Apac distributor training series 3 swift product for cancer study

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APAC Distributor Training Series:
Swift Product for Cancer Study
Jan 2021
Mengmeng Lin (mlin@swiftbio.com)

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Swift Library Prep Overview: IP families
Adaptase
2S/Amplicon
Normalase
Accel-NGS Methyl-Seq
For quant-free
library pooling
Accel-Amplicon or
SNAP Amplicon
(Single-tube multiplex PCR)
Swift 2S Turbo
Accel-NGS 2S
(PCR-free, Plus, Hyb)
Accel-NGS 1S
Accel-NGS
For Ion Torrent
Adaptase Module
(single cell methyl)
Accel-NGS XL
for PacBio
Swift RNA
Swift Rapid RNA
Done:
7th Dec Swift Product for Metagenomics Study
21st Dec Swift Product for Virus Study
More Distributor Training：
Feb 2021 Swift Product for Very Specific Application
Not covered product
Swift 2S Sonic

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Today’s Topic
Cancer Study
Genomic
Sequencing
RNA
Sequencing
Swift RNA library kit
Swift Rapid RNA library kit
After this training session:
• Able to know benefit of choosing each Swift DNA/ RNA library prep kit for cancer study
• Able to know why choose amplicon workflow and why Swift amplicon product
Amplicon
Panel
Hyb Capture
Swift DNA library prep kit+
Swift hyb capture panel / 3rd party panel
• Accel-NGS 2S kit
• 2S Turbo
• 2S Sonic
DNA
Methylation
WGS
Swift gene/cancer-
based panel
Somatic/germline
mutation detection
Fusion gene
Alternative splicing
RNA expression
Rare & novel transcripts
Disease-associate SNV
…
Hyb Capture WGBS
Swift Methyl-seq Kit+
Arbor Bio myBaits MethylCap
Swift Methyl-seq Kit
Hyb Capture RNA-seq
Swift RNA library kit or
+
Swift hyb capture panel / 3rd
party panel

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Type of Mutation in Cancer
(1) Inherited (blood spot, whole blood, buccal swab, agricultural)
An inherited gene mutation is present in the egg or sperm that formed the
child.
Germline mutation (because the cells that develop into eggs and sperm
are called germ cells) or hereditary.
Inherited mutations are thought to be a direct cause of only a small fraction
of cancers.
Germline calling where VAF = 50% or 100%
(2) Acquired (Fresh frozen tissue, FFPE, cfDNA/ctDNA)
An acquired mutation is not present in the zygote, but is acquired some
time later in life. Somatic mutation
Type:
- Substitutions of one base by another
- Insertions or deletions of small or large segment of DNA
- Rearrangement (DNA has been broken and then rejoined to a DNA segment
from elsewhere in the genome)
- Copy number increase (increases from the two copies present in the normal
diploid genome, also know as gene amplification)
- Copy number reductions
https://www.cancer.org/cancer/cancer-causes/genetics/genes-and-cancer/gene-changes.html
Landscape of somatic mutations present in a single
cancer genome
Nature. 2009 April 9; 458(7239)

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Variant classification
Ensembl Variation
https://m.ensembl.org/info/genome/variation/prediction/classification.html
SV

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Genetic
Risk
Early
Detection
Patient
Stratification
Disease
Staging
Treatment
Options
Outcomes
Relapse
Monitoring
Birth Death
Treatment
Natural History of Disease Clinical Care
Early screening for
germline mutation
NGS for Cancer Studies
Somatic mutation
or biomarker detection
“Personalized Medicine”
Molecular diagnostic
Target therapy or drug
resistance test

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https://www.illumina.com/science/customer-stories/icommunity-customer-interviews-case-studies/patton-jennings-tan-yan-ngs-oncology.html
Hyb workflow:
Swift DNA or RNA library prep kit
+ Swift or 3rd party capture panel
Swift Amplicon:
Accel or SNAP version
WGS:
Swift DNA library prep kit

