Unlock the next wave

of genomic discovery

Broader, deeper, multiomic sequencing

enabled on the NovaSeq™ X Series

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

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Table of contents

3 Revolutionizing the pace of discovery

4 The dawn of the Genome Era

5 Broader, deeper sequencing methods

6 Bigger studies to improve statistical power

6 Large-cohort data at work

7 Increasing diversity in genomic data

8 Broader studies to access missed information

9 Value of whole genomes

10 Value of whole transcriptomes

11 Multiomic studies for a wider perspective

12 Common multiomic combinations

13 Next-level multiomics

14 Higher analytical resolution to decode complex systems

14 Single-cell sequencing

15 Spatial sequencing

15 Pushing the boundaries

16 Deeper studies to find rare genetic events

16 Liquid biopsy

17 Genome-wide tumor–normal sequencing

18 How the NovaSeq X Series accelerates genomic discovery

19 Enabling broader, deeper sequencing

21 Managing big experiments and big data

24 Example NovaSeq X Series workflows

26 Unimaginable experiments, made possible

27 Abbreviations
28 References

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Revolutionizing the pace of discovery

Genomics research is expanding our understanding of biology and making personalized medicine a reality. Advancements
in next-generation sequencing (NGS) are enabling genomic visionaries to perform the studies that can answer the most
complex biological questions. Increased discovery power will come from larger studies with bigger cohorts, deeper
sequencing to identify rare genetic events, and broader sequencing methods and multiomics for a more comprehensive
view of cellular activity.

Still, this new scale of experiments has been held back by high sequencing costs and analytical complexity. Now, with the
NovaSeq X and NovaSeq X Plus Sequencing Systems, the extraordinary can become routine. Transformational throughput
and simplified informatics bring greater speed and lower costs that will be a force for democratizing sequencing and
powering the Genome Era.

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The dawn of the Genome Era

The first draft of the human genome sequence used hundreds of instruments performing capillary electrophoresis
Sanger sequencing and took over 13 years and nearly $3 billion (USD). Since the introduction of massively parallel
sequencing technologies like NGS, the throughput of genome sequencing has increased and the costs have decreased at
an incredible rate. Over the last two decades, sequencing output has grown over 10,000-fold, from less than 1 gigabase
(Gb) to 16 terabases (Tb) per run, while the number of reads has increased from millions to tens of billions. Experiments
that once required complex workflows now use simple push-button sequencing.

COST-PER-GENOME
Dramatic decrease in sequencing costs

$100 M

$2 M
Cost (USD) per human genome

Genome Analyzer™ II
$100,000
$3000/Gb
0.3 Gb/day
$10,000

$1000

NovaSeq X Series
$600 $2/Gb
6000 Gb/day

$200

2001 2003 2005 2007 2009 2011 2013 2015 2017 2019 2021 2023

Year

What if this breakneck pace of advancement was from to

applied to air travel? A hypothetical flight from
$2000 | 6 hours NY Paris $1.33 | 1 Sec
New York City to Paris, France could decrease

These incredible advancements in genome analysis are ushering in a new era of personalized medicine. Whole-genome
sequencing (WGS) has helped diagnose children with rare genetic diseases. Comprehensive genomic profiling of tumors
has been used to identify driver mutations and match them with targeted therapies. During the COVID-19 pandemic, DNA
sequencing tracked specific variants and provided the basis for rapid vaccine development. Managing future pandemics
and developing personalized therapies will depend on expanding access to high-throughput genomic sequencing.

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Broader, deeper
sequencing methods
Despite this progress, there is much work left to do
to unlock the full power of the genome. To understand
genetic variation and complex disease, we will need to
sequence millions of genomes and integrate health data.
We need to represent diverse populations, ensuring that
more people can access sequencing in more places. To
diagnose rare disease in the neonatal intensive care unit
(NICU), we need to sequence genomes at record speed
for maximum impact. To use cell-free DNA to track
disease noninvasively, we need to sequence deeply. To
see the full picture of cellular physiology, we need to look
at multiple “omes” together—genomes, transcriptomes,
epigenomes, and proteomes. And we will need powerful
bioinformatics tools to turn all that data into insight.

With the decreased costs of sequencing, scientists

can leverage budgets to perform studies of greater scale
and depth, as well as new applications. Researchers
can generate more detailed data from their samples or
interrogate their samples at multiple levels for greater
insights. This eBook highlights the types of studies that
once were unimaginable, and are now possible thanks
to breakthrough advancements in high-throughput
genomics.

