Methods Guide for Cancer Research

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

 Informatics
Informatics tools enable
critical insights. Illumina
recommends using the
DRAGEN™ pipelines on
BaseSpace™ Sequence
Hub, a DRAGEN Server,
Sequencing or onboard the NextSeq™
1000 and NextSeq 2000
Illumina offers a Systems for push-button
comprehensive portfolio analysis.
of next-generation
sequencing (NGS)
instruments that are
accessible and scalable
Library prep for virtually every lab.

Illumina library prep

kits enable a broad
range of methods and
applications.

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Illumina NGS for cancer researchers
Take your research to the next level
The introduction of NGS has transformed the way scientists study biological systems. With clear benefits, such as
reduced time and cost compared to legacy technologies and the capacity to scale from small studies to population-
level throughput, NGS opens the door to a broad range of research capabilities.

Deep sequencing provides the sensitivity to detect low-frequency molecular events, uncovering the somatic variants
behind the tumor. These studies can be instrumental to understanding how changes at the DNA, RNA, protein, or
cellular level contribute to tumor initiation, growth, and metastasis. Single-cell techniques allow researchers to go
beyond bulk measurements to understand how different cells within the tumor microenvironment promote or inhibit
cancer progression.

NGS has already expanded our knowledge of cancer as a disease of the genome. More recent techniques are poised
to shed even more light within oncology. With the advantages of speed, sensitivity, and scale, NGS can take your
cancer research to the next level.

Overview of cancer research workflows

Simple, comprehensive workflows for a broad range of cancer research applications

Step 1 Library prep

Illumina offers a comprehensive NGS sequencing workflow, from library prep to final data analysis. Library prep kits
are available for a range of applications, including exome, transcriptome, and whole-genome sequencing (WGS).

Step 2 Sequencing

Illumina offers a full portfolio of sequencing platforms, from the benchtop iSeq™ 100 System to the production-scale
NovaSeq™ 6000 System, that deliver the right level of speed, capacity, and cost for various laboratory or sequencing
center requirements. Illumina has pioneered major advances in sequencing simplicity, flexibility, and platform
performance. Experiments that once required complex workflows now use simple push-button workflows.

Step 3 Data analysis

Illumina cancer research workflows using the DRAGEN pipelines provide user-friendly data analysis tools that are
easily accessible through the web with BaseSpace Sequence Hub or onboard the NextSeq 1000 and NextSeq 2000
Systems for push-button analysis. Additionally, BaseSpace Sequence Hub enables integration of many workflow
steps, including library prep planning and sample management, run set-up and chemistry validation, and real-time
data monitoring and data transfer to computing and analysis modules.

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Methods

Mutational profiling for neoantigen prediction

Identify tumor-specific peptides that may be capable of
inducing an immune response.

Single-cell sequencing to understand the tumor

evolution and the microenvironment
Understand the molecular drivers of cancer at the DNA,
RNA, epigenetic, and protein level at single-cell resolution.

WGS to identify a comprehensive list of genetic

drivers of cancer progression
Identify cancer-driving genetic events beyond protein-
coding variants.

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Method 1: Mutational profiling
Identify tumor-specific peptides that may be capable of inducing an immune response.
Key benefits over traditional methods such as qPCR, Sanger sequencing, and small NGS panels

• Obtain a comprehensive view of the mutational profile of a tumor sample

• Detect highly expressed somatic variants in one workflow
• Identify specific neoantigens that may drive tumor immunity

Extract Prepare Variant calling and Neoantigen

Specimens Sequence
nucleic acids libraries expression analysis prediction

DRAGEN

Fresh frozen QIAGEN AllPrep Tumor and normal NovaSeq DRAGEN Platform Multiple
(FF) or formailn- DNA and RNA DNA: 6000 System methods
fixed paraffin- FFPE Kit Illumina DNA Prep DNA: Somatic Pipeline
embedded with Enrichment with NextSeq 1000
(FFPE) exome oligos and RNA: RNA-seq Pipeline
tumor samples NextSeq 2000
+ normal Tumor RNA: Illumina Systems
RNA Prep with
Enrichment

Training Service contracts Professional services

Customer site training Tiered service plans Proof-of-Concept Service

Illumina DNA Prep with Enrichment and NovaSeq 6000 Plans Standard application functional testing
Illumina RNA Prep with Enrichment NextSeq 2000 Plans with your samples

