High-throughput processing to maximize genomic analysis through simultaneous recovery of DNA and RNA...
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Angie Cheng 1 , Natalie Hernandez 1 , Rasika Sunnadeniya, Denisse Meza 1 , Jen Whiting 1 and Xingwang Fang 1 Thermo Fisher Scientific 1 2130 Woodward St. Ste 200, Austin, TX, USA, 78744 [email protected] Thermo Fisher Scientific • 5791 Van Allen Way • Carlsbad, CA 92008 • www.lifetechnologies.com High-throughput processing to maximize genomic analysis through simultaneous recovery of DNA and RNA from the same FFPE sample in separate eluates INTRODUCTION As personalized cancer care evolves, the patient’s nucleic acid becomes ever so important to provide valuable information regarding their genetic makeup and disease state. Common sample types for these analyses include biopsies, which can be very limited in material making the downstream measurement of more than one analyte rather difficult. Obtaining another biopsy, using a different section or splitting the sample can be problematic because of tumor heterogeneity. Even adjacent areas of the same tumor tissue can result in different RNA/DNA profiles so the ability to isolate multiple analytes from the same sample offer a number of benefits, which include preserving samples and data consistency eliminating any sample to sample variation. As more tests are developed to simultaneously monitor genetic alterations, there is a strong need to efficiently isolate both DNA and RNA from the same starting sample in a format compatible with high-throughput processing. MATERIALS AND METHODS Here we describe a novel chemistry using magnetic beads and robust workflow that will eliminate extraction variabilities and process a large number of samples with consistency and ease to allow isolation of DNA and RNA from the same sample; while allowing the flexibility to isolate only DNA or only RNA. We’ve developed both manual and automated protocols on the KingFisher™ Flex, Duo Prime and Presto purification systems. We have extracted from a number of different FFPE cancer tissues including breast, lung, colorectal, and melanoma from tumor resections, core needle biopsies (CNBs) and fine needle aspirates (FNAs) for various downstream applications such as real-time PCR and targeted sequencing with different panels. FFPE blocks were purchased from various vendors. Age of blocks ranged from 1981-2015 with various tumor stages. FFPE slides or curls were deparaffinized by Xylene, CitriSolv or with the use of spin cups followed by protease digestion. DNA and RNA were then isolated from the same sample following Figure 1 using the MagMAX™ FFPE DNA/RNA Ultra Kit. The obtained nucleic acids were quantified with the Quant-IT™ dsDNA and RNA assay respectively for DNA and RNA quantification. RNA was also assessed using the Agilent 2100 Bioanalyzer® instrument. A pico or nano chip was run to obtain RNA integrity information. As expected, samples were degraded with RIN values of <2.5. Real-time PCR was performed using the isolated DNA and RNA to assess functionality. Total RNA was reverse transcribed in 20ul using the SuperScript™ VILO™cDNA synthesis kit. The TaqMan™ MicroRNA Reverse Transcription kit was used for miRNA expression. 2ul of the cDNA or DNA was used in a 10ul qPCR reaction using the TaqMan™ Universal Master Mix II, with no UNG. For gene expression, the GAPDH TaqMan™ Gene Expression assay ID Hs02758991_g1 was used. Both primers and probe map within a single exon producing a 93bp amplicon. For miRNA expression, TaqMan™ MicroRNA assays for let7e was used. Reactions were run on the 7900HT real time PCR machine under standard cycling conditions. DNA and RNA AmpliSeq™ libraries were made with 10ng starting input according to the protocol for library prep. DNA libraries were made with the Ion AmpliSeq™ Cancer Hotspot panel v2 or the Oncomine™ Focus Assay. RNA libraries were made with the Ion AmpliSeq ™ RNA Cancer panel or the Oncomine Focus assay. Libraries were barcoded using the Ion Xpress™ Barcode Adapters. Final libraries were quantified by qPCR using the Ion Library Quantitation kit. We used the Ion Chef™ instrument for template preparation and chip loading. Samples were sequenced on the Ion PGM™ Sequencer on a Ion 318™ chip. After runs were completed, data was analyzed with either the coverage analysis plug in and Variant Caller on the Torrent Suite or the Ion Reporter™ Software. RESULTS Figure 1. MagMAX™ FFPE DNA/RNA Ultra Kit Isolation Workflow Figure 2a. AcroMetrix™ MultiMix FFPE Controls with known variants Chrom osom e Gene Type Expected Variant Frequency Observed Variant Frequency Reference Variant 2 ERBB4 SN P 41.2 49.3 T C 4 PD GFRA SN P 100 100 A G 4 PD GFRA SN P 96.3 100 C T 4 KIT SN P 100 100 A C 4 KD R SN P 100 100 T A 4 KD R SN P 100 100 C T 5 A PC SN P 100 100 G A 5 CSF1R 100 5 CSF1R 100 7 EGFR SN P 100 100 G A 10 RET SN P 57.4 56.5 G T 11 HRAS SN P 100 100 A G 12 KRAS SN P 100 100 C A 13 FLT3 SN P 100 100 A G 17 TP53 SN P 100 100 C T 17 TP53 SN P 100 100 C T 17 TP53 SN P 100 100 G C 18 SM AD 4 SN P 100 100 G C 3 PIK3CA SN P 44.3 49.1 G A 3 PIK3CA SN P 44.3 49.1 G A 3 PIK3CA SN P 44.7 49 G A 4 FGFR3 SN P 50.8 52.1 C A 4 PDGFRA SN P 100 100 A G 4 KIT SN P 46.5 50.6 G A 4 KDR SN P 100 100 C T 5 C SF1R SN P 100 5 C SF1R SN P 96.1 7 EGFR SN P 15.5 13.5 C T 7 EGFR SN P 49.5 47 G A 7 M ET SN P 17.2 16 C T 7 SM O SN P 46.3 51.1 A G 8 FGFR1 SN P 51 53 C A 9 NOTCH1 SN P 53 49.7 G A 11 H RAS SN P 33.6 35.8 A G 12 KRAS SN P 49.5 49.6 C T 13 FLT3 SN P 51.5 49.3 A G 17 TP53 SN P 49.8 49.6 G A 17 TP53 SN P 97.5 100 G C 22 SM ARCB1 SN P 53.5 48 G A AcroM etrix™ M ultiM ix F FFPE Control 100 TG GA A croM etrix™ M ultiM ix C FFPE Control 100 TG GA MNP DNA and RNA was extracted from 1x10um FFPE curl with the KingFisher™ Flex Purification system. 10ng of DNA was used to generate libraries with the Ion AmpliSeq Cancer Hotspot panel v2. Samples were then sequenced on the PGM on a 318™ chip. All expected variants were detected from the FFPE controls with equivalent or better allelic frequencies. The corresponding RNA was also sequenced but with the RNA Cancer panel using only 10ng of RNA. Mean read lengths were >100bp with >95% mapped. DNA and RNA was extracted from 1x10um FFPE curl with the KingFisher™ Flex Purification system. 10ng of RNA was used to generate libraries with the Ion AmpliSeq RNA fusion panel. Samples were then sequenced on the PGM on a 318™ chip. All expected fusions were detected from the FFPE control. Figure 2b. Custom solid tumor FFPE RNA control with known fusions Figure 3a. Sample Input Titration-DNA and RNA Yield To test assay sensitivity, FFPE samples were digested and subsequently diluted to as low as 10% of the sample input for the remainder of the isolation protocol from 1x10um section. Nucleic acid yield was highly linear across the full range of sample input titration, indicating high DNA and RNA recovery rates for the assay. 100% 80% 50% 20% 10% PDGFRA COSM 22413 C T 51.80% 48.40% 51.00% 50.10% 51.30% APC COSM 13125 C T 12.00% 12.30% 11.40% 10.80% 9.60% APC COSM 13123 G T 9.40% 11.70% 13.10% 6.50% 15.20% PTEN COSM 5915 C G 48.70% 52.60% 49.50% 49.70% 46.10% PTEN COSM 5218 G T 3.60% 2.80% 3.40% 3.60% 3.80% HRAS COSM 249860 A G 50.90% 53.60% 45.60% 49.50% 53.50% KRAS COSM 19404 C T 11.30% 13.20% 9.80% 11.70% 15.20% TP53 COSM 44321 A G 8.20% 9.70% 10.00% 8.30% 10.30% SM ARCB1 COSM 1090 G A 48.50% 47.60% 50.00% 50.70% 51.70% Allele Nam e Reference Variant Heterozygous SNP AllelicFrequency Sam ple input Allele Call VariantType Gene ID Figure 3b. Sample Input Titration-Variant Table Following Proteinase K digestion, the FFPE sample was diluted in