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2S Turbo 2S Sonic
Differentiators: Enzymatic fragmentation, fast and easy prep,
competitive price point, flexible adapter options
Key Applications: WGS (complex and/or small genomes), Targeted
(exome, panels), Genotyping by NGS, CNVs, Metagenomics
Target Customer: Production scale laboratories, such as: large PI,
core lab and service providers
Sample Types: gDNA, high quality FFPE, RNA (1st strand cDNA)
Input Range: 1-250 human or microbial (3rd party adapters),
50-250ng human or 1-250ng microbial (Swift adapter)
Differentiators: Mechanical Shearing, fast and easy prep, competitive
price point, flexible adapter options
(exome, panels), Genotyping by NGS, CNVs, Metagenomics
Target Customer: Production scale laboratories, such as: large PI, core
lab and service providers
Sample Types: gDNA, high quality FFPE, cfDNA
Input Range: 1ng-1ug human or microbial
1S Plus Adaptase 2S (Plus, Hyb, PCR-Free)
Differentiators: ssDNA or ss/dsDNA mix, nicked DNA, RNA (1st
strand cDNA), viral genome, highly degraded/ancient DNA, short
fragments >40 bp…WHEN 2S FALLS SHORT!
Key Applications: WGS/ Hyb, ChIP-Seq, Metagenomics
Target Segment: Infectious Disease, Agriculture,
Archeology/Museum, Forensics, Biotechnology, Biopharma, Academic
and Government Labs
Sample Types: highly degraded FFPE, Ancient DNA, degraded
cfDNA, Very Low Input ChIP samples
Input Range: >10pg
Differentiators: Mechanical Shearing, degraded or dilute
samples such as FFPE and cfDNA
(exome, panels), ChIP-Seq, low frequency variant detection, CNV
Target Customer:
Sample Types: gDNA, FFPE, cfDNA, RNA (1st strand cDNA) amplicons
Input Range: >10 pg (w/ PCR)
10 ng (cfDNA PCR-Free)
100 ng (gDNA PCR-Free)
Swift Product Selection and Positioning Guide

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Turbo Turbo
New!! Swift 2S Turbo DNA Library Kits
for Illumina® platforms
With Enzymatic Shearing
Tissue or Blood Sample Type for Cancer Study

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Custom adapter guide(IDT): https://sfvideo.blob.core.windows.net/sitefinity/docs/default-source/supplementary-product-info/idt-custom-ngs-adapters-product-sheet.pdf?sfvrsn=45d71f07_6
Minimal enzymatic incubation
&
purification steps
Indexing PCR
Time varies
Optional PCR
Terminal primers (included)
to increase library yield
Time varies
Ligation
3’ and 5’ ligation of P7 and p5
full-length adapters(Your choice of adapter)
truncated adapters
Flexible
✔️1-250 human
or microbial
✔️ PCR-free
Flexible
New! Swift 2S Turbo V2 DNA Library Kits will
launch soon
Choice of Kits: Standard & flexible
Standard
✔️50-250 human
or 1-250ng microbial
✘ PCR-free

Targeted sequencing
2S Turbo DNA library kit

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Swift 2S Turbo Library Kit was
evaluated for enrichment using Swift
Exome and Pan-Cancer Hyb Panels.
Comprehensive target coverage and
high complexity was observed with
multiple sample types, input
quantities, and platforms. Coverage
uniformity and complexity was higher
with low input Turbo libraries
compared to competitors (see
highlighted region of table).
Turbo’s efficient library provide
more data at low input !
*FFPE and HD200 sample integrity quantified by qPCR using the Swift ALU repeat assay provide with the Swift library prep kits, 0.34 ratio obtained for both samples.
Benchmarking: Hyb Capture Better Coverage
Uniformity with low input

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*FFPE and HD200 sample inputs quantified via qPCR of smaller ALU 115 repeat.
+Based on 247/115 ALU repeat fragment ratios
Sample
Integrity+
Input
(ng)*
%Duplicates
Mean Bait
Coverage
%Covered
≥ 20X
%Covered
≥ 50X
%Covered
≥ 100X
% Bases On-Target
0.24 25 6 176X 99.5 98.3 90.7 73.8
0.26 25 8 173X 99.6 98.5 94.6 74.9
0.34 24 4 163X 99.1 98.1 89.7 70.3
0.34 79 6 151X 99.4 98.5 90.2 69.6
Uniform Coverage for Somatic Mutation Detection
Targeted Sequencing of FFPE using Swift Exome Panel (MiSeq Run)
Sequencing metrics of FFPE samples match control samples
(e.g. NA12878 % bases on target = 68-75%).

