules, natural competition minimizes incorporation bias.
Furthermore, reversible termination ensures single base incorporation
per template strand per sequencing cycle, which greatly improvessequencing accuracy in homopolymeric regions and greatly reduces
raw error rates compared to other technologies.2
To see an animated video of Illumina SBS technology, visit
www.illumina.com/SBSvideo.

For Research Use Only. Not for use in diagnostic procedures.

Figure 1: Illumina SBS Chemistry with One-Channel Detection—(A) The iSeq
100 System uses an innovative one-channel chemistry method. Each sequencing
cycle includes two chemistry steps and two imaging steps. The first chemistry step
exposes the flow cell to a mixture of nucleotides that have fluorescently labeledadenines and thymines. During the first imaging step, the light emission from eachcluster is recorded by the CMOS sensor. The second chemistry step removes thefluorescent label from adenine and adds a fluorescent label to cytosine. In bothchemistry steps, guanine is dark (unlabeled). The second image is recorded. (B)The combination of Image 1 and Image 2 are processed by image analysis
software to identify which bases are incorporated at each cluster position. Thissequencing cycle is repeated "n" times to create a read length of "n" bases.

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Figure 2: Four-, Two-, and One-Channel Chemistry—Four-channel chemistry uses a mixture of nucleotides labeled with four different fluorescent dyes. Two-channel
chemistry uses two different fluorescent dyes, and one-channel chemistry uses only one dye. The images are processed by image analysis software to determine
nucleotide identity.

Four-Channel SBS Chemistry
In four-channel SBS, bases are identified using four different
fluorescent dyes for each base and four images per sequencing
cycle (Figure 2A). The sequencing cycle begins with a chemistry step
where all four differentially labeled bases are added to the flow cell.
Following nucleotide incorporation, the imaging cycle begins andincludes the capture of four distinct images using four different
wavelength bands. The images are processed with image analysis
software to determine which nucleotides were incorporated at eachcluster position across the flow cell. Therefore, with four-channelsequencing, every sequencing cycle requires four dyes and four
images to determine the DNA sequence. The MiSeq™ and HiSeq™
Series Systems currently use four-channel SBS.

Unlike four-channel SBS chemistry, where sequencers use a
different dye for each nucleotide, the iSeq 100 System uses one dye,two chemistry steps, and two imaging steps per sequencing cycle. In
one-channel chemistry, adenine has a removable label and is labeledin the first image only. Cytosine has a linker group that can bind a label
and is labeled in the second image only. Thymine has a permanent
fluorescent label and is therefore labeled in both images, and guanine
is permanently dark. Nucleotides are identified by analysis of thedifferent emission patterns for each base across the two images(Figure 2C).

Two-Channel SBS Chemistry
Rather than using a separate dye for each base, two-channel SBS
simplifies nucleotide detection by using two fluorescent dyes and twoimages to determine all four base calls (Figure 2B). Images are takenusing red and green filter bands. Thymines are labeled with a green
fluorophore, cytosines are labeled with a red fluorophore, andadenines are labeled with both red and green fluorophores. Guanines
are permanently dark. The MiniSeq™, NextSeq™, and NovaSeq™
Systems use two-channel chemistry.

Figure 3: Illumina CMOS Flow Cell—A sequencing library is loaded into the iSeq
100 reagent cartridge, which contains a patterned flow cell fabricated over aCMOS chip. Each well in the flow cell is aligned over a CMOS photodiode. Duringcluster generation, proprietary ExAmp chemistry ensures that each well in theflow cell generates a single, clonal cluster. During each imaging step, lightemissions are detected by the CMOS photodiodes.

One-Channel SBS Chemistry
The iSeq 100 System combines proven Illumina SBS chemistry withCMOS technology to deliver one-channel sequencing chemistry. The
system uses a patterned flow cell with nanowells fabricated over a
CMOS chip (Figure 3). Clustering and sequencing occur in thenanowells with direct alignment of single clusters over eachphotodiode (pixel). Using a CMOS sensor embedded in theconsumable is a simple and fast detection method.

For Research Use Only. Not for use in diagnostic procedures.

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Table 1: Instrument Comparison of Variant Analysis
Systems

Percent On-Target
Aligned Reads

Percent Mismatches

Percent Uniformity
Of Coveragea

Percent Precision (SNP)

Percent Recall (SNP)

Panel 1 (BRCA) biSeq 100

96.0

0.23

100

90.5100

MiniSeq

95.8

0.21

100

89.1

100

MiSeq

95.80.20

100

89.1100

Panel 2 (CHP2)
iSeq 10097.30.20

100100100MiniSeq

97.1

0.17

99.0

100

100MiSeq

96.80.15

10098.6

100

Panel 3 (Focus)
iSeq 100

96.50.23

99.595.2

100

MiniSeq96.60.20

98.9

92.9

100

MiSeq

96.1

0.19

99.094.0100

a. Percent > 0.2× meanb. The BRCA panel includes 265 amplicons spanning across all exon regions of the BRCA1 and BRCA2 genes; the CHP2 panel includes 207 amplicons covering
50 oncogenes and tumor suppressor genes; the Focus panel includes 269 amplicons that span 53 genes for multivariant analysis.

