Post on 16-Apr-2017
Sample to Insight
Innovative NGS library prep methods 1
Innovative QIAseq FX technology for construction of NGS libraries for use with Illumina instruments
Ioanna Andreou Ph. D.
Senior Scientist, NGS Life Sciences Team, QIAGEN
Sample to Insight
2
Overview of NGS technologies and innovative NGS library prep methods
Part 1: Introduction to next-generation sequencing (NGS) technology
Part 2: Innovative NGS library construction technology
Part 3: Advanced NGS library prep for challenging samples
Welcome to a 3-part series: NGS technology and applications
Intro to NGS, 11.30.2016
Sample to Insight
Innovative NGS library prep methods 3
Legal disclaimer
• QIAGEN products shown here are intended for molecular biology
applications. These products are not intended for the diagnosis,
prevention or treatment of a disease.
• For up-to-date licensing information and product-specific
disclaimers, see the respective QIAGEN kit handbook or user
manual. QIAGEN kit handbooks and user manuals are available
at www.qiagen.com or can be requested from QIAGEN
Technical Services or your local distributor.
Sample to Insight
Innovative NGS library prep methods 4
Agenda
1 Introduction
FX technology for NGS library construction
Single cell analysis
Single cell DNA sequencing
Single cell RNA sequencing
2
3
4
5
Sample to Insight
Innovative NGS library prep methods 5
Agenda
1 Introduction
FX technology for NGS library construction
Single cell analysis
Single cell DNA sequencing
Single cell RNA sequencing
2
3
4
5
Sample to Insight
Innovative NGS library prep methods
NGS technology overview and applications series: Part 2
6
Overview of NGS technologies and innovative NGS library prep methods
Part 1: Introduction to next generation sequencing (NGS) technology
Part 2: Innovative NGS library construction technology
Part 3: Advanced NGS library prep for challenging samples
Sample to Insight
Innovative NGS library prep methods
Typical challenges in whole genome sequencing
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Faster turnaround
Higher library complexity
Control per-sample costs
Nucleic acid isolation
Data analysis and
interpretation
Library preparation
and QCSequencing
Sample collection
and stabilization
→ Permits process scale-up
→ Maximizes clinical utility of data
→ To preserve sample information content
→ To enable high-quality downstream analysis
→ Time, consumables, first-pass success rate
Sample to Insight
Innovative NGS library prep methods 8
Current options for DNA fragmentation are suboptimal
Mechanical shearing Enzymatic shearing
FragmentDNA
Addadapters
Amplify and QC
library
Typical NGS Library Prep Process
High costs (instrumentation lab space, hands-on-time)
Harder to scale-up Intermediate cleanup steps
Poor data quality, introduction of sequence bias
Inflexible protocol Not easy to adapt for different
DNA input
Sample to Insight
Innovative NGS library prep methods 9
Agenda
1 Introduction
FX technology for NGS library construction
Single cell analysis
Single cell DNA sequencing
Single cell RNA sequencing
2
3
4
5
Sample to Insight
Innovative NGS library prep methods 10
QIAseq FX: Gold-standard quality from an enzymatic workflow
Single-use barcoded adapters can be used all at once or in batches of 10–12
Faster and easier to automate without the need for fragmentation instrumentation
Outperforms other enzymatic shearing procedures on genomic coverage
Flexible fragment size, batch size and input DNA amount
Complete kit that includes reagents for fragmentation, ligation, library amplification and 96-plex dual-barcoded adapters
Suitable for whole genome sequencing, whole exome / hybrid capture, metagenomics
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Innovative NGS library prep methods 11
QIAseq FX – the ideal balance of speed and high data quality
QIAseq FX uses only a standard thermocyler and 96-well PCR plates for truly scalable library prep 2.5 hour total workflow <20 min hands-on time Single-tube enzymatic reaction workflow Easy manual implementation or adaptation for automated liquid handling QIAGEN high-fidelity library amplification and plate format 96-plex adapters included
Adapter ligation Cleanup
Library amplification
(optional)
Fragmenta-tion, end-