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Bigger studies to improve

statistical power

“
Increase statistical power from larger sample sizes
and more diverse cohorts
A measure of the success
of our mission will be
Even decades after the first human genomes were
sequenced, a majority of gene–disease connections whether we can convince
remain a mystery. To understand the significance of
human genetic variation in relation to complex disease,
similar healthcare
genome-wide association studies (GWAS) aim to systems around the world
connect genotype to phenotype. Most GWAS have
been performed using microarray genotyping. As the to adopt approaches
cost of genome sequencing drops, researchers can
generate higher quality, more comprehensive sequence
developed within the
data at population scale. A shared global knowledge base 100,000 Genomes Project
that integrates genomic data with longitudinal health
records is needed to clearly identify key genes and to transform the application
disease mechanisms.
of genomic medicine in
healthcare and bring
Large-cohort data at work

”
better outcomes to
The 100,000 Genomes Project in the United Kingdom patients worldwide.
(UK), supported by Genomics England, is the archetype
for integrating genomics with a large-scale health system Mark Caulfield, Chief Scientist,
and demonstrates what routine, genomic-informed Genomics England
medicine might look like.1–3 The UK BioBank has also built
one of the largest biomedical databases in the world,
containing genetic, lifestyle, and health information
from 500,000 individuals.4 The population-scale WGS
data coming from these projects has yielded incredible
insights for cancer and increased diagnostic yield for
rare disease.3 For example, WGS of over 12,000 matched
tumor–normal samples revealed mutational signatures
that could help direct personalized cancer treatments.5
The UK is undertaking an even larger initiative, Our
Future Health, which aims to sequence five million more
genomes.6

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Increasing diversity in genomic data

Increased statistical power for these studies will also come from larger, more diverse cohorts. Expanding access to
NGS across the globe, especially in regions with underrepresented populations, will increase the diversity of our genome
data sets. Following in the footsteps of the pioneering work in the UK, population genomics projects are growing in other
countries and regions around the world.7–32

Population genomics projects across the globe with numbers of participants

United Kingdom6 United Kingdom1

5 million 100,000

United Kingdom4
500,000 France13
1 million
Ireland 14
Denmark16
400,000
50,000
Finland17
Iceland15
500,000
200,000
Estonia18
100,000
Turkey19
100,000

United Arab
Emirates20
1 million

Japan23
Saudi Arabia21 100,000
100,000
Mexico 27 Asia10,11
Brazil28 100,000
1 million
7000
Israel22
100,000
United States29 Hong Kong24
1 million Africa7 Singapore12 50,000
United States31 118,000+ 100,000
1 million
United States30 Australia8 China25
1 million 200,000 100,000
United States 32
Australia9 China26
100,000
7000 100,000

Many of these efforts focus on increasing the diversity to train artificial intelligence to use information from
in the pools of genome data to represent underserved natural selection to better classify variants of unknown
groups,7–12,19–27,31 because, as of 2021, 86% of genomics significance (VUS).35,36 Together, these efforts will help
studies have been conducted on individuals of European us realize a more complete view of the genome for all
descent.33 Additional tools are helping draw out the full populations and develop ways to stop disease outbreaks,
value of these large-cohort projects. The “pan genome” decrease early deaths due to sudden heart attacks, and
reference genome includes 47 diverse references to reduce the strain of chronic diseases like Alzheimer’s
uplevel variant calling.34 The Primate AI-3D resource and diabetes.
mines data from other primate genomes (233 species)

Learn more

Population genomics
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Broader studies to access missed information

See the whole breadth of variation with whole-genome or whole-transcriptome sequencing

Historically, when sequencing is expensive and data analysis is burdensome, many labs have chosen targeted
sequencing approaches and narrowed their study focus. With lower costs and streamlined informatics, it becomes
more practical to query more broadly—to shift from gene panels to exomes or from exomes to whole genomes—and
gather the most data possible from each sample. The scale of broader research minimizes bias from experiments
by extending analyses beyond a few preselected targets.

Whole-genome sequencing Whole-exome sequencing Targeted sequencing

Sequencing region Coverage Sequencing region Coverage Sequencing region Coverage

Entire genome ~30×* Protein-coding regions Specific genes or regions
~50×–100×* > 500×*
(~2% of the genome) of interest

Pros Cons Cons Cons

Pros Pros
Captures all DNA variants in Higher per-sample cost, Reliable detection of Manipulation of the sample, Resources focused on Limited resolution
the least biased way more demanding analysis coding variants misses 98% of the genome specific areas of interest

* Coverage requirements vary depending on use-case, such as rarity of factor being measured.

Beyond the exome

Structural variation Mitochondrial variants

Exome: 2% Noncoding genome: 98% Repeat expansions Noncoding SNVs and indels

Copy number variants Regulatory elements

“ In our view, for deciphering the molecular causes of any genetic disorder,

”
we need the variants of the entire genome—especially when various
disorders exhibit overlapping symptoms and consequences.
Dr. Kamran Shazand, Director,
Shriners Genomics Institute

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Value of whole genomes

When NGS is more accessible, studies can cast a are frequently key biomarkers for disease. Unlike other
“wider net” to survey more genes. For example, labs methods, WGS robustly captures all common variant
that routinely do exome sequencing can now afford types.37–39,44 WGS captures copy number variants (CNVs)
to switch to WGS. WGS offers multiple advantages for with greater resolution than chromosomal microarrays
finding variants. WGS examines the entire genome and (CMAs).37,43,45,46 WGS also captures some variants in
has the capability to assess variants in both coding and exomes with greater accuracy than whole-exome
noncoding regions of the genome.37–43 Noncoding variants sequencing (WES).2,3,37,44,46–49