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Swift Deceleration Module to control fragmentation
reaction time for automation
15 uL
10 uL
3 uL
1.5 uL
5 uL
Reagent DE
into frag
reaction
Fragment
Mode (bp)
1.5 uL 382
3 uL 434
5 uL 467
10 uL 600
15 uL 750
Automation Request
• Slow fragmentation to 15 min for 350-400bp
• Prepare reaction at room temp
• Sample placed on cycler 10 min after adding fragmentation mix
Actions
• Tested various volumes of Reagent DE in fragmentation reaction
• Ensured reproducible fragmentation, and no influence in Exome Capture
Will be added to Turbo protocol
☺︎ large insert size (>350bp) (ie, metagenomics)
☺︎ Automation
☺︎ SOP
☺︎ Similar fragment time for various condition of degraded
samples

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Accel-NGS® 2S DNA Library Kits
With Mechanical Shearing
For balanced coverage of dsDNA, even at low input

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Swift 2S: Superior Data Quality
High adapter ligation efficiency
 Unique 3’ end repair that efficiently converts all 3’-P into 3’-OH and at
the same time prevents chimera formation
 Unique 5’ end repair that replaces bases damaged by physical
shearing
 No A-tailing
 No adapter titration for low inputs maintains high ligation efficiency
 Very low adapter dimers
 Introduces Molecular ID tags (MIDs)
Best Data Quality
 WGS and exome sequencing
 Metagenomic sequencing (ultra-low inputs <1ng down to 10 pg)
 ChIP-Seq (including ultra-low inputs down to 10 pg)
 FFPE and cfDNA/ctDNA (>10 ng PCR-Free)
 HiC-Seq (as low as 5000 cells)

FFPE Sample
Accel-NGS® 2S DNA Library Kits

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2S Hyb Performance with Fixed Samples
of Various Fixation Times and Input Levels
Fixation Time Course with the Pan-Cancer Panel
Accel-NGS® 2S Hyb libraries were constructed with 100, 10, and 1 ng of DNA. DNA extracted from the same
normal kidney sample which had either been fresh-frozen or fixed for 6, 24, or 48 hours before being paraffin-
embedded. Amplified libraries were enriched with the IDT xGen® Pan-Cancer Panel. The xGen Pan-Cancer Panel is
0.9Mb and all samples were normalized to 0.6Mb reads.
INPUT
QUANTITY
SAMPLE
TYPE
%
ALIGNED
%
DUPLICATION
MEAN BAIT
COVERAGE
% COVERED
> 1X
% COVERED
> 20X
% BASES ON
TARGET
100 ng
Frozen 96 1 42X 99 91 80
6 Hr. Fix 96 1 43X 99 93 81
24 Hr. Fix 97 1 44X 99 93 82
48 Hr. Fix 97 1 45X 99 88 82
10 ng
Frozen 96 3 42X 99 90 80
6 Hr. Fix 96 5 41X 99 92 80
24 Hr. Fix 97 4 42X 99 93 81
48 Hr. Fix 97 8 42X 99 86 81
1 ng
Frozen 95 18 33X 99 85 77
6 Hr. Fix 94 32 26X 99 77 74
24 Hr. Fix 95 31 27X 100 79 76
48 Hr. Fix 95 44 22X 99 53 73

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Choice of Index- MID
Enable accurate de-duplication from single read sequencing,
and distinguishing PCR duplicates from fragmentation and
strand duplicates.
Posters
9 base
N sequence
6 or 8 base

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Choice of Index- MID
Only 1x coverage
was retained post-
deduplication
increase in data
retention up to 6x
coverage post
deduplication.

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MID Enable Ultra-Low Frequency Variant
Detection
cfDNA was extracted from blood of four individuals
with unique genetic background and Coriell gDNA
samples from different genetic backgrounds were
obtained. To determine the effect of MIDs on low
frequency variant calling, sample spike-ins were
performed at 1% or 0.5% frequency into 10 ng
cfDNA or 100 ng gDNA. Libraries were prepared
with the Swift 2S Hyb kit with MIDs, enriched with
the Swift Pan-Cancer Hyb Panel , and sequenced
on an Illumina HiSeq ® to a minimum of 8000x
coverage. A consensus sequence was generated
for each MID family (BMFtools) and data were
analyzed for homozygous SNPs present in the
spike-in sample only. 6/6 known variants were
present in all three 1% cfDNA samples and 27/27
known variants were present in both 1% and 0.5%
gDNA samples depicting the power of MIDs for low
frequency variant calling.

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Patient- derived xenografts (PDX)
CTC-derived xenografts (CDX)
Cancer Discov; 6(3); 286–99.
Cancer Research UK Manchester Institute
WES + multiplex PCR panel, long term monitoring
response of sequential targeted, immuno- and
chemotherapy of one melanoma patient.
10-25 ng cfDNA WES) swift 2S
Cancer Research UK Manchester Institute
• 214 patients (364 samples) with stage II, III, IV
melanoma
• 13ng cfDNA (WES on Miseq) swift 2S )
• Application:
Facilitate individualized treatment decisions for
patients with advanced melanoma
Publication for Clinical-based