repair and A-tailing
2.5 h
Single tube
Purified gDNA
1 ng – 1 µg
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Innovative NGS library prep methods 12
PCR-free option from as little as 10 ng input DNA
QIAseq FX (50 ng PCR-Free)QIAseq FX (100 ng PCR-Free)Mechanical shearing (100 ng) with PCRQIAseq FX (100 ng) with PCR
0 20 40 60 80 1000
0.5
1
1.5
2
Excellent G/C coverage with or without PCR
GC% over 100 bp regions
Nor
mal
ized
cov
erag
e
PCR-free library yield (nM) vs. input (ng)
Yields as low as 2 nM can typically be sequenced
10 nM PCR-free yield from just 100 ng input
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Compatible with downstream hybrid capture technologies
QIAseq FX
Library preparation:
- gDNA fragmentation
- Barcoded adapter ligation
- Library amplification
Purified gDNA1 ng–1 µg
Library QC
Innovative NGS library prep methods
Hybrid capture
Target enrichment
(IDT xGen, Agilent SureSelect)
QIAseq FX library
Sequence selected library
http://eu.idtdna.com/pages/products/nextgen/target-capture#
Sample to Insight
Mechanical-quality fragmentation from an enzymatic workflow
Innovative NGS library prep methods 14
Sample-to-sample fragmentation reproducibility
5 min 10 min0
200
400
600 Frag. #1Frag. #2Frag. #3Frag. #4
Fragmentation time
Ave
rage
frag
men
t siz
e (b
p)
Customize fragment size by adjusting incubation time
5 min 10 min0
200
400
600Input DNA species
Bacterial MixHuman
Fragmentation timeA
vera
ge fr
agm
ent s
ize
(bp)250 bp
450 bp
1000 bp
Sample to Insight
QIAseq FX exhibits less G/C bias than comparable methods
Innovative NGS library prep methods 15
QIAGEN QIAseq FXSupplier N – Enzymatic shearingMechanical shearing + Standard LP(Tagmentation not possible at 100 ng input)
QIAGEN QIAseq FXSupplier N - EnzymaticMechanical shearing + Standard LPSupplier I - Tagmentation
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Innovative NGS library prep methods 16
0 50 100 1500
10
20
30
40
50
Supplier N - 100 ngA T C G
read position (nt)
% in
divi
dual
nuc
leot
ide
0 50 100 1500
10
20
30
40
50
Supplier I - 1 ngA T C G
read position (nt)
% in
divi
dual
nuc
leot
ide
0 50 100 1500
10
20
30
40
50
QIAseq FX 100 ngA T C G
read position (nt)
% in
divi
dual
nuc
leot
ide
0 50 100 1500
10
20
30
40
50
QIAseq FX 1 ngA T C G
read position (nt)
% in
divi
dual
nuc
leot
ide
QIAseq FX generates a purer random base composition
Sample to Insight
Superior genomic coverage and duplication rate
Innovative NGS library prep methods 17
0 10 20 30 40 50 60 70 80 90 1000
0.02
0.04
0.06
0.08
0.1
0.12
Coverage distribution
Coverage depth (X)
Frac
tion
of ta
rget
gen
ome
QIAseq FX (100 ng)Supplier N – Enzymatic (100 ng)Mechanical shearing + Standard LP (100 ng)Supplier I – Tagmentation (1 ng)
QIAseq FX (1 ng and 100 ng*)Supplier N – Enzymatic (1ng)Mechanical shearing + Standard LP (1 ng)Supplier I – Tagmentation (1 ng)
100n
g
Duplication rate, 1 ng input
Gold-standard
% duplication
Mechan
ical s
heari
ng
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Innovative NGS library prep methods 18
Agenda
1 Introduction
FX technology for NGS library construction
Single cell analysis
Single cell DNA sequencing
Single cell RNA sequencing
2
3
4
5
Sample to Insight
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Cells differ on the genome level
Genome variations occur in health and disease
(1) Iourov, I.Y. et al. (2010) Somatic Genome Variations in Health and Disease, Curr Genomics 11(6)
Somatic genome variations consist of :
Aneuploidy
Structural rearrangements
Copy number variations
Gene mutations
Somatic genome variations
Occur during normal development/aging
Contribute to pathogenesis
Are the cause of diseases such as cancer, autoimmune, brain and other diseases
Examples (1)
Aneuploidy in pre-implantation embryos occurs in 15–91% of samples
Aneuploidy in skin fibroblasts occurs in adults Middle age: in 2.2% of cells Aged: in 4.4% of cells
Almost all cancers are caused by different types of genome variations including aneuploidy/polyploidy, structural rearrangements, gene amplifications, gene mutations
Innovative NGS library prep methods
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Innovative NGS library prep methods 20
Seemingly identical cells – unique transcriptional patterns