WGS provides the most comprehensive analysis of genomic variant types†

Capable Limited capabilities

Sanger Targeted NGS PCR FISH Karyotype CMA WES WGS

Singie nucieotide
variants (SNVs)37

Insertion-deletions
(Indels)

Copy numbers variants

(CNVs)37,50

Repeat
expansions39,40,51,52

Structural variants
(SVs)38,48

Mitochondria37

Paralogs41

† Variant detection may vary depending on the particular laboratory and performance limits of validated variant types. Detection of repeat expansions by PCR is typically limited
to single-gene analysis, compared to multigene capabilities of WGS. Improvements to SV callers and increased success with long-read whole-genome approaches suggest fully
capable SV insights from WGS. PCR, polymerase chain reaction; FISH, fluorescence in situ hybridization.

In the case of rare genetic disease research, many disease-causing variants identified with WGS would have been
missed by WES, including those caused by repeat expansions or mutations in noncoding regions.2,3,39,40,47,48,51–53 Because
WGS provides better coverage and higher yield across the genome, including GC-rich regions, the European Society
for Human Genetics (ESHG) guidelines recommend use of WGS, even if only the exome or specific genes are examined
bioinformatically.28

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Value of whole transcriptomes

Even whole genomes don’t tell the whole story. Certain variants, like gene fusions, may not be readily apparent in the
DNA sequence and can be overlooked by WGS.53 RNA sequencing (RNA-Seq) enables scientists to identify and confirm
the presence of novel gene fusions and alternate transcript isoforms. Because RNA is dynamic between cell types and
states, RNA-Seq also offers critical information about biological activity.

Whole-transcriptome sequencing (WTS), or total RNA-Seq, delivers a high-resolution, base-by-base view of coding
and multiple forms of noncoding RNA activity. This provides a comprehensive picture of gene expression across the
full transcriptome at a specific moment in time. With total RNA-Seq, the whole transcriptome—including both known
and unknown regions—is captured.54–56 The increased affordability of high-throughput NGS, paired with push-button
analysis tools to decipher richer data sets, make WTS accessible for more routine use.

RNA-Seq for cancer research

In cancer research, RNA-Seq is a critical tool for direct measurement of the functional consequences of mutations.
Despite the average cancer containing about 46 mutations, only five to eight are necessary for initiation.57 Genomic
profiling alone is insufficient to differentiate these driver mutations from passenger mutations, or those mutations that do
not influence cancer initiation or progression. Measurement of gene expression patterns and mutation consequences
using RNA-Seq enables large-scale, unbiased differentiation of factors crucial for cancer progression, resulting in
more thorough and accurate cancer modeling.58–62 Growing evidence demonstrates the value of combining whole-
genome and transcriptome sequencing (WGTS) to collect broader information from cancer samples.63

RNA-Seq for genetic disease research

RNA-Seq offers a complementary approach to GWAS for genetic disease research that increases diagnostic yield.64–66
Measuring expression abundance in specific tissues can reveal the functional impact of pathogenic mutations and identify
which genes mediate the genotype’s effect on phenotype.64–67 RNA-Seq can also validate computational predictions of
splicing and increase confidence in the reclassification of VUS.

Many studies have shown that GWAS risk variants co-localize with genes that regulate expression.67,68 These genes,
known as expression quantitative trait loci (eQTLs), suggest that regulation is an important molecular mechanism used
by GWAS risk variants, most of which lie in noncoding regions of the genome. Polygenic risk scores based on eQTLs are
helping scientists better understand complex phenotypes.69

Learn more

Whole-genome sequencing
Whole-transcriptome sequencing

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Multiomic studies for a wider perspective

Multidimensional insights with sequencing provide a single readout from multiple omes across DNA, RNA, and protein

Biology is multilayered and complex. The central dogma outlines the intertwined relationship between DNA, RNA, and
protein. Genetic variation at the DNA level can impact RNA expression or protein function in diverse and unpredictable
ways. Environmental factors can also alter regulatory pathways and cellular metabolism to affect biology and human health.

Multiomics provides a perspective to power discovery across multiple levels of biology. This biological analysis approach
combines genomic data with data from other modalities, measuring gene expression, gene activation, and protein levels
to enable a more comprehensive understanding of molecular changes contributing to normal development, cellular
response, and disease.

Multiple layers of information connect genotype to phenotype

Combining DNA, epigenetics, RNA, protein, or other molecular

measurements into a full cellular readout provides researchers
with novel scientific insights that cannot be found from single
omic methods alone.