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Part A (100 patients,22 tumor type) Part B (450
patients)
Workflow establishment & test its feasibility
Workflow for ctDNA test：
4x 10mL Streck
↓
Manual purification: QIAmp Circulating Nucleic Acid Kit (Qiagen)
Automation purification: ：QIAsymphony (Qiagen)
↓
0.5 to 25 ng ctDNA for Accel-NGS 2S DNA Library Kits
↓
SureSelectXT Reagent Kits (Agilent) 641-gene panel, 2.1Mb
↓
Illumina NextSeq 500, 2 × 150 bp
TARGET
(Tumor chARacterisation to Guide Experiment Target therapy)
Aim:
Use ctDNA to identify clinically actionable mutations in early
phase clinical trail patients with a range of advanced-stage
cancers.

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Accel 2S Plus – Best Coverage and Superior Variant Detection in ccfDNA
• Prostate cancer plasma sample
• Prepared 10ng ccfDNA with Accel 2S Plus,
along with 3 other library prep kits.
• Sequenced on Illumina HiSeqX
(2 x 150bp PE)
• Analyzed CNV, SNV and Indels
• Accel 2S Plus kit detected more variants
and specific SNVs, INDELs and CNVs.
• Accel 2S Plus provided the best coverage
Mauger, F., et al. Comparison of commercially available whole-genome sequencing kits for variant detection..., Sci Rep 10, 6190 (2020)
Publication for Methodology Comparison

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Accel-NGS® 1S Plus DNA Library Kit
For Difficult-to-Process Samples Including Damaged,
Denatured, or ssDNA

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Accel-NGS® 1S Plus Specifications
• Broad input range from 10 pg to 250 ng
• Adapt strands of DNA ≥ 40 bp long
• Simple, 2-hour protocol
• Offered for Ion Torrent™ and Illumina
®
• No end polishing step
Features
• Capture previously unattainable sequences
• Process more samples per day
• Use platform of choice
• Preserve input fragmentation patterns for
precise mapping of DNA insert ends
Benefits
Ion Torrent is a trademark of Thermo Fisher Scientific. Illumina is a registered trademark of Illumina, Inc.
Adaptase tail
Truncated P7 adapter
ssDNA

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Cancer Research UK Cambridge Institute
200 patients (18 different cancer types)
65 plasma samples
90–150 bp
180–220 bp
250–320 bp
High ctDNA: breast, ovarian, lung, melanoma, colorectal, and cholangiocarcinoma
Low ctDNA: glioma, renal, pancreatic, and bladder cancers.
Mutant ctDNA is generally more fragmented than non-
mutant cfDNA (19 cancer patients)
Mutant DNA in plasma of patients with advanced cancer is consistently
shorter than predicted mono-, and di-nucleosomal DNA fragment lengths

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6.4倍富集
4倍富集
Longitudinal plasma samples of a colorectal cancer patient
Detect tumor progression 60 and 87 days before by
imaging or unselected t-MAD analysis.
Improve the detection of response or disease progression
60 days
87 days

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AUC = 0.97
(sens: 90% & spec:98%)
AUC = 0.64
4.19X
SNVs
Conclusion：Size selection of shorter plasma DNA fragments enriches ctDNA and assists in the identification of a
greater number of genomic alterations with both targeted and untargeted sequencing at minimal additional cost.
Increased number of mutations detected in plasma
(samples from 6 patients with HGSOC)

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Swift RNA Library Kits

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Feature Specification Benefit
Input Quantity
10 ng – 1 g total RNA
100 pg – 100 ng mRNA
Supports a Wide Range
Consistent Library Output
RNA Types
Supported
Poly(A)-enriched mRNA
Ribodepleted RNA
Total RNA
Supports Most RNA
Applications
Technology
Adaptase® tailing and ligation
of 1st strand cDNA
No 2nd Strand cDNA
No Adapter Titration
Lowest Dimers & Duplicates
Maintains Strandedness,
High Mapping & Detection
Time 4.5 hours
Save 1 hour vs. NEBNext®
Half the time of TruSeq®
Kit Reaction
Sizes
24 • 96 Evaluation and Adoption
Indexing
Options
Single • Combinatorial Dual
Unique Dual • Normalase
Flexible to Sequencers,
Workflows and Applications
Multiplexing
Capability
Up to 768 libraries Save Sequencing Costs
Automation
Compatible with Liquid
Handlers
Custom Packaging Available
Adopt at Scale
Fast
Lowest Inputs
Low Cost
Feature Specification Benefit
Input Quantity
100 ng – 1 g total RNA
5 ng – 100 ng mRNA
Supports a Wide Range
RNA Types
Supported
Poly(A)-enriched mRNA
Ribodepleted RNA
Total RNA
Supports Most RNA
Applications
Technology
Adaptase® tailing and ligation
of 1st strand cDNA
High Strandedness
No 2nd Strand cDNA
No Adapter Titration
Time 3.5 hours Fastest on Market
Kit Reaction
Sizes
24 • 96 Evaluation and Adoption
Indexing
Options
Single • Combinatorial Dual
Unique Dual • Normalase
Flexible to Sequencers,
Workflows and Applications
Multiplexing
Capability
Up to 768 libraries Save Sequencing Costs
Automation
Compatible with Liquid
Handlers
Custom Packaging Available
Adopt at Scale
Fastest
Lowest Cost
Swift Rapid RNA Library Kit
Accelerate Your RNA-Seq Discovery
Swift RNA Library Kit
Deepen Your RNA-Seq Discovery