fresh digestion buffer to various input percentages. Extractions were carried out according to the protocol, and libraries were prepared with the Ion AmpliSeq Cancer Hotspot Panel v2 for Ion PGM sequencing. The exact same cancer hotspot variants were detected from 100% down to 10% of input, verifying the sensitivity and functionality of the assay across the different input titrations. Figure 4a. CitriSolv vs. Xylene A less toxic alternative, CitriSolv, is compatible and comparable to the gold standard Xylene. Figure 4b. Spin cups eliminate the need for chemical solvent deparaffinizations and save time Alternatively, the use of spin cups can be used to de-wax samples. This eliminates the need for chemical solvents, ethanol washes and hoods while saving time especially when having to deparaffinize FFPE curls. Figure 5a. Core Needle Biopsies and Fine Needle Aspirates, Real-time PCR dat DNA and RNA were isolated from various biopsy or aspirate samples. Nucleic acid functionality was tested by real-time PCR yielding good Ct values. Figure 5b. Core Needle Biopsies and Fine Needle Aspirates, Sequencing data Isolated nucleic acids were then sequenced with the Oncomine Focus assays on the PGM. High sequencing metrics were achieved for DNA and RNA such as mean read lengths, % uniformity, % no strand bias, % end to end reads and % reads on target. Figure 6. Manual vs. Automated Extractions Equivalent yields were achieved by manual extracting versus automating purifications on the Duo or Flex purification systems. CONCLUSIONS In summary, we have a robust workflow that allow for the extraction of DNA and RNA from the same FFPE sample. Samples can be processed manually or with automation on the KingFisher purification systems with concordant variant detection. From tiny amounts of material such as CNB’s or FNA’s to bigger tissues such as tumor resections, samples generated quality sequencing data meeting all of our core sequencing metrics. TRADEMARKS/LICENSING For Research Use Only. Not for use in diagnostic procedures. ©2016 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. TaqMan is a registered trademark of Roche Molecular Systems, Inc., used under permission and license. Bioanalyzer is a trademark of Agilent Technologies, Inc.
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Transcript of High-throughput processing to maximize genomic analysis through simultaneous recovery of DNA and RNA...
Angie Cheng1, Natalie Hernandez1, Rasika Sunnadeniya, Denisse Meza1, Jen Whiting1 and Xingwang Fang1 Thermo Fisher Scientific1 2130 Woodward St. Ste 200, Austin, TX, USA, [email protected]
Thermo Fisher Scientific • 5791 Van Allen Way • Carlsbad, CA 92008 • www.lifetechnologies.com
High-throughput processing to maximize genomic analysis through simultaneous recovery of DNA and RNA from the same FFPE sample in separate eluates
INTRODUCTIONAs personalized cancer care evolves, the patient’s nucleic acid becomes ever so important to provide valuable information regarding their genetic makeup and disease state. Common sample types for these analyses include biopsies, which can be very limited in material making the downstream measurement of more than one analyte rather difficult. Obtaining another biopsy, using a different section or splitting the sample can be problematic because of tumor heterogeneity. Even adjacent areas of the same tumor tissue can result in different RNA/DNA profiles so the ability to isolate multiple analytes from the same sample offer a number of benefits, which include preserving samples and data consistency eliminating any sample to sample variation. As more tests are developed to simultaneously monitor genetic alterations, there is a strong need to efficiently isolate both DNA and RNA from the same starting sample in a format compatible with high-throughput processing.