Through the combined lens of multiomics, researchers can witness the complicated interplay between the molecules of
life. Integrating these complementary metrics into multiomic data sets brings a more comprehensive picture of cellular
phenotypes and helps pull more high-quality information from each sample. The transformational power in the latest
sequencing systems makes it easier to query multiple omes in parallel.

Bigger picture biology through multiomics

Multiomics goes beyond the genome to unlock deeper

biological insights. Using every piece of molecular data available
can accelerate biological discoveries and transform our
understanding of human health.

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Common multiomic combinations

Genomics + transcriptomes ties between gene regulation and gene expression,
instead of simply inferring those connections. Integrating
epigenetics and RNA-Seq can help researchers identify
GWAS have successfully identified genetic variants
candidate genes and understand the mechanisms
associated with complex diseases. The genotype offers
controlling interesting phenotypes. This holistic, non-
information on susceptibility to the disease; however,
biased multiomics approach can uncover new regulatory
determining the specific genes and pathways affected
elements for biomarkers and therapeutic targets.
by those variants is more difficult. Incorporating
RNA-Seq can help researchers annotate and prioritize
variants uncovered in GWAS for functional analysis to Transcriptomics + proteomics
understand mechanisms of disease. Gene expression
analysis informs if and when the genes of interest are RNA-Seq offers unparalleled discovery power to
down- or upregulated in the disease samples. This interrogate the transcriptome without prior knowledge.
multiomic approach to functional genomics can help Incorporating protein detection with RNA-Seq can tie
power drug target identification and biomarker discovery. new discoveries back to known canonical markers
and historical clinical outcomes. Antibodies tagged
Genomics + epigenetics with oligonucleotide barcodes enable analysis of cell
surface proteins with results read by sequencing, which
scales to a much higher number of parameters than
The majority of human variation identified by GWAS is in
flow cytometry or mass cytometry. Single-stranded
the noncoding regions of the genome, including introns,
nucleotide aptamers with selective protein binding
promoters, or enhancers. Comprehensive epigenetic
targets can be used in a similar way. Methods like
profiling can reveal patterns of gene regulation to help
cellular indexing of transcriptomes and epitopes by
find the function of those variants. NGS-based epigenetic
sequencing (CITE-Seq) combine single-cell RNA-Seq
techniques include chromatin immunoprecipitation
with cell surface protein analysis. Bulk epitope and
(ChIP-Seq), assay for transposase-accessible chromatin
nucleic acid sequencing (BEN-Seq) is performed at the
(ATAC-Seq), and chromosome conformation capture (HiC).
bulk level. Spatial transcriptomics interrogates RNA and
DNA methylation patterns are also conserved and can
proteins in context with tissue morphology.
represent a new class of biomarkers. Multiomic approaches
that combine methylation, or other epigenetic profiling,
with genetic information can connect functional layers to Genomics + proteomics
decipher complex pathways and disease mechanisms.
This multiomic approach directly connects genotype to
Epigenetics + transcriptomics phenotype for more informed research on disease and
therapeutics development. Linking genetic variation
to protein expression at the single-cell level can reveal
Epigenetics and transcriptomics offer complementary
the functional impact of somatic mutations on human
information to study the details of cellular differentiation
cancers to better understand tumor evolution and
and response. Combining epigenetic and RNA-Seq
disease progression.
methods allows researchers to directly measure the

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Next-level multiomics
The multiomic methods highlighted here represent a
holistic approach to understanding biology. Additional
discovery power comes from studies that measure
three or more modalities.70,71 Combining WGS, RNA-Seq,
and methylation sequencing is improving diagnostic
yield for neurodevelopmental disorders and other rare
diseases.70,72 Multiomic studies have also focused on
complex diseases. For example, integrated genome,
epigenome, and transcriptome data from population-
scale data sets helped identify genes and biological
mechanisms associated with blood pressure regulation.71

As the cost of sequencing continues to decrease and

the technology advances, multiomic assays will become
more comprehensive and better integrated. Labs will be
able to study more samples under different conditions
to reveal the dynamic properties of cells and systems.
Sequencing will support proteomic studies with oligo-
tagged antibodies or aptamers for hundreds to thousands
of markers. More assays will incorporate multimodal
measurements at higher resolution.

One of the biggest challenges for multiomic research

is how to integrate different molecular data sets
in a standardized way. Researchers need robust
computational strategies to extract biologically
meaningful insights from these vast amounts of data.
Sophisticated bioinformatics tools enable normalization
and integration of multimodal single-cell sequencing
experiments. Machine learning methods will also help
organize and filter complex multiomic data.

