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

+

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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