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Katherine E. Varley , Randy C. Bachmeyer , Irina A. Vasenkova , Tatiana Shvetsova , Katie Jansen Spayd , Richard M. Myers , D. Troy Moore 1,21,2
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TargetRich™ and Nested PatchPCR™ are trademarks of Kailos Genetics, Inc.
Nested PatchPCR™ for Highly Multiplexed Targeted Sequencing in Cancer SamplesNested PatchPCR™ for Highly Multiplexed Targeted Sequencing in Cancer Samples
Kailos Genetics Inc., Huntsville, AL 35806HudsonAlpha Institute for Biotechnology, Huntsville, AL 35806
Abstract
The ability to deeply sequence hundreds to thousands of targeted genomic regions across a large number of patient samples is crucial for adopting next-generation sequencing in clinical research and clinical practice settings. Various targeting/capture methods have been devel-oped for next-generation sequencing, but the limitations of each approach become conspicuous when applied to large-scale clinical research applications: large input DNA quantities, wasted o�-target sequencing, high cost of reagents, low sample throughput, specialized equipment, and in�exible content design. To address these limitations, we have developed Nested PatchPCR™ to perform targeted sequencing across hun-dreds of genomic regions in large numbers of patient samples to fully utilize next-generation sequencing capacity for clinical re-sequencing. We have used Nested PatchPCR™ to simultaneously amplify 760 genomic regions, covering the exons of 62 genes that are frequently mutated in cancer. The procedure required only a standard PCR machine and the patient samples were processed in parallel in 96-well plates. Nested PatchPCR™ was performed from 250 ng genomic DNA, including the fragmented DNA obtained from formalin �xed para�n embedded samples (FFPE). The Nested PatchPCR™ products are compatible with standard library construction/barcoding for sequencing on various plat-forms.
We used Nextera library construction and barcoding to sequence 96 samples on a single Illumina GAIIx �owcell. Nested PatchPCR™ was highly speci�c; >90% of sequencing reads align to the targeted regions. The high percentage of on-target sequence data and focused content, allowed us to achieve high coverage across the loci (average of 400X read depth) for the sensitive detection of cancer mutations.
We have also created a more focused cancer panel (157 regions covering exons from 10 genes) that is scaled down to take advantage of the throughput of the Life Technologies Ion Torrent PGM and the Illumina MiSeq. Platform speci�c primers can be added during the Nested Patch-PCR™ reaction so that the products can be sequenced directly without time-consuming library construction.
Nested PatchPCR Work�ow
TargetRich™ Kits
ConclusionsNested PatchPCR provides highly speci�c multiplexed PCR of a large number of targeted loci for next-generation sequencing.
Now available as Kailos Genetics TargetRich™ Kits:
• SPECIFIC - high on-target alignment rate means no wasted sequence
• SENSITIVE - improved uniformity provides high coverage across targets for sensitive mutation detection
• LOW STARTING MATERIAL - requires just 250 ng DNA
• SCALABLE - samples can be prepared in parallel in 96-well plates
• EASY - addition-only reactions on standard thermocyclers
• FAST - single day work�ow
PARTICIPATE IN THE EARLY ACCESS PROGRAMFor additional information, call 866-833-6865 or email [email protected]
kailosgenetics.com/targetrich
Multiplexed low-cycle PCR defines
targeted regions
Enzymes digest primers and blunt ends
Patch oligos direct ligation of universal
primers with protecting group to correct