Learn more

Multiomics
CITE-Seq

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Higher analytical resolution to

decode complex systems
Transition to high-resolution approaches, including single-cell sequencing
and spatial analysis, for insights into complex tissues

High-resolution methods like single-cell sequencing and spatial analysis offer

another layer of fundamental detail to examine heterogeneity in complex
cell populations.73 These approaches have enabled researchers to look at
cancer, development, and infectious disease at the single-cell level within tissue
context.74–82 Studies have combined single-cell and spatial RNA-Seq to map
the architecture of skin cancer,83 characterize human intestinal development,84
and track COVID-19 pathology.74,75

Single-cell sequencing
Single-cell sequencing is a popular approach used to characterize hundreds
to millions of individual cells from a tissue. This method reveals cellular
heterogeneity and provides a more comprehensive understanding of tissue
composition. Significant advances in the area of single-cell characterization
include technologies for cell isolation and new methods and applications
for single-cell sequencing. These advances have stimulated the launch of
accessible commercial solutions for every step of the single-cell sequencing
workflow, from tissue preparation through data analysis.

Single-cell RNA sequencing (scRNA-Seq) has become a powerful tool in

immunology, cancer, and developmental biology studies. As part of the Human
Cell Atlas, a large-scale effort to map human development, researchers used
single-cell combinatorial indexing to profile the transcriptomes of ~2 million
cells derived from 61 embryos staged between 9.5 and 13.5 days of gestation
in a single experiment.85 Cell atlas studies often have implications in genetic
disease research. For cystic fibrosis, scRNA-Seq of human bronchial epithelial
cells helped uncover a rare cell type, pulmonary ionocytes, that accounts for
the majority of CFTR expression in the lungs.86

Cancer researchers use scRNA-Seq to better understand cancer biology,

as traditional bulk RNA-Seq does not address the heterogeneity within
and between tumors. scRNA-Seq has aided the development of targeted
therapy and immunotherapy treatments. Researchers often use scRNA-Seq
in conjunction with cell-surface protein expression and immune repertoire
sequencing to characterize the inflammatory response.

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Spatial sequencing
Typical NGS methods using dissociated samples can lose key spatial information present in vivo. Traditionally,
immunohistochemistry and in situ hybridization have been the tools of choice to reveal spatial gene expression in
tissue sections. But the throughput of these procedures is limited, analyzing only a few genes at a time. By combining
high-throughput imaging and sequencing technologies, spatial RNA-Seq provides a previously inaccessible view of
the full transcriptome in morphological context. Spatial RNA-Seq methods that retain the precise location of biological
molecules in tissue samples can further our understanding of mechanisms in health and disease.

Pushing the boundaries

As single-cell and spatial technologies advance,
researchers are increasing the scale, scope, and
dimensions of their experiments. One consortium, the
BRAIN Initiative Cell Census Network, coordinated
large-scale multiomic analyses from multiple labs of
the mammalian primary motor cortex with single-cell
and spatial resolution.87 Cross-modal computational
analysis integrated data with single-cell transcriptomes,
chromatin accessibility, DNA methylation, and
phenotypic properties, across species, to establish
a framework for neuron organization.87

A team of researchers at the Howard Hughes Medical

Institute performed genome-scale Perturb-Seq (a
CRISPR-based functional genomics screen with scRNA-
Seq readouts) across 2.5 million human cells.88 From the
single-cell transcriptional phenotypes, the team was
able to dissect complex cellular pathways and develop a
map of gene and cellular function.88 The researchers note
ways to increase the power of future studies, including
greater numbers of cells or sequencing depth, full-length
RNA-Seq or protein-level readouts, and sampling a wider
range of time points or cell types.88

Learn more

Single-cell RNA sequencing

Spatial transcriptomics

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Deeper studies to find rare events

NGS assays can be designed to either target a large However, recent improvements in NGS instrumentation
number of genes with low sequencing depth (more provide options for sequencing samples at extremely
comprehensive, less sensitive) or a relatively small high depth of coverage for large portions of the genome
number of genes with higher sequencing depth in a single sample. This enables analysis of hundreds
(less comprehensive, more sensitive). In cases of of genes with the sequencing depth required for ctDNA
heterogenous samples such as blood or tumors, high analysis. Furthermore, these technologies enable
sequencing depth is necessary to provide the sensitivity hypothesis-free interrogation of biofluid analytes,
required to detect low-abundance variants accurately. providing discovery power to assay genes and pathways
Scientists can take advantage of the latest advancements that were not considered prior to experimental design.
in high-throughput sequencing to perform deeper
sequencing experiments, more rapidly, at production-scale. Liquid biopsy combined with comprehensive
genomic profiling (CGP) can identify tumor-specific