MATERIALS AND METHODSHere we describe a novel chemistry using magnetic beads and robust workflow that will eliminate extraction variabilities and process a large number of samples with consistency and ease to allow isolation of DNA and RNA from the same sample; while allowing the flexibility to isolate only DNA or only RNA. We’ve developed both manual and automated protocols on the KingFisher™ Flex, Duo Prime and Presto purification systems. We have extracted from a number of different FFPE cancer tissues including breast, lung, colorectal, and melanoma from tumor resections, core needle biopsies (CNBs) and fine needle aspirates (FNAs) for various downstream applications such as real-time PCR and targeted sequencing with different panels.
FFPE blocks were purchased from various vendors. Age of blocks ranged from 1981-2015 with various tumor stages. FFPE slides or curls were deparaffinized by Xylene, CitriSolv or with the use of spin cups followed by protease digestion. DNA and RNA were then isolated from the same sample following Figure 1 using the MagMAX™ FFPE DNA/RNA Ultra Kit. The obtained nucleic acids were quantified with the Quant-IT™ dsDNA and RNA assay respectively for DNA and RNA quantification. RNA was also assessed using the Agilent 2100 Bioanalyzer® instrument. A pico or nano chip was run to obtain RNA integrity information. As expected, samples were degraded with RIN values of <2.5.
Real-time PCR was performed using the isolated DNA and RNA to assess functionality. Total RNA was reverse transcribed in 20ul using the SuperScript™ VILO™cDNA synthesis kit. The TaqMan™ MicroRNA Reverse Transcription kit was used for miRNA expression. 2ul of the cDNA or DNA was used in a 10ul qPCR reaction using the TaqMan™ Universal Master Mix II, with no UNG. For gene expression, the GAPDH TaqMan™ Gene Expression assay ID Hs02758991_g1 was used. Both primers and probe map within a single exon producing a 93bp amplicon. For miRNA expression, TaqMan™ MicroRNA assays for let7e was used. Reactions were run on the 7900HT real time PCR machine under standard cycling conditions.
DNA and RNA AmpliSeq™ libraries were made with 10ng starting input according to the protocol for library prep. DNA libraries were made with the Ion AmpliSeq™ Cancer Hotspot panel v2 or the Oncomine™ Focus Assay. RNA libraries were made with the Ion AmpliSeq™ RNA Cancer panel or the Oncomine Focus assay. Libraries were barcoded using the Ion Xpress™ Barcode Adapters. Final libraries were quantified by qPCR using the Ion Library Quantitation kit. We used the Ion Chef™ instrument for template preparation and chip loading. Samples were sequenced on the Ion PGM™ Sequencer on a Ion 318™ chip. After runs were completed, data was analyzed with either the coverage analysis plug in and Variant Caller on the Torrent Suite or the Ion Reporter™ Software.
RESULTS Figure 1. MagMAX™ FFPE DNA/RNA Ultra Kit Isolation Workflow
Figure 2a. AcroMetrix™ MultiMix FFPE Controls with known variants
Chromosome Gene TypeExpected Variant
Frequency
Observed Variant
FrequencyReference Variant