Cells change their transcription pattern:
(1) Kumar L.M. et al. (2014) Deconstructing transcriptional heterogeneity in pluripotent stem cells. Nature 4;516
The transcriptome of a cell is not fixed but dynamic
The transcriptome reflects the Function of the cell Type of the cell Cell stage
Gene expression is influenced by intrinsic or extrinsic factors (signaling response, stress response)
Only on single cell level you obtain: Real (not average) transcriptome gene
expression data Allelic expression data A deeper understanding of the
transcription dynamics within a cell
Heat map of single cell RNA-seq data for selected pluripotency regulators (1)
Sample to Insight
Single cell analysis enables new insights
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CTC = Circulating tumor cells, PGD = Pre-implantation genetic diagnosis
Cellular heterogeneity
Detection and analysis of rare cells (example: CTC from liquid biopsy)
Identification of cell subpopulations based on genomic structure or gene expression (tumors, tissues, immune cells, cell cultures)
Limited availability of cells Analysis of limited sample
material (example: embryo biopsy for PGD, fine-needle aspirates)
ApplicationReason
Biological insights instead of data from an aggregate
No data
Bulk result Single cell data
Innovative NGS library prep methods
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Innovative NGS library prep methods 22
Agenda
1 Introduction
FX technology for NGS library construction
Single cell analysis
Single cell DNA sequencing
Single cell RNA sequencing
2
3
4
5
Sample to Insight
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Discover the QIAseq FX Single Cell DNA Library Kit
Maximize coverage
Superior and more uniform genome coverage
compared to other kitsHigh sequence
fidelityMDA-based amplification
technology, proven for higher fidelity compared to
PCR-based methods
Enables biobanking
Excess amplified DNA can be stored for follow-up use, perfect for confirming novel
mutations or structural variants.
Higher diversity libraries
PCR-free workflow, better library diversity by
eliminating PCR-duplicates
Less GC-biasSuperior presentation of
GC-rich regions, perfect for bacteria with high GC-
content genomes
Robust and streamlined
workflowEverything in one package,
single-use adapters cut down on contamination possibilities, no need for
extensive QC
Minimize hands-on-timeUnder 4 h workflow from
single cell to library, without any additional kits
Superior sensitivity
The only kit sensitive enough for single bacterial genomes
QIAseq FX Single Cell
DNA Library Kit
Innovative NGS library prep methods
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Innovative NGS library prep methods 24
For single-cell DNA sequencing
Ideally suited for
The analysis of inter-cellular genome heterogeneity The analysis of aneuploidy and
sub-chromosomal copy number variations Sequence variation analysis (SNV, structural variants) in single cells Whole genome sequencing from rare samples Resequencing or de-novo sequencing of unculturable microorganisms For new type of experiments such as low-pass sequencing, consensus-based
variant calling
QIAseq FX Single Cell
DNA Library Kit
Sample to Insight
QIAseq FX Single Cell DNA Library Kit
Complete cell-to-library solution
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Primary sample isolation
Single cell isolation
NGS library construction NGS run Data
analysis InterpretationSample Insight
Single eukaryotic cell Single bacterial cell Picogram levels of purified DNA
Whole genome NGS Library Illumina-compatible Sequence variants Structural variants Aneuploidy Bacterial genomes
Innovative NGS library prep methods
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Innovative NGS library prep methods 26
QIAseq FX Single Cell DNA Library Kit: kit contents
Both kits contain: Cell lysis reagents Enzymes and buffers for whole genome amplification Enzymatic DNA fragmentation Single-step NGS library preparation Single-use, disposable Illumina Adapters in 96-well
format Multiple reagent aliquots to reduce contamination risk
and freeze-thaw cycles
What is not included: AMPure XP beads for library purification PCR reagents for library amplification: not needed as the