Liquid biopsy mutations in patient samples93 and provide information

of cancer type, stage, and vascularization.94–97 Recent
studies that performed liquid biopsy paired with
Liquid biopsy detects and characterizes various tumor- corresponding tissue biopsy from tumor samples have
derived biomarkers present in the blood of an individual demonstrated that, when comprehensive assays are
with cancer. These biomarkers are absent in healthy used, ctDNA analysis detected a significant number of
individuals and patients who are cancer free. Liquid guideline-recommended biomarkers and resistance
biopsy is a noninvasive approach that offers a potential alterations not found in matched tissue biopsies.98,99
alternative to invasive tissue biopsies to detect targetable Furthermore, ctDNA sequencing identifies variants in
oncogenic drivers and resistance mutations.89 the tumor from which it originates, including both driver
and passenger mutations, respectively.90 Given the rapid
Tumors release circulating tumor DNA (ctDNA) into turnover of cfDNA and ctDNA in the bloodstream, liquid
the bloodstream through various cellular mechanisms, biopsy can be a powerful tool for cancer research to
including apoptosis, necrosis, phagocytosis, and active assay tumor burden in real time and monitor response to
secretion.90 Even so, ctDNA represents a small fraction therapy.100 Studies also demonstrate how liquid biopsy-
of total cell-free DNA (cfDNA) in the blood,91 the majority based detection of molecular residual disease (MRD) can
of which is released by erythrocytes, leukocytes, and predict recurrence with high sensitivity and specificity,
endothelial cells.90,92 The rarity of ctDNA combined with with lead times that precede standard imaging.101–103
the fact that different cancer types at different stages
shed ctDNA at different rates complicates analysis.

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Next-level liquid biopsy

“
Circulating analytes include more than just ctDNA and can
apply to more than just cancer. As technology continues to
We've known for many
advance, there is the potential to apply multiomic approaches years that there is tumor-
that integrate analyses of the genome, transcriptome,
epigenome, proteome, and microbiome to liquid biopsy.104 related material in the
For example, cell-free RNA (cfRNA) is indicative of early-
stage cancer and other conditions like preeclampsia.105–111
bloodstream, we just
Methylation patterns in ctDNA can also recapitulate the didn't have the
abnormal methylation patterns that are a hallmark of many

”
cancers.112–116 The ability to combine multiomics with liquid technologies to detect
biopsy will provide unique discovery power for deeper insights
and comprehensive answers to the mechanisms of cancer.
it for it to be meaningful.
Minetta C. Liu, MD, Mayo Clinic

Genome-wide tumor–
normal sequencing
Deep sequencing of tumor exomes or genomes can provide
rapid and accurate CGP.117,118 Through tumor–normal WGS,
researchers can compare tumor mutations to a matched
normal sample. Tumor–normal comparisons are crucial for
identifying the somatic variants that act as driver mutations
in cancer progression. Tumor–normal sequencing typically
requires a minimum of 80× sequencing depth.

WGS for cancer studies offers base-pair resolution of the

unique mutations present in a tumor. Cancer genomes typically
contain unpredictable numbers of point mutations, gene
fusions, and other aberrations. As a hypothesis-free approach,
cancer WGS offers unbiased insights into a variety of genomic
aberrations, including emerging biomarkers.119,120 It enables
discovery of novel cancer-associated variants, including
single nucleotide variants (SNVs), copy number changes,
indels, and structural variants.

Learn more

Circulating tumor DNA sequencing

Comprehensive genomic profiling
Cancer whole-genome sequencing

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How the NovaSeq X Series accelerates

genomic discovery
The potential of broader, deeper sequencing can now be realized, thanks to the latest advancements in Illumina NGS
systems. The NovaSeq X and NovaSeq X Plus Sequencing Systems are built with breakthrough technological innovations
to transform the economics of high-throughput sequencing.

The NovaSeq X Series is powered by XLEAP-SBS™ chemistry—a faster, higher fidelity, and more robust version of proven
Illumina sequencing by synthesis (SBS) chemistry. XLEAP-SBS reagents are optimized for performance and speed, to
maximize throughput without sacrificing data quality. Ultrahigh-resolution optics were developed to match the capabilities
of the chemistry. Ultrahigh-density patterned flow cells with tens of billions of nanowells enable up to 16 Tb output (or up
to 52 billion single reads) per dual flow cell run on the NovaSeq X Plus System.

In addition to advancements in chemistry and optics, the NovaSeq X Series is built with DRAGEN™ hardware
onboard the instrument to accelerate and streamline secondary analysis and compress data by 80% without loss.
The NovaSeq X Series also sets a new standard in operational simplicity with a software ecosystem built specifically
to support the Illumina NGS workflow.

NovaSeq X Series specification sheet

High-accuracy NGS data with the NovaSeq X Series application note

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Enabling broader, deeper sequencing

The NovaSeq X Series will allow scientists to perform broader, more ambitious studies at a new level of scale.
Researchers can increase the scope of their studies without increasing their budgets. Lower costs and streamlined
informatics enable more analyses per sample and more data per analysis.

With the ability to sequence three-fold more samples, population genomics researchers can add statistical power with
larger cohorts—up to tens of thousands of genomes per year. Cancer or genetic disease researchers can sequence
three-fold deeper to detect low-frequency signals and rare variants. Other researchers can easily adopt multiomic
interrogation techniques with more omes and multiple simultaneous analyses. The comprehensive, high-resolution view
of biological systems will expand the discovery power of genomic scientists.