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Why not Poly(A)?
 10ng RNA for Swift RNA Library Kit
 100ng RNA for Swift Rapid RNA Library Kit
 100pg RNA for Swift RNA Library Kit
 5ng RNA for Swift Rapid RNA Library Kit

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Ribodepletion VS. Capture
Sample
Library
yield
(nM)
STAR
mapping
rate (%)
Genes
detected
Exonic
rate (%)
rRNA
rate (%)
ERCC
(R2
)
UHR RNA
44.2 86.2 17,132 46.0 3.4 0.94
33.6 87.5 17,180 46.2 3.2 0.92
FFPE
Sample 1
14.0 83.6 15,095 22.0 6.7 0.91
19.8 85.7 15,112 21.2 6.0 0.92
FFPE
Sample 2
24.9 84.2 15,274 24.6 4.8 0.92
23.9 85.5 15,325 25.7 4.5 0.94
UHR
FFPE
Sample 1
FFPE
Sample 2
UHR
Ribo-
depleted
UHR FFPE
Sample 1
FFPE
Sample 2
Sample
Library
yield
(ng/uL)
STAR
mapping
rate (%)
Genes
detected
Exonic
rate (%)
rRNA
rate
(%)
Selected
bases
(%)
UHR
RNA
82 89.6 17,758 91.2 1.3 96.7
FFPE
Sample 1
93 88.8 17,009 89.2 0.9 96.5
FFPE
Sample 2
73 87.9 17,024 88.1 1.0 95.8
Sample
Library
yield
(ng/uL)
STAR
mapping
rate (%)
Genes
detected
Exonic
rate (%)
rRNA
rate
(%)
Selected
bases
(%)
FFPE
Sample 3
23.0 89.4 16,089 87.4 1.5 95.1
FFPE
Sample 4
54.0 88.0 16,065 88.7 1.3 95.6
FFPE
Sample 5
33.4 87.5 15,653 87.6 1.3 94.6
FFPE
Sample 6
56.0 87.6 16,246 88.4 1.2 95.4
DV200 = 60
DV200 = 61
DV200 = 54
DV200 = 54
Ribodepletion (100ng of total RNA input)
Capture (100ng of total RNA input)
Capture (10ng of total RNA input)
https://swiftbiosci.com/wp-content/uploads/2020/01/APP-004-THE-SWIFT-RNA-LIBRARY-KIT-OPTIMIZES-RNA-SEQ-DATA-QUALITY-AND-COSTS-FOR-FFPE-
SAMPLES-Rev-1.pdf

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Capture Workflow for Lower Inputs
Swift RNA on FFPE samples
100 pg total RNA
20 PCR cycles
Yields > 20 nM
1:10 dilution loaded on Bioanalyzer HS DNA chip
10 ng total RNA
16 PCR cycles
Yields > 100 nM
HER2- Sample 1
DV200 = 54
HER2+ Sample 4
DV200 = 61
HER2- Sample 3
DV200 = 54
Confidential
HER2- Sample 1
DV200 = 54
HER2+ Sample 4
DV200 = 61 HER2- Sample 3
DV200 = 54

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HER2-
Sample 4
DV200 = 73
327 bp
93 ng/uL
HER2+
Sample 6
DV200 = 48
296 bp
73 ng/uL
100 ng FFPE RNA input directly into Swift library prep kits
1:10 dilution loaded on Bioanalyzer HS DNA chip
Library details:
• 100 ng total RNA
• 2 min @ 94 C adjusted
fragmentation time
• Relaxed 1.8X SPRIs
throughout
• 12 PCR cycles
HER2-
Sample 4
DV200 = 73
318 bp
58 ng/uL
HER2+
Sample 6
DV200 = 48
282 bp
66 ng/uL
Capture Workflow for Both Swift Kits
Swift RNA& Rapid RNA Library Kits on FFPE RNA (pre-hyb)
Swift RNA Swift Rapid RNA

