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![Exome sequencing
• Affordable approach to interrogate protein
coding portion of the genome and identify
disease-causing variants
• Mendelian disorders
– Prenatal diagnosis, preimplementation
genetic diagnosis, carrier/mutation detection
of heterogeneous disorders (e.g., hearing
loss), prognosis of preclinical individuals
• Personalized cancer therapy
– Diagnosis and prognosis, treatment,
discovery of new cancer-related genes,
address tumor heterogeneity and evolution
[http://www.dnalinkus.com/us/service/exome_sequencing.html]](https://image.slidesharecdn.com/analyzingtheexomewebinarkgiorda041917-170420142832/75/Analyzing-the-exome-focusing-your-NGS-analysis-with-high-performance-target-capture-3-2048.jpg)
![Exome Aggregation Consortium (ExAC)
• APOE is typically poorly represented in exome capture
27
[http://exac.broadinstitute.org/gene/ENSG00000130203]](https://image.slidesharecdn.com/analyzingtheexomewebinarkgiorda041917-170420142832/75/Analyzing-the-exome-focusing-your-NGS-analysis-with-high-performance-target-capture-27-2048.jpg)
Uploaded byIntegrated DNA Technologies
Analyzing the exome—focusing your NGS analysis with high performance target capture
The document discusses exome sequencing and compares the performance of the xGen Exome Research Panel to other commercial exome sequencing panels. Key points: 1) An independent study directly compared the xGen panel to 3 other commercial exome panels and found that the xGen panel had a higher on-target rate and more uniform coverage than the other panels. 2) When deeply sequenced, the xGen panel was able to achieve greater than 20x coverage of over 94% of bases in the RefSeq database with only 40 million reads, which is 2.5-4 times fewer reads than the other panels tested. 3) The coverage profile produced by the xGen panel more closely resembled whole genome sequencing
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Introduction by Kristina Giorda on exome sequencing, its relevance and outline of topics including NGS products and exome performance.
Exome sequencing as a cost-effective method to identify disease variants, especially in Mendelian disorders and personalized cancer therapy.
Overview of xGen® products for NGS target enrichment, including technologies, probe panels, and essential reagents for library preparation.
Details on xGen Lockdown Probes, their synthesis, quality control, and hybridization techniques for effective target enrichment in NGS.
Comparative analysis of different exome capture panels, focusing on gene coverage metrics and design efficiency of probe databases.
Performance metrics of the xGen Exome Research Panel in terms of on-target rate, uniformity, and coverage for GC-rich regions.
Insights into exome deep sequencing processes, showcasing the efficiency of the xGen Exome Research Panel in terms of necessary reads and coverage.
Discussion of the Exome Aggregation Consortium's findings on APOE coverage and the advantages of the xGen panel in achieving uniform coverage.
Wrap-up and acknowledgments, concluding the presentation on exome sequencing analysis and NGS target capture.
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Analyzing the exome—focusing your NGS analysis with high performance target capture
- 1. Analyzing the exome—focusingyour NGS analysis with high performance target capture Kristina Giorda, PhD 1
- 2. Outline • Review – Exomesequencing – xGen® product portfolio – Library preparation and target enrichment • Exome performance – Multiplexed capture – Deep sequencing – Exome sequencing vs. WGS 2
- 3. Exome sequencing • Affordableapproach to interrogate protein coding portion of the genome and identify disease-causing variants • Mendelian disorders – Prenatal diagnosis, preimplementation genetic diagnosis, carrier/mutation detection of heterogeneous disorders (e.g., hearing loss), prognosis of preclinical individuals • Personalized cancer therapy – Diagnosis and prognosis, treatment, discovery of new cancer-related genes, address tumor heterogeneity and evolution [http://www.dnalinkus.com/us/service/exome_sequencing.html]