entire workflow is PCR-free qPCR reagents for library quantification: recommended
for accurate flow-cell loading
Cat No./ID: 180713QIAseq FX Single Cell DNA Library Kit (24)
Cat No./ID: 180715QIAseq FX Single Cell DNA Library Kit (96)
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Innovative NGS library prep methods 27
QIAseq FX Single Cell DNA Library Kit: the workflow
Cell lysis From single eukaryotic or bacterial cells, or small amounts (pg–ng) of intact gDNA Starting with 4 µl cell material in PBS (included) Prepare lysis buffer, mix with cells, incubate for 10 minutes at 65°C. If using purified DNA as
input, incubate for 3 minutes at room temperature Hold at 4°C
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Innovative NGS library prep methods 28
QIAseq FX Single Cell DNA Library Kit: the workflow
Whole genome amplification Prepare WGA master mix, mix with lysed cells, incubate for 2 h
Amplified gDNA can be used directly or frozen until needed There will be an excess of amplified gDNA, this can be stored for later use or follow-up
studies (i.e. confirming deletions detected with NGS via PCR or Sanger sequencing) Library preparation accepts a wide range of inputs, so quantification of the amplified DNA
is not needed
Sample to Insight
Innovative NGS library prep methods 29
QIAseq FX Single Cell DNA Library Kit: the workflow
NGS library preparation Prepare FX master mix, add to diluted WGA product and incubate for ~15 min. Insert size
can be set by user. Hold at 4°C Add adapters from single-use adapter plate Prepare ligation master mix, add to samples and incubate for 15 min to produce library
Cell lysis15 min
WGA2 h
FX library preparation
70 min
Purification20 min
ILLUMINA sequencing
3h 45 min with ~40 min hands-on time
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Innovative NGS library prep methods 30
QIAseq FX Single Cell DNA Library Kit: the workflow
Library purification Remove excess adapters with double-sided Agencourt AMPure XP cutoff
No PCR amplification necessary: protocol generates sufficient library without enrichment Library quantification via qPCR (i.e. QIAseq Library Quant) is highly recommended to
ensure accurate clustering on sequencer
Cell lysis15 min
WGA2 h
FX library preparation
70 min
Purification20 min
ILLUMINA sequencing
3h 45 min with ~40 min hands-on time
Sample to Insight
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Discover complete genome coverage
Perfect for low-pass sequencing
Don’t miss out on variants or structural features due to low coverage or locus
drop-outs
See more of the genome with the same sequencing
depth
Innovative NGS library prep methods
Libraries generated from single PBMC using the QIAseq FX DNA Library Kit or kits from two other suppliers and sequenced at low depth using MiSeq. Data were analyzed according to Zhang C.Z. et. al “Calibrating genomic and allelic coverage bias in single cell sequencing“, (2015) Nat. Commun. 6, 6822.
Comprehensive genome coverage
Genome coverage of various kits
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More uniform coverage, even with GC-rich regions
Perfect for bacteria with high GC-
content genomes
Coverage of traditionally
difficult-to-sequence regions
Innovative NGS library prep methods
Coverage versus GC content
Libraries were generated from single PBMC using the QIAseq FX DNA Library Kit or kits from two other suppliers and sequenced at low depth on the MiSeq. Data was analyzed using the CLC Genomic workbench 8.5.1.
Sequence GC-rich regions
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Highest fidelity sequence data: Have confidence in your results
Perfect for low-pass consensus variant
calling
Lower background when analyzing
sequence variants or mutations and
small indels
Fewer spurious sequence errors in
your dataset
Fewer false positives
Innovative NGS library prep methods
Single cell libraries from isolated PBMCs were sequenced with an Illumina MiSeq. Reads were mapped to the human genome (hg19) and sequence mismatches between NGS data and the reference were computed using the CLC Genomic workbench 8.5.1. Data plotted are the mean proportion of sequence differences +/– standard deviation for 3 replicates.