EXAMPLE PROJECT
Single-cell gene expression study with fixed $50K reagent budget‡

450

400
~2×
350
Single-cell samples per $50K reagent budget

300

250

200

150

100

50

25B 10B 1.5B S4 S2 S1 SP NextSeqTM

NovaSeq X Series NovaSeq 6000 System 2000 P3

‡ A nalysis based on 20,000 reads per cell and 10,000 cells per sample.
All pricing is in USD, based on United States list prices.

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Setting a new bar in every sequencing metric

NovaSeq 6000 System NovaSeq X Series

65 Gb–6 Tb Output 165 Gb–16 Tb

~2.5×
Throughput
800M–20B Read number 1.68B–52B

~1 genome/hr Genomes per hour

~2×
~2.5 genomes/hr

Faster

2–48 genomes Genomes per run 4–128 genomes

The NovaSeq X Series more than doubles the throughput of the NovaSeq 6000 System, while taking up the same lab footprint—less than
one square meter of floor space. NovaSeq X and NovaSeq X Plus Systems provide massive throughput and productivity gains with the
ability to sequence ~2.5 genomes/hour and up to 128 genomes (30× coverage) per run and 96 human genomes (40× coverage) per dual
25B flow cell run.

$183
SHRINKING THE GAP
The NovaSeq X Series decreases cost/Gb
WGS and WES by up to 50% and shrinks the cost gap between
WGS and WES by 2.5× to a difference of only
$183 (USD).

Sequencing cost

Library prep cost

WGS WES

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Managing big experiments and big data

with the NovaSeq X Series
Day-to-day operational simplicity

Illumina believes that genomics should be available to the many, not the few. We have an obligation to make our
technology as affordable and accessible as possible while setting the highest standard for data quality and security.
To democratize access to the power of the genome, the NovaSeq X Series was designed to be especially easy to
operate, from run planning through analysis. The simple workflow requires fewer touchpoints and fewer steps than the
NovaSeq 6000 System, reducing the learning curve and the chance of user error.

Expanded global access to NGS

The stability of XLEAP-SBS reagents allows for

ambient temperature shipping, without ice packs or
dry ice. Eliminating the need for cold-chain transport
expands access to NGS globally, especially for hard-
to-reach geographies. This key innovation will speed
adoption of high-throughput sequencing for more
diverse populations.

Flexible scalability

The choice of three flow cell types with individually

addressable lanes offers flexible scalability. The
NovaSeq X 10B and 25B flow cells enable larger
projects and deeper sequencing. The NovaSeq X
1.5B flow cell eases the transition from benchtop to
higher-throughput sequencing. The fast turnaround
times for the 1.5B flow cell are ideal for small batch
sizes and data-lite applications.

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Designed with the user in mind

4K ultra System prompts

high-definition to guide user
touch screen along workflow

Adjustable screen height with position memory

Easy-to-handle flow cell with illuminated flow cell stage

Less time and effort with established

workflow applications

Push-to-access hidden keyboard

Reduced chance of error with intuitive
user interface with clear visualization
of run status and lane-level metrics,
and immediate view of secondary
analysis reports and QC metrics

> 90% reduction in reagent packaging

waste/weight and 50% reduction in
Light-guided consumable loading plastic for easier handling and reduced
disposal costs§

§ In comparison to the NovaSeq 6000 System.

Assisted open doors and waist-height reagent drawers

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“
Integrated, cohesive bioinformatics ecosystem The physical reduction in kit
The NovaSeq X Series streamlines genomic data management size has greatly decreased
with onboard secondary analysis and integration with a our cold storage needs
comprehensive suite of bioinformatics solutions. The
Illumina Connected Software suite includes some of the in lab and elimination of
fastest, most accurate,94 and advanced solutions for data
analysis, interpretation, and aggregation. With flexible local dry ice shipping has made

”
and cloud-based options for lab operations, run planning, unpacking larger orders
and data analysis, the NovaSeq X Series enables users to
run high-throughput sequencing without creating a much easier.
bioinformatics bottleneck.
Eric Chow, Assistant Adjunct Professor
and Director, University of California
Plug-and-play onboard DRAGEN workflows enable variant San Francisco (UCSF) Center for Advanced
calling directly on the system. Up to four DRAGEN applications Technology
can run in parallel, speeding multiomic analysis. Integrated
DRAGEN ORA (original read archive) data compression reduces
data storage needs five-fold. The savings on server, licenses,

“
and cloud storage can total more than $1 million (USD) over
five years of ownership.** Access to a broader menu via the
cloud with highly configurable pipelines will accelerate larger, One of the big benefits
more data-intensive sequencing projects.
I can see in the new
DRAGEN onboard analysis
NovaSeq X Series software ecosystem technical note on the NovaSeq X Plus
System is the considerable
reduction in file size now
** Assuming FASTQ files are archived 30 days after cloud upload
and throughput of 10,000 WGS/year for the NovaSeq X System and being compressed to
20,000 WGS/year for the NovaSeq X Plus System.
approximately one-fifth of
what would be a traditional
file, which allows us to

