Maximizing your NGS sequencing with IDT
Transcript of Maximizing your NGS sequencing with IDT
Maximizing your NGS sequencing with IDT
Adam Chernick, PhDField Applications Manager, Functional Genomics
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Contents
• Expanding our NGS portfolio – what’s next?
• xGen® technology and Lockdown® probe advantages
– Exome case study
• Adapter strategy considerations
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Expanding our NGS product portfolio
Amplicon Sequencing
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What is RnaseH2 PCR (rhPCR)?
• rhPCR is RNase H2-dependent PCR with blocked primers
• rhPCR is different from standard PCR– Primers blocked at 3’
– Primers have a single RNA base near the 3’ end
– It requires both Taq and RNase H2
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Dobosy et al. BMC Biotechnology (2011) 11:80
https://doi.org/10.1186/1472-6750-11-80
rhPCR
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rN
RNase H2
Cleavage
rN : RNA base
: Mismatch base
: 3’ Blocker
Forward rhPrimer
Reverse rhPCR primer
PCR with DNA Pol
Forward PCR primer
Reverse PCR primer
rN
gDNA target
PCR Product
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rhPCR
technology
Amplicon
sequencing
rhAmpSeq™
rhAmpSeq™ workflow
Step 1: Multiplex
rhPCR
Step 3: Universal
PCR
DNA
Step 2: Dilution
Step 4: Pool samples and
single SPRI purification
Sequence
Applications of rhAmpSeq™ for AgBio
• Molecular breeding and genetic analysis
• Analysis of CRISPR editing events
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# markers*
10’s 1000’s
Marker assisted selection
Marker assisted backcrossing
Genomic selection
* Initial launch of rhAmpSeq will target
hotspot SNP and short indel markers
Animal parentage and breeding
Development data 1000-3000 plex
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% On-target % Uniformity
rhPCR chemistry greatly reduces primer-dimers and other
non-specific amplification
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10 ng NTC 10 ng NTC
PCR Primer rhPCR Primer
• 96-plex
• 10 ng human gDNA
Chemistry enables high multiplex-
ability and reduces complexity of
IDT’s amplicon sequencing system
Support for primer design with degenerate bases
Primers with N-base
Solution
Flexibility of panel designs
Expanding panel
• Add to existing content
Sub-panels
• Select subsets from existing
panel
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No change to
existing panel
primer design
Features and Performance
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Feature Specification
Input DNA 10-100 ng
Amplicon size Flexible – 40-200+
Panel size Up to 5,000 amplicons per panel
Sample indexing
capability
96 index sequences – up to 9216
combinations
Sequencing
performance
>90% mapped reads
>90% on-target
>90% coverage uniformity
Compatible platforms Illumina, Ion Torrent
Supported workflows Standard and HT
xGen® Lockdown® Probes
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Target capture with Lockdown probes
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NGS workflow
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xGen® Hybridization
and Wash Kit
xGen® Lockdown® Probes
Available manufactured under
21 CFR Part 820 (GMP)
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QC failures are excluded
and resynthesized
Individually normalized
Individually quality
controlled by ESI-MS
Individually synthesized biotinylated
120mer
• Additional target content can be added quickly and inexpensively to IDT panels
• Mix and match gene capture pools, stocked panels and custom pools
• Customers can “tune” the capture as needed to adjust for the desired coverage depth
• 6–8 weeks lead time required for other vendors to accomplish the same
Modularity of xGen® Panels
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Adapters for NGS library prep
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Indexing strategies using ligation
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Ligation
Library amplification
Considerations when choosing adapters
• Application
• Sensitivity and specificity
– molecular barcoding
– sample cross-talk/index hopping
• Sample quality
– low input, degraded material, high quality, etc.
• Sample number and sequencing platform
– barcode selection
• Analysis pipeline
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Growing awareness of sample crosstalk
• Index hopping with Illumina
patterned flow cells blasted into
the spotlight with a bioRxiv
publication in April 2017
• Consequences can be
enormous: worst case scenario,
incorrect mutation status
reported for patients
• Index contamination may occur
on any sequencing platform
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Index hopping during multiplexed sequencing
• Patterned flow cells utilize exclusion amplification (ExAmp) chemistry,
associated with more index mis-assignment than bridge amplification
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Sinha R, Stanley G, et al. (2017) Index switching causes “spreading-of-signal” among multiplexed samples in
Illumina HiSeq 4000 DNA sequencing. bioRxiv.
www.illumina.com/science/education/minimizing-index-hopping.html
Illumina’s recommendations
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www.illumina.com/content/dam/illumina-marketing/documents/products/whitepapers/index-hopping-white-paper-
770-2017-004.pdf?linkId=36607862
Index hopping during multiplexed target capture
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• There are low levels of index hopping in multiplexed target enrichment
– index hopping will increase with increase in multiplex #
Index hopping reads are effectively filtered out
with unique dual indices
• Index hopping reads are filtered
out with unique dual index
adapters
• Reads would have been mis-
assigned with combinatorial
indices
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Unique dual index adapters
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Unique dual index (UDI) + UMI experiment
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Sensitivity and positive predictive value (PPV) with
consensus analysis with 0.25% variant frequency
threshold
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TP
Total reads
TP
De-dup with start/stop
TP
With UMIs
97
98
99
100
20 40 60 80 100
PPV (%)
Se
nsitiv
ity (
%)
No UMI
(Start/Stop)
UMI
TP
De-dup with UMIs
Mean de-duped coverage
No UMI
(Start/Stop)
0
2000
4000
6000
8000
UMI
Sensitivity and positive predictive value (PPV) with
consensus analysis with 0.25% variant frequency
threshold
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TP
Consensus
97
98
99
100
20 40 60 80 100
PPV (%)
Se
nsitiv
ity (
%)
No UMI
(Start/Stop)
UMIConsensus
Mean de-duped coverage
No UMI
(Start/Stop)
UMI
0
2000
4000
6000
8000
Consensus
Consensus calling reduces false positives
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FP called FP filtered TP called TP filtered TP missing Sensitivity PPV
No UMI (Start/Stop) 641 2 241 0 1 99.59% 27.32%
UMI 368 0 239 0 3 98.76% 39.37%
Consensus Only 2 13 239 0 3 98.76% 99.17%
Levels of error correction and sensitivity
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TruGrade® DNA Oligos
Reduce barcode crosstalk in your multiplexed NGS experiments
Available as:
ssDNA dsDNA plates tubes
Delivered in:
single use plates 22
Summary
• Target enrichment is highly utilized in the sequencing arena
• Lockdown® hybridization capture technology offers several unique advantages including individual probe synthesis and QC
• There are many considerations to take into account when picking adapters – we welcome consultations to help make those decisions
• Molecular indexing (UMI) consensus reads dramatically increase variant calling accuracy
• Amplicon sequencing to be launched soon!
• IDT will continue to expand our NGS portfolio to meet the ever-evolving demands in research and in multiple other settings
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THANK YOU
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