Combined error-rate of several single cell NGS methods Highest sequence
fidelity
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Analyze copy number variants and aneuploidy
Biobanking allows follow-up
experiments: confirm structural
variations with PCR or Sanger
sequencing
With comprehensive coverage, detect structural variants
regardless of where they are in the
genome
Innovative NGS library prep methods
QIAseq FX Single Cell DNA libraries from PBMCs and Jurkat cells were sequenced to 0.1x depth on a MiSeq. Reads were mapped to human genome (GRCh38) and the copy number variation of Jurkat vs PBMCs (control diploid cells) was assessed using the methods published in: Chao Xie, Martti T Tammi, “CNV-seq, a new method to detect copy number variation using high-throughput sequencing”, BMC Bioinformatics, 2009,10:80. The plot is the Log2 ratio (Jurkat/PBMC) of coverage using a window size of 500 Kb for chromosome 2 from a cell with an approx. 25 Mbp deletion.
Detection of a 25 Mbp deletion in a single cellAnalyze
CNVs and aneuploidy
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High-yield WGA enables biobanking and confirmatory testing
Follow-up tests to confirm structural
variants or sequence variants
or novel discoveries
Store unused amplified gDNA at
–20 for later use
Innovative NGS library prep methods
Data from 4 individual PBMCs for each kit, with the kit from Supplier R, no extra DNA was available for storage.
Yields of amplified gDNA of various kits Biobanking enables follow-up
testing
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Robust FX accepts a wide range of inputs: Save time on QC
PCR-free libraries from a wide range of
amplified gDNA inputs
Go directly from amplified gDNA to library prep: simple
dilution for all samples
No need to quantitate amplified gDNA: save time on
QC
Innovative NGS library prep methods
In this experiment, libraries were prepared from different amounts of the same pool of amplified gDNA.The library yield after the final purification is shown. Any libraries over 2 nM concentration can be sequenced directly and do not need PCR amplification.
Consistent results with
less QC
Yields of generated library vs DNA input
Sample to Insight
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Highly tunable fragmentation: Determine insert size
Adjust insert size with longer or
shorter FX incubation
Insert size consistent with
varying amounts of amplified gDNA
Innovative NGS library prep methods
Libraries with a varying amount of amplified gDNA were prepared and subjected to a gradient of FX fragmentation times. The plots show that the longer the incubation times, the shorter the inserts, and that this approximately follows the same trend regardless of the amount of input used.
Mean insert size per incubation timeInsert size
determined by fragmentation
incubation time
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Innovative NGS library prep methods 38
Agenda
1 Introduction
FX technology for NGS library construction
Single cell analysis
Single cell DNA sequencing
Single cell RNA sequencing
2
3
4
5
Sample to Insight
Discover the QIAseq FX Single Cell DNA Library Kit
39
Maximize transcript discovery
Discover a greater number of transcripts with the same
sequencing depth
High sequence fidelity
High-fidelity WTA minimizes spurious sequence errors.
Ideal for viral RNA sequencing.
Enables biobanking
Excess amplified DNA can be stored for follow-up use,
perfect for confirming novel mutations or structural
variants
Higher diversity libraries
WTA technology with less dropouts and less length-
bias against long transcripts and highly efficient library
preparation with maximized conversion rate
No PCR duplicatesPCR-free workflow
eliminating PCR duplicates
Robust and streamlined
workflowEverything needed in one
package. Single-use adaptors cut down on contamination
possibilities, no need for extensive QC.
