Maximizing your NGS sequencing with IDT Adam Chernick, PhD Field Applications Manager, Functional Genomics 1
Contents Expanding our NGS portfolio what s next? xgen technology and Lockdown probe advantages Exome case study Adapter strategy considerations 2
Expanding our NGS product portfolio Amplicon Sequencing 3
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 Dobosy et al. BMC Biotechnology (2011) 11:80 https://doi.org/10.1186/1472-6750-11-80 4
rhpcr rn : RNA base : Mismatch base : 3 Blocker Forward rhprimer rn RNase H2 Cleavage rn Reverse rhpcr primer gdna target Forward PCR primer PCR with DNA Pol Reverse PCR primer PCR Product 5
rhpcr technology Amplicon sequencing rhampseq 6
rhampseq workflow DNA Step 1: Multiplex rhpcr Step 2: Dilution Step 3: Universal PCR Step 4: Pool samples and single SPRI purification Sequence
Applications of rhampseq for AgBio Molecular breeding and genetic analysis # markers* * Initial launch of rhampseq will target hotspot SNP and short indel markers 10 s 1000 s Marker assisted selection Genomic selection Marker assisted backcrossing Animal parentage and breeding Analysis of CRISPR editing events 8
Development data 1000-3000 plex % On-target % Uniformity 9
rhpcr chemistry greatly reduces primer-dimers and other non-specific amplification PCR Primer rhpcr Primer 10 ng NTC 10 ng NTC Chemistry enables high multiplexability and reduces complexity of IDT s amplicon sequencing system 96-plex 10 ng human gdna 10
Support for primer design with degenerate bases Solution Primers with N-base
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 Feature Input DNA Specification 10-100 ng Amplicon size Flexible 40-200+ Panel size Sample indexing capability Sequencing performance Compatible platforms Supported workflows Up to 5,000 amplicons per panel 96 index sequences up to 9216 combinations >90% mapped reads >90% on-target >90% coverage uniformity Illumina, Ion Torrent Standard and HT 13
xgen Lockdown Probes 14
Target capture with Lockdown probes 15
NGS workflow xgen Hybridization and Wash Kit 16
xgen Lockdown Probes Individually synthesized biotinylated 120mer Individually quality controlled by ESI-MS Individually normalized QC failures are excluded and resynthesized Available manufactured under 21 CFR Part 820 (GMP) 17
Modularity of xgen Panels 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 15
Adapters for NGS library prep 19
Indexing strategies using ligation Ligation Library amplification 20
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 21
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 22
Index hopping during multiplexed sequencing Patterned flow cells utilize exclusion amplification (ExAmp) chemistry, associated with more index mis-assignment than bridge amplification 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 23
Illumina s recommendations www.illumina.com/content/dam/illumina-marketing/documents/products/whitepapers/index-hopping-white-paper- 770-2017-004.pdf?linkId=36607862 24
Index hopping during multiplexed target capture There are low levels of index hopping in multiplexed target enrichment index hopping will increase with increase in multiplex # 25
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 misassigned with combinatorial indices 26
Unique dual index adapters 27
Unique dual index (UDI) + UMI experiment 28
Sensitivity (%) Sensitivity and positive predictive value (PPV) with consensus analysis with 0.25% variant frequency threshold TP 8000 6000 4000 Mean de-duped coverage 100 99 No UMI (Start/Stop) UMI 2000 98 0 97 De-dup Total With with with reads UMIs start/stop UMIs No UMI (Start/Stop) UMI 20 40 60 80 100 PPV (%) 29
Sensitivity (%) Sensitivity and positive predictive value (PPV) with consensus analysis with 0.25% variant frequency threshold TP 8000 6000 4000 Mean de-duped coverage 100 99 No UMI (Start/Stop) UMI Consensus 2000 98 0 97 Consensus No UMI (Start/Stop) UMI Consensus 20 40 60 80 100 PPV (%) 30
Consensus calling reduces false positives 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% 31
Levels of error correction and sensitivity 32
TruGrade DNA Oligos Reduce barcode crosstalk in your multiplexed NGS experiments Available as: Delivered in: ssdna dsdna plates tubes 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 everevolving demands in research and in multiple other settings 34
THANK YOU 35