Whole Genome Amplification (WGA):
What to Do When You Don’t Have
Enough Genomic DNA
Rob Brazas, Ph.D.
Senior Product Manager,
Lucigen
January, 2017
www.lucigen.com
Agenda
Improving Whole Genome Amplified DNA Quality
• What is whole genome amplification?
• Experimental challenges of inaccurate or incomplete WGA
• PCR- and MDA-based methods of WGA and their strengths and weaknesses
• Variation on standard MDA: Sygnis TruePrime™ WGA Kit Technology
• Overview of TruePrime™ WGA Kits
• Comparison of Sequencing Results of WGA gDNA from TruePrime™ Single
Cell WGA Kit and Other WGA Kits/Methods
• Summary
• Final Thoughts
Whole Genome Amplification:
Production of µg of gDNA from ng or Less
Perfect Whole Genome Amplification
Whole Genome Amplification
Homologous
Chromosomes
Alleles
From what and why?
• gDNA amplification from:
– Single cells
– Limiting amounts of purified gDNA samples (biopsies, metagenomic
samples, etc.)
• Produce enough material for your experiments and archiving
Whole Genome Amplification:
Real-Life Amplification is Not Perfect
Perfect Whole Genome Amplification
Whole Genome Amplification
Homologous
Chromosomes
Alleles
Real-Life Whole Genome Amplification
Whole Genome Amplification
Homologous
Chromosomes
Alleles
Whole Genome Amplification:
Multiple Types of Amplification Errors Occur
Perfect WGA
Real-Life WGA
• Uniform amplification of entire
chromosomes
• Accurate representation of each set of alleles
o AB, AA, BB
• High fidelity amplification – no errors (No
SNV, INDEL creation)
• Amplified gDNA = Starting gDNA except
there’s more of it
• Uneven amplification across chromosomes
(missing areas, uneven amplification)
• Loss of heterozygosity
o AB → AA or AB → BB
• Error introduction – false SNVs
• Introduction of contaminants
• Creation of chimeras
• Amplified gDNA ≠ Starting gDNA
Inaccurate Whole Genome Amplification Creates
Multiple Experimental Challenges
• Incomplete whole genome sequencing results
• Difficulty assembling whole genomes due to contaminating sequences
• Changes in species abundance (representation) within a population sample
• Inaccurate or difficulty identifying:
– SNV (Single Nucleotide Variants)
– CNV (Copy Number Variation)
– Structural variation
PCR-based WGA Methods
Based on Various Primer Designs
LA-PCR (Linker-Adaptor or Ligation-Anchored PCR)
1. Fragment
2. Ligate adaptors with embedded
PCR primer sites
3. Amplify by PCR
IRS-PCR (Interspersed Repetitive Sequence PCR)
1. Primers designed to known
repetitive elements
2. Amplify by PCR
PEP-PCR (Primer Extension Preamplification PCR)
1. Random 15-mer PCR primers
2. Amplify by PCR under
permissive priming conditions
From: Blainey 2013 FEMS Microb. Rev
PCR-based WGA Methods
Based on Various Primer Designs
DOP-PCR (Degenerate Oligonucleotide Primed PCR)
1. Primers with degenerate 3’ ends (~6 bp
constant at 3’ end) and constant 5’ ends
2. Primer extension at random sites, low
temp annealing
3. PCR amplification at higher temps
D-DOP-PCR (Displacement - Degenerate Oligonucleotide Primed PCR)
1. Primers with degenerate 3’ ends and
constant 5’ ends
2. Primer extension at random sites, low
temp annealing, strand displacement
of newly synthesized strands by others
3. PCR amplification at higher temps with
added 5’-end specific primers
From: Blainey 2013 FEMS Microb. Rev
Multiple Displacement Amplification WGA Methods
Based on DNA Pols with Strand Displacement Activity
MDA (Multiple Displacement Amplification)
1. Random hexamer primers
2. Extended by DNAP with strong
strand displacement activity
3. Isothermal reaction temp
Variant of MDA Available
pWGA based on a reconstituted T7 replication
system (Li, Y. et. al. Nuc. Acids Res. E79 (2008)
From: Blainey 2013 FEMS Microb. Rev
Multiple Displacement Amplification WGA Methods
Based on DNA Pols with Strand Displacement Activity
SPIA (Single Primer Isothermal Amplification)
1. Primers with specific RNA sequence fused to partially
degenerate DNA primer sequence
2. Primer extension at set temperature
3. Degradation of RNA portion of primer with RNaseH
4. Reinitiation with new RNA/DNA primer and strand
displacement extension
From: Blainey 2013 FEMS Microb. Rev
Multiple Displacement Amplification WGA Methods
Based on DNA Pols with Strand Displacement Activity
MALBAC (Multiple Annealing and Looping-based Amplification Cycles)
1. Primers with degenerate 3’ ends and constant 5’ ends
2. Primer extension (quasi-linear amp) at random sites with thermocycling
3. Products with primer sequences at both ends loop due to sequences within DNA
portion of primers
4. Conventional PCR amplification
From: Blainey 2013 FEMS Microb. Rev
Strengths and Weaknesses (Perceived and Real) of
PCR and MDA WGA Systems
MDA-based
PCR-based
10 – 100 kb
~1-2 kb
10-8
10-4 - 10-5
Higher (?)