Minimize hands-on-time5.5 h workflow from single cell to library, without any
additional kits
Uncover mRNA and
lincRNASequence
lincRNA and mRNA with a
single protocol
QIAseq FX Single Cell
RNA Library Kit
Innovative NGS library prep methods
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Innovative NGS library prep methods 40
For single cell RNA sequencing
Ideally suited for
Sensitive transcript discovery
and differential gene expression analysis from
single eukaryotic cells
Transcriptome analysis with best-in-class transcript detection
The analysis of both mRNA and long non-coding RNAs in a single dataset
Studies in inter-cellular heterogeneity
RNA-seq from limited amounts of difficult-to-obtain samples
Studies in infectious disease research
QIAseq FX Single Cell RNA Library
Kit
Sample to Insight
QIAseq FX Single Cell RNA Library Kit
41
Complete cell-to-library solution
Primary sample isolation
Single cell isolation
NGS Library construction NGS run Data
analysis InterpretationSample Insight
Single eukaryotic cell Picogram levels of purified
RNA from different species
Whole genome NGS Library Illumina-compatible Transcript discovery Gene expression Differential expression Viral RNA
Innovative NGS library prep methods
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Innovative NGS library prep methods 42
QIAseq FX Single Cell RNA Library Kit: the contents
Both kits contain: Cell lysis and gDNA degradation reagents Reverse transcription primers, buffers and enzyme Enzymes and buffers for cDNA amplification Enzymatic cDNA fragmentation Single-step NGS library preparation Single-use, disposable Illumina adapters in 96-well
format Multiple reagent aliquots to reduce contamination risk
and freeze-thaw cycles
What is not included: Agencourt AMPure XP beads for library purification PCR reagents for library amplification: not needed as the
entire workflow is PCR-free qPCR reagents for library quantification: recommended
for accurate flow-cell loading
Cat No./ID: 180733QIAseq FX Single Cell RNA Library Kit (24)
Cat No./ID: 180735QIAseq FX Single Cell RNA Library Kit (96)
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Innovative NGS library prep methods 43
QIAseq FX Single Cell RNA Library Kit: the workflow
Cell lysis From single eukaryotic or small amounts (pg – ng) of high-quality, purified RNA. Starting with 7 µl cell material in PBS (included). Prepare lysis buffer, mix with cells, incubate for 8 minutes. Cool to 4°C. Add gDNA degradation reagent, incubate for 10 min.
Cell lysis15 min
WTA3 h 45 min
FX library preparation
70 min
Purification20 min
Illumina sequencing
5.5h with ~1h hands-on-time
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Innovative NGS library prep methods 44
QIAseq FX Single Cell RNA Library Kit: the workflow
Whole transcriptome amplification Prepare RT master mix, mix with lysed cells, incubate 1 h Prepare cDNA ligation mix, incubate for 30 min Prepare cDNA amplification mix, mix with unamplified cDNA, incubate for 2 h
Amplified cDNA can be used directly or frozen until needed. There will be an excess of amplified cDNA, this can be stored for later use or follow-up studies (i.e.
confirming deletions detected with NGS via PCR or Sanger sequencing). Library preparation accepts a wide range of inputs, so quantification of the amplified cDNA is
generally not necessary.
Cell lysis15 min
WTA3 h 45 min
FX library preparation
70 min
Purification20 min
Illumina sequencing
5.5h with ~1h hands-on-time
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Innovative NGS library prep methods 45
QIAseq FX Single Cell RNA Library Kit: the workflow
Cell lysis15 min
WTA3 h 45 min
FX library preparation
70 min
Purification20 min
Illumina sequencing
5.5h with ~1h hands-on-time
NGS library preparation Prepare FX master mix, add to diluted WTA product and incubate for ~15 min. Insert size can
be set by user. Hold at 4°C. Add adapters from single-use adapter plate. Prepare ligation master mix, add to samples and incubate for 15 min to produce library.
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Innovative NGS library prep methods 46
QIAseq FX Single Cell RNA Library Kit: the workflow
Library purification Remove excess adapters with double-sided Agencourt AMPure XP cut-off
No PCR amplification necessary: protocol generates sufficient library without enrichment. Library quantification via qPCR (i.e. QIAseq Library Quant) is highly recommended to
ensure accurate clustering on sequencer
Cell lysis15 min
WTA3 h 45 min
FX library preparation
70 min
Purification20 min
Illumina sequencing
5.5h with ~1h hands-on-time
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Discover more: higher library diversity than competing workflows
See more of the transcriptome
Discover a greater number of
transcripts with the same sequencing
depth
Spend reads sequencing RNA
molecules, not PCR duplicates
Innovative NGS library prep methods
Libraries were generated from 100 pg of input from the same pool of RNA isolated from PBMCs with either the QIAseq FX Single Cell RNA Library Kit or a commonly used workflow employing separate kits for whole transcriptome amplification and library preparation from Suppliers C/I. Libraries were multiplexed and sequenced on the same MiSeq to approximately the same sequencing depth. After QC and mapping, the number of annotated transcripts with TPM >1 was computed. Data were then rarified repeatedly, where a subset of reads was selected at random, and the number of annotated transcripts with TPM >1 was computed for each sub-sampled set of reads. This was repeated at multiple read depths.