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Accel-NGS® Methyl-Seq
DNA Library Kits
Library Preparation for Bisulfite-Converted DNA

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Adaptase-based (1S, Methyl, Module, RNA)
U U U
U U U
U U U
Powered by Swift’s Adaptase®
Technology
•Single-stranded tailing and ligation
•Template-independent reaction
•>90% efficiency
•US Patent 9,896,709
•Underlying technology of Swift Methyl-Seq and
Single Cell Methyl (Adaptase Module) and 1S
Plus Library Kit
Benefits to Methyl-seq
• >40bp retention
• Lower input requirement
• Able to use ss&dsDNA as library prep template

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PreBS PostBS
2% amplifiable
fraction
- High input
- Low yield
- High dup%
- Low diversity%
PBAT
- Uneven coverage
- High dup%
- Low diversity
Target towards
CpGs

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4841 participants
CCGA Study
PATHFINDER
Study
STRIVE Study SUMMIT Study
Current Status Follow-up (>5 year) Enroll Follow-up (5 year) Enrolling
Actual/Expected
Enrollment
15254 participants 6,200 participants
99481 participants
(women)
50,000 participants
Sample Type Blood and tumor tissue A single blood draw Blood Blood
Time Perspective/
Purpose
Prospective Screening Prospective Prospective
Actual Study Start
Date
August 2016 December 2019 February 2017 April 2019
Estimated
Completion
March 2024 June 2021 May 2025 August 2030
https://grail.com/clinical-studies/
On-going Clinical Trials

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https://doi.org/10.1016/j.annonc.2020.02.011
4841 participants
Annals of Oncology
>50 Cancer type
Adrenal (cortical carcinoma and neuroendocrine),
ampulla of Vater, anus, appendix, bone, brain and spinal
cord, breast, cervical lymph nodes and unknown primary
tumors of the head and neck, cervix uteri, colon and
rectum, corpus uteri…
STRIVE study
Training set + 1587
Validation + 615
6689 total
WGBS vs. WGS vs. target sequencing
Population-level screening

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Step 5
Targeted Enrichment
Sequenced fragments are
associated back to regions
of interest
Step 6
Median depth: 113 million
median unique on-target depth: 139X
150bp PE sequencing, NovaSeq
Step 1
CCGA :8 tubes of 10mL whole blood
STRIVE: 4 tubes of 10mL whole blood
(Streck)
Centrifuge：<5days，median <2 days
Step 4
Swift Methyl-seq kit for
library preparation
WGBS; 30X depth
(n=3,508)
Twist panel (17.2Mb)
1,116,720 CpG
(～10 regions)
7.5Mb hypomethylated sites
7.4Mb hypermethylated sites
2.3Mb target both
Step 3
Bisulfite conversion：
EZ-96 DNA Methylation Kit (Zymo)
≥0.99 conversation rate
Step 2
10mL plasma， 75ng cfDNA
Manual： QIAamp Circulating Nucleic Acid kit
Automation： MagMax kit (ThermoFisher)

Enroll population: progressing advanced cancers(2011-
2016)
Sample collection： 1-4mLplasma (ETDA tube
Purification： QIAamp Circulating Nucleic Acid kit (4.5-30ng,
avg 21.1ng)
BS: EZ DNA Methylation-Lightning Kit (Zymo Research)
Library Prep： Accel-NGS Methyl- Seq DNA Library Kit
(Swift Biosciences)
Hyb Capture： llumina TruSight Rapid Capture Kit
Sequencing： Illumina HiSeq 2500 (2x100 cycle)
～10,000x mean target coverage
Targeted Methylation Sequencing of Plasma Cell-free DNA for
Cancer Detection and Classification
MD Anderson Cancer Center, Phase I Clinical Trials
9223 CpG sites， 32 cancer
type
（ hypermethylated in tumors ）
Liu. L, F.Janku, et. Al, 2018. Annals of Oncology
37 samples from late phase of CRC
Cutoff 7.52
Sens: 94.4%
Spe: 100%
Performance Evaluation

(A) Patients on therapy had lower
methylation rates
(B) Patients with lower methylation rates
(blue) had longer survival rates
(C) Patients with lower methylation rates
had longer time to treatment failure
(D) Positive correlation between
methylation score and best RECIS
(response) score
Liu. L, F.Janku, et. Al, 2018. Annals of Oncology
68 patients off therapy (83.8% sens)
14 patients on therapy (50% sens)
OS
Off-therapy
TTF
55 patients
10.4 months
4.4 months
1.6 months
2.8 months
Clinical validation(78 patients with advanced colorectal
cancer, non-small-cell lung cancer (NSCLC), breast cancer or
melanoma
Targeted Methylation Sequencing of Plasma Cell-free DNA for Cancer
Detection and Classification
MD Anderson Cancer Center, Phase I Clinical Trials