SBS Accuracy Across All SystemsIllumina SBS chemistry delivers highly accurate sequencing data at
virtually any coverage level, the highest yield of error-free reads, and267
the highest percentage of base calls above Q30* in the industry. , ,As with other Illumina platforms, the iSeq 100 System generates ahigh percentage of bases above Q30 (Figure 4). To assess how iSeq
100 System data compares to data generated on the MiniSeq andMiSeq Systems for variant identification, three amplicon panels wererun and analyzed on all three systems (Table 1). Data analysis wasperformed using Burrows-Wheeler aligner (BWA),8 Picses variant
caller,9 and Hap.py Benchmarking software.10 Precision and recall
were calculated with the Platinum Genomes reference data set.11
The results show comparable, high-quality alignment, coverage, andvariant analysis metrics across all three systems.
Because all Illumina sequencing systems use the same reversible
terminator–based SBS technology, researchers can confidently
transition their research from one Illumina sequencing system toanother. Regardless of whether data are generated by four-, two-, orone-channel SBS, results can be easily compared and analyzed in
BaseSpace™ Sequence Hub, the Illumina genomics computing
environment, or across a wide array of third- party analysis tools.

SummaryFigure 4: Quality Scores from the NextSeq 500 and iSeq 100 Systems—(A)Two-channel SBS technology on the NextSeq 550 System delivers high-quality
data, with a sequencing accuracy of 88% of sequenced bases ≥ Q30. Human cellline mixture samples were pooled and run with a 2 × 101 bp configuration. (B)One-channel SBS chemistry on the iSeq 100 System delivers comparable highquality data. A metagenomics microbial pool sequenced on a 2 × 151 bp run
provides 89% of bases ≥ Q30.

Illumina SBS chemistry offers highly accurate base-by-basesequencing and robust coverage across the genome. The latest
evolution in SBS technology can be found in the iSeq 100 System,
which combines CMOS sensor detection with one-channel SBS
technology. With simplified chemistry and optics, an entry-level
pricepoint, and the convenience of a small footprint, the iSeq 100
System enables the power of NGS to become an everyday researchtool for virtually any laboratory.

*A quality score (Q-score) is a prediction of the probability of an error in base calling. A Q-score of 30 (Q30) is widely considered a benchmark for high-quality data. 5For Research Use Only. Not for use in diagnostic procedures.

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Learn More
To learn more about the iSeq 100 System, visit
www.illumina.com/iseq
To learn more about microbial or mitochondrial sequencing on theiSeq 100 System, read the Microbial WGS with the iSeq 100 Systemor the Mitochondrial DNA Sequencing on the iSeq 100 System
Application Notes
To learn more about patterned flow cells, visit the Patterned Flow Cell
pageReferences
1.

Data calculations on file. Illumina, Inc., 2015.

2.

Bentley DR, Balasubramanian S, Swerdlow HP, et al. Accurate Whole
Human Genome Sequencing using Reversible Terminator Chemistry.

Nature. 2008;456(7218):53-59.
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Precone V, Monaco VD, Esposito MV, et al. Cracking the Code of Human
Diseases Using Next-Generation Sequencing: Applications, Challenges, andPerspectives. Biomed Res Int. 2015;161648.

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Shokralla S, Porter TM, Gibson JF, et al. Massively parallel multiplex DNA
sequencing for specimen identification using an Illumina MiSeq platform. SciRep. 2015;5:9687.
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Illumina (2011). Quality Scores for Next-Generation Sequencing. Accessed
November 30, 2017.

6.

Ross MG, Russ C, Costello M, et al. Characterizing and measuring bias insequence data. Gen Biol. 2013;14:R51.

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Nakazato T, Ohta T, Bono H. Experimental design-based functional mining
and characterization of high-throughput sequencing data in the sequence
read archive. PLoS One. 2013;22;8:e77910.

8.

Li H, Durbin R. Fast and accurate short read alignment with Burrows-Wheelertransform. Bioinformatics. 2009;25:1754-60.

9.

Pisces Variant Caller. github.com/Illumina/Pisces/wiki. Accessed December
4, 2017.

10. Hap.py Benchmarking. www.illumina.com/products/by-type/informaticsproducts/basespace-sequence-hub/apps/hap-py-benchmarking.html.
Accessed December 4, 2017.
11. Platinum Genomes. Eberle, MA et al. A reference data set of 5.4 million
phased human variants validated by genetic inheritance from sequencing athree-generation 17-member pedigree. Genome Res 2017;27:157-164.

Illumina, Inc. • 1.800.809.4566 toll-free (US) • +1.858.202.4566 tel • techsupport@illumina.com • www.illumina.com© 2018 Illumina, Inc. All rights reserved. All trademarks are the property of Illumina, Inc. or their respective owners. For specific trademark information,
see www.illumina.com/company/legal.html. Pub. No. 770-2013-054-B QB 5474

For Research Use Only. Not for use in diagnostic procedures.

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