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Potential research applications of neoantigen prediction

Identify responders and nonresponders of immune checkpoint blockers

Immune checkpoint blockade has been shown to induce durable responses in
patients across multiple types of cancer. Unfortunately, only a fraction of patients
respond to this type of therapy. Multiple biomarkers have been proposed to stratify
responders from nonresponders, though none have been shown to be completely
effective. As neoantigens play a direct role in inducing antitumor immunity, accurate
prediction of these molecules may be the most effective biomarker for predicting
response to emerging immunotherapies.

Drive personalized vaccine development

Recent studies have illustrated the promise of neoantigen-based vaccines.
Unfortunately, while clinical trials are ongoing in several different tumor types, multiple
challenges remain. The first step in the development of a vaccine is the accurate
identification of putative neoantigens. Optimizing this technique is key to developing
personalized vaccines and realizing the hope of this promising new therapeutic
approach.

Identify tumor-reactive T-cells

Another emerging therapeutic approach across multiple types of cancers is adoptive
cell transfer of neoantigen-targeting tumor-infiltrating lymphocytes (TILs) into patients.
The key to this approach is the identification of the appropriate neoantigen-reactive
T-cells that can mediate a durable antitumor immune response. The first step to
identifying this possibly disease-altering population of cells is the identification of the
neoantigens that can drive this response.

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 Step 1 Recommended extraction methods

There are several DNA and RNA extraction methods that can be used with fresh frozen (FF) samples. For formalin-
fixed, paraffin-embedded (FFPE) samples, Illumina recommends the QIAGEN AllPrep DNA/RNA FFPE Kit. In Illumina
internal studies, this kit extracts DNA and RNA with high-quantity and -quality in the same workflow.

Step 2 Recommended library prep methods

Product Illumina DNA Prep with Enrichment Illumina RNA Prep with Enrichment
Most important to me Minimal input requirements, fewest number of steps, and high uniformity of coverage
Minimum input requirements 10 ng 10 ng
Total library prep time ~6.5 h 9.5 ha
Sample type FF and FFPE compatible FF and FFPE compatible
Sample index sets 384 unique dual indexes 384 indexes availableb
a. Turnaround time for 24 samples with 3-plex enrichment
b. Up to 192 unique dual indexes are currently supported; 384 indexes will be available later in 2020.

Step 3 Recommended sequencing systems

NextSeq 1000 and

Product NovaSeq 6000 System
NextSeq 2000 Systems
Most important to me Instrument affordability and desktop footprint Low cost/sample
4–5 (SP flow cell)
2–3 (P2 flow cell) 8–10 (S1 flow cell)
Samples/flow cella
6–10 (P3 flow cell) 20–26 (S2 flow cell)
52-64 (S4 flow cell)
Recommended read length 2 × 101 bp 2 × 101 bp
a. Illumina recommends 200M reads for tumor DNA, 75M reads for normal DNA, and 30M reads for tumor RNA.

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 Step 4 Secondary analysis

Illumina recommends using the DRAGEN pipelines on BaseSpace Sequence Hub, a DRAGEN Server, or onboard
the NextSeq 1000 and NextSeq 2000 Systems to obtain somatic variant calls and gene expression data. When
using BaseSpace Sequence Hub, users can monitor runs in real time while securely streaming data directly from the
instruments into the ecosystem for push-button analysis.

Pipeline Application Input

Rapid alignment of reads from targeted
DRAGEN Enrichment Pipeline Tumor DNA FASTQ
enrichment experiments
Somatic variant detection in tumor samples; Tumor DNA FASTQ
DRAGEN Somatic Pipeline
includes tumor-only and tumor-normal modes Normal DNA FASTQ
Rapid alignment, splice junction mapping and
DRAGEN RNA Pipeline Tumor RNA FASTQ
quantification, and fusion detection
DRAGEN RNA Differential Pairwise differential gene expression analysis.
Tumor RNA FASTQ
Expression Available on BaseSpace Sequence Hub only.