Discovery plot High library diversity
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Discover more: high library diversity from single cells
Usually, single cell libraries are easily
saturated
Sequence more, discover more
Innovative NGS library prep methods
Libraries were generated from single isolated PBMCs the QIAseq FX Single Cell RNA Library Kit. After QC and mapping, the number of annotated transcripts with TPM >1 was computed. Data were then rarified repeatedly, where a subset of reads was selected at random, and the number of annotated transcripts with TPM >1 was computed for each sub-sampled set of reads. This was repeated at multiple read depths.
High library diversity: even
from single cells
Discovery plot
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Discover more: high number of transcripts detected
High diversity libraries maximize transcript detection in each single cell
Innovative NGS library prep methods
Libraries were generated from 3 single isolated HeLa cells or from 100 pg of bulk RNA isolated from the same cells using the QIAseq FX Single Cell RNA Library Kit. After QC and mapping, the number of annotated transcripts with FPKM>1 was computed.
Maximize transcript detection
Number of transcripts
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High dataset diversity provided by PCR-free library preparation
PCR-free workflow eliminates
PCR duplicates
Frees read-depth for discovering new
transcripts
Innovative NGS library prep methods
Libraries were generated from single isolated PBMCs using the QIAseq FX Single Cell RNA Library Kit and a kit from Supplier C. Libraries were multiplexed and sequenced on the same run of a MiSeq instrument to equal sequencing depth. Duplicates were calculated using FastQC. PCR duplicates represent reads that provide no additional insight into the sample, and detract from the sensitivity of the experiment by consuming valuable sequencing depth.
Duplicate levelSequence
new transcripts not PCR
duplicates
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Uncover linc and mRNA with a single technique
Sequence mRNA and lincRNA in a single experiment
Examine protein-coding gene
expression and regulatory RNAs simultaneously
Innovative NGS library prep methods
Libraries were prepared from single PBMCs or HeLa cells using the QIAseq FX Single Cell RNA Library Kit or a commonly used workflow employing separate kits for whole transcriptome amplification and library preparation from Suppliers C/I. Libraries were multiplexed and sequenced on the same MiSeq to approximately the same sequencing depth. After QC and mapping, the proportion of reads mapping to annotated lincRNA for each library was calculated.
Sequence lincRNA and mRNA with a
single protocol
% of detected lincRNA
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High proportion of mRNA reads
Discover the insights only a
combined mRNA and lncRNA
dataset can bring
Use single cell sensitivity to
analyze mRNA expression
Innovative NGS library prep methods
Libraries were prepared from single isolated PBMCs. The QIAseq FX Single Cell RNA Libraries were multiplexed and sequenced on an Illumina NextSeq. After QC and mapping via STAR, the proportion of reads mapping to each GO annotation was calculated with HTSeq.
Maintains a high proportion
of protein-coding reads
RNA biotypes
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Consistent, robust cDNA amplification enables biobanking
Protocol consistently
generates excess cDNA
Consistent WTA produces consistent
libraries
Freeze excess amplified cDNA for follow-up studies or confirmatory testing
Innovative NGS library prep methods
Whole transcriptome amplification yield from single cells from the QIAseq FX Single Cell RNA Library Kit and a competing, PCR-based method (Supplier C). 8 Replicates from different single cells are shown.
Yield of amplified cDNA in ng Robust WTA enables
biobanking
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Innovative NGS library prep methods 54
Summary
QIASeq FX Kits for NGS library construction use an innovative technology that:
Uses a fast and streamlined workflow, without the need of additional kits and highly
specialized instrumentation
Delivers high yields of high-quality libraries ready for sequencing on Illumina platforms
Addresses different applications
DNA libraries from a wide range of input DNA
PCR-free DNA libraries from single eukaryotic and bacterial cells and very limited purified DNA
material
PCR-free RNA libraries from single eukaryotic cells, limited purified RNA material and viral RNA
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Upcoming webinars
“Overview of NGS technologies and innovative NGS library prep methods”
Part 1: Introduction to next-generation sequencing (NGS) technology
Part 2: Innovative NGS library construction technology
Part 3: Advanced NGS library prep for challenging samples
Innovative NGS library prep methods
Sample to Insight
56
Questions?
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Innovative NGS library prep methods