”
run more projects at
population scale.
Lachlan Morrison, Senior Technical Officer
Biomolecular Resource Facility (BRF),
Australian National University (ANU)

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Example NovaSeq X Series workflows

WGS

Prepare libaries Sequence Analyze data

Illumina DNA
NovaSeq X 1.5B flow cell DRAGEN Germline
PCR-Free Prep
~4 samples per flow cell (onboard or in the cloud)
25–300 ng DNA input
NovaSeq X 10B flow cell
~1.5-hour workflow
~24 samples per flow cell

NovaSeq X 25B flow cell

~64 samples per flow cell

400M reads per sample

2 × 150 bp read length

Illumina Complete Long

Read Prep, Human NovaSeq X 10B flow cell Illumina Complete
4 samples per flow cell Long Read WGS App
50 ng DNA input (in the cloud)

NovaSeq X 25B flow cell

~1-day workflow
10–11 samples per flow cell

750 Gb per sample

2 × 150 bp read length

WES

Prepare libaries Sequence Analyze data

Illumina DNA Prep with

NovaSeq X 1.5B flow cell DRAGEN Enrichment
Exome 2.0 Plus Enrichment
~41 samples per flow cell (onboard or in the cloud)
50–1000 ng DNA input
NovaSeq X 10B flow cell
~6.5-hour workflow
~250 samples per flow cell

NovaSeq X 25B flow cell

~750 samples per flow cell

8 Gb per sample
2 × 100 bp read length

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WTS

Prepare libaries Sequence Analyze data

Illumina Stranded Total

NovaSeq X 1.5B flow cell DRAGEN RNA
RNA Prep
~32 samples per flow cell (onboard or in the cloud)
1–1000 ng high-quality
DNA input NovaSeq X 10B flow cell
~200 samples per flow cell
~7-hour workflow
NovaSeq X 25B flow cell
~520 samples per flow cell

50M reads per sample

2 × 100 bp read length

Illumina Stranded
NovaSeq X 1.5B flow cell DRAGEN RNA
mRNA Prep
~64 samples per flow cell (onboard or in the cloud)
25–1000 ng high-quality
RNA input NovaSeq X 10B flow cell
~400 samples per flow cell
~7-hour workflow
NovaSeq X 25B flow cell
~1040 samples per flow cell

25M reads per sample

2 × 100 bp read length

Illumina RNA Prep

NovaSeq X 1.5B flow cell DRAGEN RNA
with Enrichment
~64 samples per flow cell (onboard or in the cloud)
20 ng FFPE RNA input
NovaSeq X 10B flow cell
~9-hour workflow ~400 samples per flow cell

NovaSeq X 25B flow cell

~1040 samples per flow cell

25M reads per sample

2 × 100 bp read length

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Unimaginable experiments,
made possible
The NovaSeq X Series is driven by your vision to help moonshot
projects become a reality. Increase statistical power with
broader study designs and larger sample cohorts. Maximize
read numbers and increase sequencing depth for a higher
resolution view that can detect low-frequency signals and
“ [The NovaSeq X Series]
now allows us to provide
much larger scale projects
variants. Answer the most complex questions in human for both human health and
genomics, with larger sample cohorts, deeper sequencing,
and more data-intensive methods—from whole-genome
nonhuman scenarios in
sequencing to multiomics. the research world, and
Genomics visionaries are ready to usher in the Genome Era, to be able to translate
that into more meaningful

”
and the NovaSeq X Series can help make it happen.

outcomes.
Joe Baini, CEO, Australian Genome Research
Facility (AGRF)

NovaSeq X and NovaSeq X Plus Sequencing Systems

NovaSeq X Series virtual tour

NovaSeq X Series overview video

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Abbreviations
ATAC-Seq: assay for transposase-accessible chromatin Indel: insertion–deletion

BEN-Seq: bulk epitope and nucleic acid sequencing MRD: molecular residual disease

CGP: comprehensive genomic profiling NGS: next-generation sequencing

cfDNA: cell-free DNA NICU: neonatal intensive care unit

ChIP-Seq: chromatin immunoprecipitation sequencing RNA-Seq: RNA sequencing

CITE-Seq: cellular indexing of transcriptomes and SBS: sequencing by synthesis

epitopes by sequencing
scRNA-Seq: single-cell RNA sequencing
CMA: chromosomal microarray
SNV: single nucleotide variant
CNV: copy number variant
SV: structural variant
CRISPR: clustered regularly interspaced short palindromic
repeats Tb: terabase

ctDNA: circulating tumor DNA VUS: variant of unknown significance

eQTL: expression quantitative trait loci WES: whole-exome sequencing

FFPE: formalin-fixed, paraffin-embedded WGS: whole-genome sequencing

Gb: gigabase WGTS: combined whole-genome and transcriptome

sequencing
GWAS: genome-wide association studies
WTS: whole-transcriptome sequencing
HiC: chromosome conformation capture

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