Step 5 Tertiary analysis

Before neoantigen prediction, users must use the somatic variant calls and gene expression data for human
leukocyte antigen (HLA) typing, peptide processing, and major histocompatibility complex (MHC)-binding prediction.
There are many options for the user in each of these steps. For a review of these options and instructions for use,
read “Best practices for bioinformatic characterization of neoantigens for clinical utility” at https://genomemedicine.
biomedcentral.com/articles/10.1186/s13073-019-0666-2.

Analysis step Description

HLA typing The use of exome data to determine a patient’s HLA alleles and corresponding MHC complexes
Peptide processing A generation of small peptides using a sliding window that is applied to the mutant protein sequence
MHC-binding prediction An analysis of peptide affinity toward the MHC complexes identified
Neoantigen prioritization The prioritization of selected peptides based on variant frequency, binding affinity, and other factors

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Method 2: Single-cell sequencing
Understand the molecular drivers of cancer at single-cell resolution.
Key benefits over bulk sequencing

• Detect functional cell populations in the tumor microenvironment

• Uncover the B-cell receptor (BCR) and T-cell receptor (TCR) sequences of individual tumor lymphocytes
• Understand the effects of epigenetic heterogeneity in cancer progression
• Construct the evolution of somatic variants from tumor samples

Analysis,
Tissue Single-cell isolation
Specimens Sequencing visualization, and
preparation and library prep
interpretation

Easy: Fresh Mechanical Single-cell isolation: NovaSeq Multiple

Droplet 6000 System methods
Enzymatic
Difficult: FF and FFPE fluidics platforms
Combinatorial NextSeq 1000 and
Library prep: multiple NextSeq 2000
solutions Systems

Training Service contracts Professional services

Customer site training Tiered service plans N/A

N/A NovaSeq 6000 Plans
NextSeq 2000 Plans

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Potential applications of single-cell cancer studies

Identify specific cell and immune states associated with immunotherapy

response
Immune checkpoint blockade has been shown to induce durable responses in patients
across multiple types of cancer. Unfortunately, only a fraction of patients respond to
this type of therapy. The antitumor immune response is a complex mechanism involving
multiple cell types. A deeper understanding of each of these cell types in patients
treated with checkpoint blockers may yield new biomarkers differentiating responders
from nonresponders.

Identify specific tumor-reactive T-cell profiles

Immunosurveillance is mediated through the CD8+ T-cell recognition of neoantigens
presented on the surface of tumor cells. This recognition is mediated by T-cell
receptors whose diversity, originating though V(D)J recombination, is a hallmark of
the adaptive immune response. Using single-cell RNA-Seq techniques techniques to
elucidate specific TCR sequences that drive neoantigen recognition, along with clonal
expansion and TCR diversity, may help drive an understanding of the central players in
immunosurveillance and immunotherapy.

Elucidate the role of epigenetics in drug resistance

While the key role of epigenetics on tumor progression has been known for quite some
time, cell-to-cell heterogeneity of the epigenome may have profound effects on tumor
progression and most notably, drug resistance. Drug-resistant tumor clones have been
known to co-opt epigenetic pathways to suppress gene expression that drives drug
sensitivity. Single-cell assays for transposase-accessible chromatin using sequencing
(ATAC-Seq) can be used to detect this kind of heterogeneity to drive a deeper
understanding of drug resistance mechanisms.

Uncover the genetic drivers of relapse

One barrier to effective cancer treatment paradigms is the failure to prevent relapse
after the initial treatment, even after complete remission. Using targeted single-cell DNA
sequencing (scDNA-Seq) of clones present in both pretreatment and relapsed sample
settings, the molecular hallmarks of relapse can be elucidated.

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Single-cell RNA Sequencing (scRNA-Seq)
This guide presents an overview of scRNA-Seq in cancer research. For a complete overview of single-cell sequencing
techniques, download the single-cell eBook https://www.illumina.com/single-cell-rna-sequencing.

Step 1 Single-cell tissue preparation

• Tissue preparation from solid tumors can be difficult, as the goal is to obtain viable, individualized fresh cells
• For RNA-Seq, this difficulty can translate into artifacts, as tissue manipulation can lead to transcriptional changes
• For cancer research methods, isolation of nuclei only has been employed
• In general, after dissociation, fluorescence-activated cell sorting (FACS) to isolate single cells has been used,
especially with tumor samples

Step 2 Single-cell isolation

Advances in microfluidics technologies have enabled high-throughput single-cell profiling, where researchers can
cost-effectively examine hundreds to tens of thousands of cells per experiment. This guide only covers these high-
throughput technologies; for low-throughput options, consult the single-cell eBook.