amplicons
Enzymes digest unprotected off-target
amplicons
Universal PCR amplifies all targets
simultaneously in the same reaction
250ng gDNA per sample in a 96-well
PCR plate
1 hr 25 min
1 hr 40 min
2 hr 20 min
1 hr 55 min
1 hr 50 min
Targeting PCR
Primer Removal
Patch Ligation
O�-Target AmpliconDegradation
Universal PCR
Cancer Research
(CRX)
Cancer Survey (CSP)
coming soon
Genes 10 62
Exons 161 760
Amplicons 157 760
Genome Size 38.8 Kb 165 Kb
Amplicon Size 100-1,100 bp 100-1,100 bp
Gender Controls
SRY: 624 bases USP9Y: 703 bases
SRY: 624 bases USP9Y: 703 bases
Cancer Research CRX v1.4 Content
Gene Number of Amplicons
Base-pairs Covered
BRAF 18 4,483 EGFR 28 5,347 FLT3 23 4,979 JAK2 22 6,233 KIT 20 4,375
KRAS 5 1,249
PIK3CA 16 5,509
PTEN 9 3,062 TP53 8 1,792
VEGFA 8 1,812
Total: 157 38,841
Custom designed panels available by request
*100Mbp of Sequence = 1 Million Paired-End 50bp Reads = 1/5 of a MiSeq = 1/30 of a GAIIx Lane =
1/180 of HiSeq Lane ~= 1/10 of an Ion PGM
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Amplicons Ordered By Coverage
CSP Kit Performance 760 Amplicons Covering 62 Cancer Genes
CSP-Rep1 CSP-Rep2 CSP-Rep3 CSP-Rep4
Results
Sample Percent of Passed Filter Reads That Align to Targets
CSP-Rep1 92.53% CSP-Rep2 92.45% CSP-Rep3 93.07% CSP-Rep4 93.39%
Method Percentage of Reads That Align to Targets
Kailos Genetics CRX 92.04% Ion AmpliSeq 14.74%
Kailos Genetics’ high on-target alignment rate results in higher coverage per amplicon
Platform Content Method Time Starting Material
Percent of Reads Aligning
to Targets
Kailos Genetics
TargetRich Targeted PCR 9 hrs 250ng 92%
Illumina TruSeq6 Exome Hybridization 19 hrs 500ng 62%1
Agilent SureSelect7 Exome Hybridization 94 hrs 3ug 54%3
Nimblegen SeqCap8 Exome Hybridization 82 hrs 1ug 46%2
Ion AmpliSeq Targeted PCR 3.5 hrs 10ng 15%9
Spec Comparison
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Amplicons Ordered By Coverage
Performance Comparison: Kailos CRX versus Ion AmpliSeq Cancer Panel
Kailos CRX
Ion AmpliSeq
*100Mbp of Sequence = 1 Million Paired-End 50bp Reads = 1/5 of a MiSeq = 1/30 of a GAIIx Lane = 1/180 of HiSeq Lane ~= 1/10 of an Ion PGM
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Amplicons Ordered By Coverage
CRX Kit Performance 157 Amplicons Covering 10 Cancer Genes
CRX-Rep1 CRX-Rep2 CRX-Rep3 CRX-Rep4
Sample Percent of Passed Filter Reads That Align to Targets
CRX-Rep1 92.04% CRX-Rep2 91.00% CRX-Rep3 91.46% CRX-Rep4 91.43%
*100Mbp of Sequence = 1 Million Paired-End 50bp Reads = 1/5 of a MiSeq = 1/30 of a GAIIx Lane =
1/180 of HiSeq Lane ~= 1/10 of an Ion PGM
KRAS Design:
Targeting PCR Primers
and Patch Oligos
Example of Uniform and Reproducible Amplicon Coverage
KRAS Design:
CRX-Rep1 CRX-Rep2 CRX-Rep3 CRX-Rep4
KRAS
Ampli
con
KRAS
Am
plico
nKR
AS
Ampli
con
KRAS
Am
plico
n
1
KRAS
Ampli
con 5
KRAS
Ampli
con 4
KRAS
Ampli
con 3
KRAS
Ampli
con 2
KRAS
Ampli
con 1
1944
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111Katherine E. Varley , Randy C. Bachmeyer , Irina A. Vasenkova , Tatiana Shvetsova , Katie Jansen Spayd , Richard M. Myers , D. Troy Moore
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1. Clark, M.J. & Snyder, M. Nature Biotech. 2011 Vol 29, No. 10 908-916 2. Kiialainen, A. & Syvanen, A-C PlosOne 2011 Vol. 6, Issue 2, e16486:1-10 3. Sulonen, A-M & Saarela, J. Genome Biology 2011 12:R94 4. Teer, J. K. & Biesecker, L. G. Genome Res. 2010 20:1420-1431 5. Varley, K. E. & Mitra, D. Genome Res. 2008 18:1844-1850 6. Illumina TruSeq™ Enrichment Guide (June 2011) 7. Agilent SureSelect™ Guide (May 2010) 8. Roche SeqCap EZ Guide (April 2010) 9. Shotgun Nextera Library on GAIIx