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Publication for Research-based
A large integrated study of WGS, WGBS and whole-transcriptome, first study to look at comprehensive methylation
landscape in metastatic prostate cancer.
Observed that 22% of tumors exhibited a novel epigenomic subtype associated with hypermethylation and somatic mutations
in these four driver genes: TET2, DNMT3B, and BRAF.

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Publication for Research-based
Wu et al., 2020, The Journal of Clinical Investigation
Characterize the plasma methylome in mCRPC and identify
prostate cancer–specific methylation signatures.
University College London Cancer Institute

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Publication for Methodology Development
Uses the methylation profiles of adjacent CpG sites on an individual read to
accurately identify the tumor cfDNA fraction in plasma.
CancerDetector-generated tumor burden percentages correlated well with
tumor size (0.87) and can thus be used to monitor disease progression and
treatment.
This publication describes CancerLocator, a method that
simultaneously infers the proportion and tissue of origin of ctDNA in a
blood sample using WGBS.
CancerLocator performance was evaluated on both simulated data and
real data, and compared to that of 2 established multi-class
classification methods

swiftbiosci.com
Publication for Methodology Comparison
NanyangTechnological University, Singapore;UCL
The Accel-NGS Methyl-Seq kit had the
best GC coverage across both genomes
TruSeq Methyl-Seq
NEBNext Ultra
SPLAT (home brew)
TruSeq Methyl-Seq
QIAseq Methyl Library kit

swiftbiosci.com
Swift Solution for Target Methyl Study
Swift Methyl-Seq + Arbor Bio myBaits MethylCap
+
https://swiftbiosci.com/wp-content/uploads/2020/10/AppNote_Arbor-Swift_MethylSeq_2020-3.pdf
Webinar: https://www.youtube.com/watch?v=iZydUU-DZgI

swiftbiosci.com
Accel-Amplicon Panels™
Normalase™ Amplicon Panels (SNAP)

swiftbiosci.com
• Consists of individually synthesized and quality controlled
probes that have been validated to provide the highest level
of performance
• ~9-hour workflow
• 500 ng per library, up to 12-plex
• 150-350 bp fragment size
• Customizable
Swift Hybridization Capture Kits
Accel-Amplicon™ Panels
• Accel-Amplicon and SNAP Panels combine the
easiest, fastest workflow with flexible content to
accelerate your variant discovery and screening.
Simply leverage our pre-designed, validated core
content and add your own targets.
• ~2-hour (Accel)/ ~3-hour (SNAP) workflow
• 10 ng input sample
• Average amplicon size ~130-140bp
• Customizable
Panel size
Gene Panels
(<50genes)
Gene Panels
(>50genes)
DAR
(Disease associate regions)
Exome
20-30%Probes
70-80%Amplicon
50%Probes
50%Amplicon
70-80%Probes
20-30%Amplicon
80-90%Probes
10-20%Amplicons

swiftbiosci.com
Swift Amplicon NGS Panels
Format
• Core + customizable gene content
• Single-tube, highly multiplexed assay
• DNA to library in 2 hours for Accel-amplicon
3 hours for SNAP panel workflow
• 10 ng input of cfDNA or FFPE DNA
• Illumina®, Ion Torrent™ for Accel-amplicon
Only Illumina® for SNAP Panels
Performance
• Limit of detection of 1% VAF
• On-target > 90%
• Coverage uniformity > 90%
• Compatible with cfDNA and FFPE
• Supports limited amounts of DNA
• Cost-effective
Accel-amplicon SNAP amplicon

swiftbiosci.com
• Low uniformity and lack of coverage
• Multiple primer pools increase the input requirement
• You need large contiguous stretches of DNA in order to make library
• Inflexibility to add primers to increase the size of the panel
• Low library complexity
• Inability to cover entire coding regions
Traditional two-primer strategy

swiftbiosci.com
Competitors of Two-Primes Panel
https://www.youtube.com/watch?v=D3Dv7QHpLcs

swiftbiosci.com
• OVERLAPPING AMPLICONS IN A
SINGLE REACTION
• Low total input
• Few reagents, less time
• Fast workflow
• ~2hrs or 3hrs
• Accel-Amplicon™ or SNAP Amplicon panels
may be used to interrogate the following types
of variants in samples:
- SNV
- Insertions and deletions
- CNV
Why are Swift Amplicon Products Special?
Multiplex PCR with
overlapping target
specific-primers