Lower (?)
Completeness of Genome Coverage
High
10 – 70%
Variability of Amplification
High
Low
CNV Detection
Poor
Good
Lower
Higher
5 -50% (?)
?
OK
+/-
Simple
Often multi-step
Amplified Fragment Lengths
Nucleotide Error Rate
Chimera Formation
Duplicate Formation
Allelic Dropout (ADO) (AB → AA or BB)
SNV Detection
Protocol
Focus On MDA Due to Completeness of
Genome Coverage
Kits/Methods Used
• REPLI-g Single-Cell Kit (Qiagen)
– Commercially available MDA kit utilizing random hexamers as primers
• TruePrime™ WGA Kits (Sygnis Kits, Single Cell and general purified gDNA Kits)
– Primase enzyme synthesizes primers in place of random primers
• Generic MDA WGA Kit
– TruePrime components with random hexamers substituted for Primase enzyme
• MALBAC™ Single Cell WGA Kit (Yikon Genomics)
Majority of data shown are published in:
Picher, AJ et al. Nat. Comm. 7:13296 (2016)
or provided to Lucigen by Sygnis
Sygnis TruePrime™ Kit Methodology
Primase Enzyme Synthesizes Initial Primers
1. TthPrimPol (Primase) binds denatured DNA at
random sites
2. Primase synthesizes short DNA primers
3. Phi29 DNA pol displaces Primase and begins
polymerization
4. Phi29 DNA pol performs strand displacement
5. Primase binds to newly formed DNA and
synthesizes new DNA primers
6. Phi29 DNA pol displaces Primase, binds
DNA primers and begins polymerization
Protocols for Sygnis TruePrime™ Kits
Simple Isothermal Amplification Reactions
TruePrime™ Single Cell WGA Kit v2 Protocol
TruePrime™ WGA Kit Protocol
Size of Products Amplified from a Single HEK293 Cell
MALBAC Produced Small Fragments
MALBAC
0.5 – 1.5 kb
TruePrime
1.5 – 12 kb
REPLI-g
9 – 19 kb
0.8% Agarose Gel
Picher, AJ et al. Nat. Comm. 7:13296 (2016)
Tapestation Plots
Yield of Amplified DNA with Primase vs. RPs
100X Greater Sensitivity with TruePrime Kit (Primase)
Generic WGA (TruePrime with Random Primers)
TruePrime WGA Kit
•
•
•
•
Human gDNA (Promega) input at indicated amounts
TruePrime™ WGA Kit and protocol (or with Random Primers substituted for Primase)
3 hr incubation at 30°C
DNA quantitation using Quant-iT PicoGreen dsDNA Assay Kit (ThermoFisher)
Picher, AJ et al. Nat. Comm. 7:13296 (2016)
Decreased Creation/Amplification of Random
Primer Artefacts with TruePrime™ WGA Kit
TruePrime™ WGA Kit
Generic WGA (TruePrime with Random Primers)
• 1 pg human gDNA (Promega)
• TruePrime™ WGA Kit and protocol (or with Random Primers substituted for Primase)
• Subjected to next gen sequencing and mapped back to known genomes
At 1 fg of Input, 95% of TruePrime™ WGA Kit
Amplified gDNA is Target Derived
• Varied inputs of human gDNA (Promega)
• Amplified with TruePrime™ WGA Kit for 6 hr
• Subjected to next gen sequencing and mapped back to known genomes
WGA with Random Primers is More Sensitive to
Contaminating DNA than TruePrime™ WGA Kit
TruePrime™ WGA Kit
Generic WGA (TruePrime with Random Primers)
• 1 pg denatured human gDNA (Promega) + 1 ng non-denatured yeast gDNA
• TruePrime™ WGA Kit and protocol (or with Random Primers substituted for Primase)
• Subjected to next gen sequencing and mapped back to known genomes
Sequencing Analysis
WGA Followed by Illumina Sequencing
• Single HEK293 cells were amplified by WGA using various kits/methods




TruePrime™ Single Cell WGA Kit
Generic MDA WGA Kit (TruePrime Kit with random primers in place of primase)
REPLI-g Single Cell WGA Kit (Qiagen)
MALBAC™ Single Cell WGA Kit (Yikon Genomics)
• Libraries were made and sequenced by:




Shearing using Covaris Focused-Ultrasonicator
Constructing libraries using NEBNext® DNA Library Prep Kit (NEB) which includes PCR
Deep sequencing on a HiSeq 2500, 2 x 125 bp, v4 chemistry
Sampling and analysis of specific number of reads based on experimental goals
Picher, AJ et al. Nat.