- 4. xGen® products forNGS target enrichment 4 NGS workflow
- 5. Library construction 5 Fragmentation End repairand A-tailing Adapter ligation Bead cleanup Library amplification Bead cleanup
- 6. xGen products forNGS target enrichment • xGen® Lockdown® Probe Technology – Predesigned Lockdown Panels – Predesigned Gene Capture Pools – Custom probe panels • xGen Blocking Oligos • xGen Lockdown Reagents for hybridization & wash • gBlocks® Gene Fragments for NGS spiking controls 6
- 7. NGS target captureenrichment xGen Lockdown Probes • Individually synthesized • Individual QC on every probe • Individually normalized • Pooled 7
- 8. Target enrichment byhybridization 8
- 9. xGen Lockdown Probesare individually synthesized and quality controlled Each xGen Lockdown Probe receives an individual ESI-MS analysis Failed Remade Full length Truncated Full length 9
- 10. Inventoried xGen targetcapture panels 10 Panel No. of genes Target capture size (Mb) No. of probes Exome 19,396 51.0 429,826 AML 264 1.4 11,743 Pan-Cancer 127 0.8 7,816 Human ID 0.03 229 Human mtDNA 0.02 138
- 11. Batch-to-batch consistency 30,895 Enjoy thesame highly uniform coverage for custom xGen® Lockdown Panels 11
- 12. Outline • Review – Exomesequencing – xGen® product portfolio – Library preparation and target enrichment • Exome performance – Multiplexed capture – Deep sequencing – Exome sequencing vs. WGS 12
- 13. Comparative study design 13 1.An independent large genome center compared 4 exome capture panels 2. 12 libraries were made with KAPA Hyper Prep kit 3. Libraries were split into each capture 4. Captures were completed by each vendor’s protocol 5. Captures were sequenced on a lane of the HiSeq® 2500 instrument in high output mode and subsampled to 17M PE reads or 5.1 Gb of sequencing
- 14. Probe design overlapwith exon databases 14 % Database covered Database / probe space xGen® panel Vendor R Vendor A Vendor I (Size bp) (51 Mb) (45 Mb) (54 Mb) (45 Mb) RefSeq (34 Mb) 98% 97% 97% 100% CCDS (32 Mb) 99% 98% 98% 100% Ensembl (35 Mb) 93% 92% 96% 99% GENCODE (35 Mb) 96% 95% 97% 99% Vega (26 Mb) 95% 96% 97% 100%
- 15. xGen Exome ResearchPanel: Higher on-target rate 15 On-target (%) = (on bait bases + near bait bases) / aligned bases
- 16.
- 17. xGen Exome ResearchPanel: Higher coverage uniformity 17
- 18. First exons areGC rich 18 Kalari, K.R. et al. (2006) First exons and introns - a survey of GC content and gene structure in the human genome. In Silico Biology, 6(3):237-242
- 19. xGen Exome ResearchPanel: RB1 1st exon performance 19
- 20. xGen Exome ResearchPanel: Best 1st exon coverage 20 Coverage
- 21. Outline • Review – Exomesequencing – xGen® product portfolio – Library preparation and target enrichment • Exome performance – Multiplexed capture – Deep sequencing – Exome sequencing vs. WGS 21
- 22. Exome deep sequencing 22 1.1 library made with KAPA Hyper Prep kit 2. Each library was sequenced on a lane of the HiSeq 4000 instrument 3. Sequencing was subsampled to matched reads
- 23. xGen Exome ResearchPanel: More coverage with fewer reads 23
- 24.
- 25. xGen Exome ResearchPanel: More coverage with fewer reads 25 Genescovered≥X(%) Genescovered≥20X(%) Number of reads (M) Minimum coverage
- 26. Outline • Review – Exomesequencing – xGen® product portfolio – Library preparation and target enrichment • Exome performance – Multiplexed capture – Deep sequencing – Exome vs. WGS 26
- 27. Exome Aggregation Consortium(ExAC) • APOE is typically poorly represented in exome capture 27 [http://exac.broadinstitute.org/gene/ENSG00000130203]
- 28. xGen Exome ResearchPanel: APOE is uniformly covered 28
- 29. xGen Exome ResearchPanel: Better resemblance to WGS data 29
- 30. xGen Exome ResearchPanel • Exhibits superior uniformity • Yields the most complete coverage of RefSeq database • The only commercial exome panel without reduced first exon coverage • Achieves ≥20X coverage of >94% of bases in RefSeq database with only 40M reads, ~2.5 – 4X fewer than any other exome panel tested • Coverage profile resembles whole genome data 30
- 31.