Method Example commercial offering Advantages Disadvantages

Unique molecular identifiers (UMIs)
• 10x Genomics Chromium Controller and cell barcodes enable cell- and
Requires specialized equipment
Droplet fluidics platforms • Mission Bio Tapestri Platform gene-specific identification, low
and poses technical challenges
• Bio-Rad ddSEQ Single-Cell Isolator cost per cell, and extensive support
from commercial providers
• BD Rhapsody Single-Cell Supports imaging and short-term Uses emerging technology
Microwells Analysis System culture of cells; ideal for and offers limited commercial
• Celsee Genesis System adherent cells solutions

Step 3 Library prep

Single-cell method Description Example commercial offering
• 10x Genomics Chromium Single Cell Gene
Capture of mRNA by 3‘ polyadenylation (poly(A)) tails
Expression Solution (3’ WTA)
RNA-Seq enables sequencing of the coding transcriptome with
• Bio-Rad SureCell™ WTA 3’ Library Prep Kit for the
strand-specific information
ddSEQ System
IR-Seq is a targeted sequencing method used to
Immune-repertoire-seq (IR- • 10x Genomics Chromium Single Cell Immune
quantify the composition of B- or T-cell antigen
Seq) Profiling Solution
receptor repertoires
Understand genome-wide chromatin accessibility • 10x Genomics Chromium Single Cell ATAC Solution
ATAC-Seq
within each cell • Bio-Rad SureCell ATAC-Seq Library Prep Kit
Simultaneously detect single nucleotide variants and
Targeted DNA-Seq • Mission Bio Tapestri Platform
copy number variants from the same cells

An emerging single-cell method, called RNA expression and protein sequencing (REAP-Seq) or cellular indexing of
transcriptomes and epitopes by sequencing (CITE-Seq), uses antibodies conjugated to DNA barcodes to measure
protein levels at the single-cell level. For a deeper insight into this method, consult a recent publication at
www.nature.com/articles/nbt.3973.

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 Step 4 Sequencing

The number of cells being evaluated varies depending on the experimental study design. For illustrative purposes,
this guide uses 5000 cells per experiment.

Single-cell method Reads per nucleus

RNA-Seq 20K
IR-Seq 5K
ATAC-Seq 50K
Targeted DNA-Seq Depends on the panel size

NextSeq 1000 and

Product NovaSeq 6000 System
NextSeq 2000 Systems
Most important to me Instrument affordability and desktop footprint Low cost/sample
No. of experiments/flow cell
6-8 (SP flow cell)
4 (P2 Flow Cell) 13-16 (S1 flow cell)
RNA-Seq
12 (P3 Flow Cell) 33-41 (S2 flow cell)
80-100 (S4 flow cell)
26-32 (SP flow cell)
16 (P2 flow cell) 52-64 (S1 flow cell)
IR-Seq
48 (P3 flow cell) 132-164 (S2 flow cell)
320-400 (S4 flow cell)
2–3 (SP flow cell)
1 (P2 flow cell) 5–6 (S1 flow cell)
ATAC-Seq
4 (P3 flow cell) 13–16 (S2 flow cell)
32–40 (S4 flow cell)
7–9 (SP flow cell)
4 (P2 flow cell) 15–18 (S1 flow cell)
DNA-Seq a
14 (P3 flow cell) 38–48 (S2 flow cell)
94–117 (S4 flow cell)
a. Using the Mission Bio Tapestri Single-Cell DNA ALL Panel as an example (includes 107 genes with 283 amplicons)

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 Step 5 Single-cell analysis and visualization

After the single-cell sequencing run is complete, downstream analysis can be performed. Generally, the analysis
pipeline for single-cell sequencing experiments involves three phases: primary analysis (base calling), secondary
analysis (demultiplexing, alignment, and genetic characterization), and tertiary analysis (data visualization and
interpretation). There is no one correct way to carry out an analysis pipeline for single-cell sequencing experiments.
Many approaches and software programs are available for each step in the pipeline. The research objective, single-
cell isolation platform, and general lab considerations will largely determine the specific pipeline used. For more
information around analysis, consult the single-cell eBook.