Comm. 7:13296 (2016)
Genomic Coverage
TruePrime™ Kit Coverage is Closest to Non-Amplified
X
Y
1
22
21
2
Non-amplified
TruePrime™ SC WGA Kit
20
19
REPLI-g SC WGA Kit
18
3
MALBAC™ SC WGA Kit
Generic MDA WGA Kit
17
16
4
15
14
5
13
6
12
7
11
10
9
8
• Analyzed 12 million read
pairs per sample
• 50 kb bin size
• Averaged coverage at
each 50 kb interval
Picher, AJ et al. Nat.
Comm. 7:13296 (2016)
Genomic Coverage of Chromosome 3
TruePrime™ Kit Coverage is Closest to Non-Amplified
Chromosome 3 Position
Coverage (0 - 50X)
Non-amplified
TruePrime™ SC WGA Kit
REPLI-g SC WGA Kit
MALBAC™ SC WGA Kit
Generic MDA WGA Kit
• Analyzed 12 million read pairs per sample
Picher, AJ et al. Nat.
Comm. 7:13296 (2016)
Zoomed Genomic Coverage of Chromosome 3
TruePrime™ Continues to Match Non-Amplified
Non-amplified
TruePrime™ SC WGA Kit
Coverage (0 - 50X)
REPLI-g SC WGA Kit
MALBAC™ SC WGA Kit
Generic MDA WGA Kit
~25M bp
Picher, AJ et al. Nat.
Comm. 7:13296 (2016)
Zoomed Genomic Coverage of Chromosome 3
TruePrime™ Continues to Match Non-Amplified
Non-amplified
TruePrime™ SC WGA Kit
Coverage (0 - 50X)
REPLI-g SC WGA Kit
MALBAC™ SC WGA Kit
Generic MDA WGA Kit
~500K bp
Picher, AJ et al. Nat.
Comm. 7:13296 (2016)
Average Breadth (%) of Coverage
Similar TruePrime™ Coverage to NA Except Chr19,22
Breadth of Coverage (%)
Non-amplified
• Analyzed 12 million
read pairs per
TruePrime™ SC WGA Kit
sample
REPLI-g SC WGA Kit
• Drop in TruePrime™
coverage for chr19
and chr22
• Other MDA
approaches as well
MALBAC™ SC WGA Kit
Chromosome
Picher, AJ et al. Nat.
Comm. 7:13296 (2016)
TruePrime™ Kit Coverage is Highly Reproducible
and Parallels Non-amplified Well
X
Y
1
Non-amplified
TruePrime™ SC WGA Kit
(Replicates 1-4)
22
21
2
20
19
18
• Analyzed 5 million
read pairs per sample
3
17
16
4
15
14
5
13
6
12
7
11
Chromosome
10
9
8
Picher, AJ et al. Nat.
Comm. 7:13296 (2016)
TruePrime™ Kit Chr4 Coverage is Highly
Reproducible and Parallels Non-amplified
Chromosome 4 Position
Chromosome 4 Position
Non-amplified
Coverage (0 - 20X)
TruePrime™ SC WGA Kit
Replicate 1
Replicate 2
Replicate 3
Replicate 4
• Analyzed 5 million read pairs per sample
Picher, AJ et al. Nat.