Swift Panel Design base on OncoKB and market
research
Included
OncoKB Introduction
https://www.youtube.com/watch?v=XqoKrrm2Boc&feature=youtu.be
Swift used:
FDA-recognized biomarkers
Standard of care biomarkers

Available Panels
56G and 57G Pan-Cancer Profiling
EGFR Pathway (KRAS/NRAS/BRAF)
BRCA1 and BRCA2
BRCA1, BRCA2 and PALB2
TP53 (all exons)
CFTR (all exons, UTRs, some introns)
Sample_ID
Colorectal Cancer
Lynch Syndrome
Myeloid Disease
Lung Cancer
Gene-Based Disease-Based

swiftbiosci.com
• ERASE-Seq (Elimination of Recurrent Artifacts and Stochastic Errors) functions by
quantitatively modeling the background error profile of normal controls that have
gone through the library prep and sequencing process in order to establish the
inherent noise distribution for each possible variant
• ERASE-Seq delivers superior detection sensitivity to 0.05% allele frequency (AF),
with a false positive rate 10-100X lower than leading molecular barcode.
Benefits:
• Superior sensitivity and 10-100X fewer false positives than molecular barcodes (UMIs)
• Excels in the 0.05%-0.5% AF range where molecular barcodes struggle
• Any targeted panel can be adapted easily to ERASE-Seq with software validation- and no barcodes needed
• Provides similar detection performance to digital PCR, but can test for thousands of variants per test
• Validated in numerous sequencing labs globally

swiftbiosci.com
Swift Amplicon HS Panels
Confident Detection of Low Frequency Variants
in Liquid Biopsy Sequencing

swiftbiosci.com
Swift Amplicon HS – Features, Benefits, Pricing
Features and Benefits
• Sensitive and Specific
Robustly detect SNVs and indels down to 0.2%
• Compatible with cfDNA
Amplifies from 10-20 ng of cfDNA
• Fast workflow, high quality data
From DNA to Illumina® libraries within 3 hours
• Paired Data Analysis Tools
Cloud-based via Genialis or open-source
Ordering Information
Swift Amplicon HS EGFR Panel, Cat. No. HS-51024, 24 rxns
A focused cfDNA-compatible panel covering clinically-relevant oncology
hotspots including EGFR T790, KRAS G12/G13,NRAS Q61 and BRAF V600

swiftbiosci.com
Turbo flexible
+ IDT Probes
10-100 amplicons/tube
15-1,500 amplicons/tube

Using a custom panel targeting 30 hotspot amplicons in
these four genes, NGS was performed on cell-free DNA
extracted from plasma at the time of hysterectomy and the
matching tumor DNA from 48 patients with endometrioid
endometrial carcinomas.
These results demonstrate that mutations in genes
relevant to endometrial cancer can be identified in the
peripheral blood of patients at the time of surgery

swiftbiosci.com
Conclusion: ctDNA could be a powerful tool for therapy decisions and patient management in a large number of cancers across a variety of stages.
Summary:
• The data demonstrate that ctDNA can be a
reliable marker of the solid tumor in a large
number of tissue types in non‐metastatic cancers.
• These presurgical ctDNA can serve a surrogate
marker for patient prognosis.

swiftbiosci.com
Liquid Biopsy Publications with Swift Amplicons
56G panel
56G panel
56G panel
56G panel
56G panel
56G panel

swiftbiosci.com
Today’s Topic
Cancer Study
Genomic
Sequencing
RNA
Sequencing
Swift RNA library kit
After this training session:
• Able to know benefit of choosing each Swift DNA/ RNA library prep kit for cancer study
• Able to know benefit of Swift amplicon product
Amplicon
Panel
Hyb Capture
Swift DNA library prep kit+
Swift hyb capture panel / 3rd party panel
• 2S Turbo
• 2S Sonic
DNA
Methylation
WGS
Swift gene/cancer-
based panel
Somatic/germline
mutation detection
Fusion gene
Alternative splicing
RNA expression
Rare & novel transcripts
Disease-associate SNV
…
Hyb Capture WGBS
Swift Methyl-seq Kit+
Arbor Bio myBaits MethylCap
Swift Methyl-seq Kit
Hyb Capture RNA-seq
Swift RNA library kit or
+
Swift hyb capture panel / 3rd
party panel

swiftbiosci.com
THANKS!
We need your feedback!

Apac distributor training series 3 swift product for cancer study

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