Primary analysis: 1 File conversion

File conversion Raw data files (BCL) are converted to FASTQ
.bcl file .fastq file format for downstream analysis.

2 Demultiplexing
Secondary analysis:
If the samples were multiplexed for sequencing,
Demultiplexing
resulting read files are demultiplexed prior to
(if applicable)
downstream analyses.

3 Sequence aignment
Sequence alignment The reads are mapped and aligned to a
reference genome.

4 Data set QC and filtering

Noncellular barcodes and low-quality cells are
Data set QC and filtering
excluded from downstream analysis by various
metrics

5 Genetic characterization
Initial genetic Quality-controlled data sets are analyzed for
characterization genomic variants, gene expression, chromatin
accessibility, protein expression, etc.

6 Data visualization
Tertiary analysis: Multidimensional data plots enable the
Data visualization and interpretation clustering of cells and identification of
subpopulations.

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Method 3: WGS
Identify cancer-driving genetic events beyond protein-coding variants.
Key benefits over targeted or exome sequencing

• Obtain a complete view of the mutational profile of a tumor sample, including noncoding and structural variants
• Identify mutational signatures linked to cancer progression
• Differentiate driver from passenger mutations

RNA Expression
Specimens Library prep Sequencing
extraction analysis

DRAGEN

FF and FFPE QIAGEN AllPrep DNA Illumina DNA Prep NovaSeq 6000 DRAGEN Bio-IT
Kit System Platform

Training Service contracts Professional services

Customer site training Tiered service plans Proof-of-Concept Service

Illumina DNA Prep NovaSeq 6000 Plans Standard application functional testing
with your samples

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Potential applications of WGS cancer studies

Link somatic mutational signatures with cancer progression

Multiple processes can lead to a buildup of somatic mutations in cancer genomes.
Each process may lead to a distinct mutation signature. WGS is the most ideal way
to identify these mutational signatures. Understanding these signatures can lead to a
better understanding of the underlying mutational processes leading to novel insights
into cancer progression and possible therapeutic targets.

Elucidate the role of noncoding mutations in cancer

The link between somatic mutations and cancer progression has largely focused
on the role of coding mutations on cellular processes. While most noncoding
mutations may simply be passenger mutations, some may be in regulatory elements
that themselves exert effects on cancer progression. WGS can identify coding and
noncoding somatic mutations and, using computational techniques, classify the
importance of these mutations on cancer progression.

Identify the role of viral integration in cancer

The driving role of genomic viral integration in cancer progression has been known
for many years. Unfortunately, systematic studies understanding the role of viral
integration are lacking. WGS can be used to identify viral integrations across the
genome. This hypothesis-free approach can elucidate associations between certain
viruses and cancer progression, beyond what is known today.

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 Step 1 Recommended extraction methods

There are several DNA extraction methods that can be used with FF samples. For FFPE samples, Illumina recommends
the QIAGEN AllPrep FFPE Kit. In Illumina internal studies, this kit gives a high quantity and quality of DNA.

Step 2 Recommended library prep methods

Product Illumina DNA Prep

Input requirements 100–500 ng
Total library prep time 3–4 hrs
Sample type gDNA from blood, FFPE, and FF samples
Sample index sets 384 unique dual indexes

Step 3 Recommended sequencing systems

Product NovaSeq 6000 System

1 (S1 flow cell)
Samples/flow cella 2–3 (S2 flow cell)
5–6 (S4 flow cell)
Recommended read length 2 × 101 bp
a. Illumina recommends 220 Gb reads for tumor DNA and 85 Gb reads for normal DNA.

Step 4 Secondary analysis

Illumina recommends using the DRAGEN pipelines on BaseSpace Sequence Hub or on a DRAGEN Server to obtain
somatic variant calls and gene expression data. When using BaseSpace Sequence Hub, users can monitor runs in
real time while securely streaming data directly from the instruments into the ecosystem for push-button analysis.

Pipeline Application Input

Somatic variant detection in tumor samples; Tumor DNA FASTQ
DRAGEN Somatic Pipeline
includes tumor-only and tumor-normal modes Normal DNA FASTQ

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