Comm. 7:13296 (2016)
Even Zoomed-in, TruePrime™ Chr6 Coverage is
Highly Reproducible & Parallels Non-amplified
Chromosome 6 Position
Non-amplified
TruePrime™ SC WGA Kit
Replicate 1
Replicate 3
Coverage (0 - 20X)
Coverage (0 - 30X)
Replicate 2
Replicate 4
• Analyzed 5 million read pairs per sample
Picher, AJ et al. Nat.
Comm. 7:13296 (2016)
One Last Look at Coverage Using Deep Sequencing
Non-amplified
TruePrime™
SC WGA Kit
REPLI-g SC
WGA Kit
MALBAC™ SC
WGA Kit
No significant differences in errors
Picher, AJ et al. Nat. Comm. 7:13296 (2016)
Making CNV Calls with WGA Amplified Material
CNV Calling is Possible with TruePrime™ Results
Non-amplified
Copy Number
TruePrime™ SC
WGA Kit
REPLI-g SC
WGA Kit
MALBAC™ SC
WGA Kit
Generic MDA
WGA Kit
• Used deep sequencing
results
• Calculated # of reads
per 500 kb bin
• Deduced ploidy level
using Gingko analysis
software and reads per
bin
• More reads = greater
copy number and vice
versa
Highly varied coverage
from REPLI-g, MALBAC
and Generic MDA
makes CNV calling
difficult.
Picher, AJ et al. Nat. Comm. 7:13296 (2016)
Making SNV Calls with WGA Amplified Material
Better SNV Overlap of TruePrime™ Samples with NA
SNV #
Median #
Overlapping NonAmplified (NA)
Median %
Overlapping
NA
Het>Hom SNV
Conversion
(ADO)
Non-amplified
3.02M
3.02M
100%
0%
TruePrimeTM SC WGA Kit
2.72M
2.42M
80%
5.95%
REPLI-g SC WGA Kit
1.65M
1.37M
45%
29.65%
MALBACTM SC WGA Kit
2.55M
0.82M
30%
31.05%
• Used deep sequencing results
• Used 4 different SNV callers to identify/analyze SNV and Het>Hom conversion
• ISAAC, Samtools / Bcftools, Varscan2, CLC Low Frequency Caller
• Median results are shown
• Numbers varied considerably depending the analysis program used
Better SNV overlap and lower Het>Hom conversion rates with TruePrime™ amplified
samples when compared to non-amplified sample results.
Picher, AJ et al. Nat. Comm. 7:13296 (2016)
Summary: TruePrime™ WGA Kits
More Uniform Amplification Improves NGS Results
• High sequencing breadth of coverage nearly equal to non-amplified samples
 91.27% at ~19X depth vs. 91.64% at ~19X depth for non-amplified
 Significantly better than REPLI-g and MALBAC results (85.6%, 58.9% respectively)
• More uniform sequencing depth that parallels non-amplified sequencing the best
• High quality SNV calling is possible with TruePrime™ amplified samples
 80.6% overlap with SNVs called in the non-amplified samples
• Decreased heterozygous SNV to homozygous SNV conversions with TruePrime™
amplified samples
Sygnis TruePrime™ WGA Kits are Available
from Lucigen in the U.S.*
Lucigen Cat.
Size (rxn)
No.
Sygnis TruePrimeTM WGA Kits
TM
Sygnis TruePrime Single Cell WGA Kit version 2.0
U.S. List
Price
SYG370025
25
$248
SYG380100
100
$675
SYG351025
25
$560
SYG351100
100
$1890
Visit the TruePrime™ WGA Kits webpages
TruePrime WGA Kit: https://goo.gl/HvJKtq
TruePrime Single Cell WGA Kit: https://goo.gl/WR4K3T
*Lucigen is a Sygnis distributor for the United States.
For those outside the U.S., please contact Lucigen
Customer Service ([email protected]) and we will
connect you with Sygnis/Expedeon
One Last Thought
PCR Amplified NGS Libraries Were Used…
• PCR introduces its own bias within the library
• Could using PCR-free library prep improve the results even more?
• WGA produces enough material for PCR-free library prep
• The Lucigen NxSeq® AmpFREE Low DNA Library Kit requires only 75 ng
sheared gDNA and produces the most efficient PCR-free libraries
Learn more about the
NxSeq® AmpFREE Low DNA Library Kit
https://goo.gl/3cBYmb
Questions? www.lucigen.com
Lucigen Tech Support
[email protected]
1 (608) 831-9011
8 am – 5 pm central time
Contact me.
Rob Brazas, Ph.D.
Sr. Product Manager
[email protected]
Thank You
and
Our Friends at Sygnis!