2 ERBB4 SNP 41.2 49.3 T C4 PDGFRA SNP 100 100 A G4 PDGFRA SNP 96.3 100 C T4 KIT SNP 100 100 A C4 KDR SNP 100 100 T A4 KDR SNP 100 100 C T5 APC SNP 100 100 G A5 CSF1R 1005 CSF1R 1007 EGFR SNP 100 100 G A10 RET SNP 57.4 56.5 G T11 HRAS SNP 100 100 A G12 KRAS SNP 100 100 C A13 FLT3 SNP 100 100 A G17 TP53 SNP 100 100 C T17 TP53 SNP 100 100 C T17 TP53 SNP 100 100 G C18 SMAD4 SNP 100 100 G C3 PIK3CA SNP 44.3 49.1 G A3 PIK3CA SNP 44.3 49.1 G A3 PIK3CA SNP 44.7 49 G A4 FGFR3 SNP 50.8 52.1 C A4 PDGFRA SNP 100 100 A G4 KIT SNP 46.5 50.6 G A4 KDR SNP 100 100 C T5 CSF1R SNP 1005 CSF1R SNP 96.17 EGFR SNP 15.5 13.5 C T7 EGFR SNP 49.5 47 G A7 MET SNP 17.2 16 C T7 SMO SNP 46.3 51.1 A G8 FGFR1 SNP 51 53 C A9 NOTCH1 SNP 53 49.7 G A11 HRAS SNP 33.6 35.8 A G12 KRAS SNP 49.5 49.6 C T13 FLT3 SNP 51.5 49.3 A G17 TP53 SNP 49.8 49.6 G A17 TP53 SNP 97.5 100 G C22 SMARCB1 SNP 53.5 48 G A
AcroMetrix™
MultiMix F FFPE Control
100 TG GA
AcroMetrix™
MultiMix C FFPE Control
100 TG GAMNP
DNA and RNA was extracted from 1x10um FFPE curl with the KingFisher™ Flex Purification system. 10ng of DNA was used to generate libraries with the Ion AmpliSeq Cancer Hotspot panel v2. Samples were then sequenced on the PGM on a 318™ chip. All expected variants were detected from the FFPE controls with equivalent or better allelic frequencies. The corresponding RNA was also sequenced but with the RNA Cancer panel using only 10ng of RNA. Mean read lengths were >100bp with >95% mapped.
DNA and RNA was extracted from 1x10um FFPE curl with the KingFisher™ Flex Purification system. 10ng of RNA was used to generate libraries with the Ion AmpliSeq RNA fusion panel. Samples were then sequenced on the PGM on a 318™ chip. All expected fusions were detected from the FFPE control.
Figure 2b. Custom solid tumor FFPE RNA control with known fusions
Figure 3a. Sample Input Titration-DNA and RNA Yield
To test assay sensitivity, FFPE samples were digested and subsequently diluted to as low as 10% of the sample input for the remainder of the isolation protocol from 1x10um section. Nucleic acid yield was highly linear across the full range of sample input titration, indicating high DNA and RNA recovery rates for the assay.
100% 80% 50% 20% 10%PDGFRA COSM22413 C T 51.80% 48.40% 51.00% 50.10% 51.30%
APC COSM13125 C T 12.00% 12.30% 11.40% 10.80% 9.60%APC COSM13123 G T 9.40% 11.70% 13.10% 6.50% 15.20%PTEN COSM5915 C G 48.70% 52.60% 49.50% 49.70% 46.10%PTEN COSM5218 G T 3.60% 2.80% 3.40% 3.60% 3.80%HRAS COSM249860 A G 50.90% 53.60% 45.60% 49.50% 53.50%KRAS COSM19404 C T 11.30% 13.20% 9.80% 11.70% 15.20%TP53 COSM44321 A G 8.20% 9.70% 10.00% 8.30% 10.30%
SMARCB1 COSM1090 G A 48.50% 47.60% 50.00% 50.70% 51.70%
Allele Name Reference Variant
Heterozygous SNP
Allelic FrequencySample inputAllele Call Variant Type Gene ID
Figure 3b. Sample Input Titration-Variant Table
Following Proteinase K digestion, the FFPE sample was diluted in fresh digestion buffer to various input percentages. Extractions were carried out according to the protocol, and libraries were prepared with the Ion AmpliSeq Cancer Hotspot Panel v2 for Ion PGM sequencing. The exact same cancer hotspot variants were detected from 100% down to 10% of input, verifying the sensitivity and functionality of the assay across the different input titrations.
Figure 4a. CitriSolv vs. Xylene
A less toxic alternative, CitriSolv, is compatible and comparable to the gold standard Xylene.
Figure 4b. Spin cups eliminate the need for chemical solvent deparaffinizations and save time
Alternatively, the use of spin cups can be used to de-wax samples. This eliminates the need for chemical solvents, ethanol washes and hoods while saving time especially when having to deparaffinize FFPE curls.
Figure 5a. Core Needle Biopsies and Fine Needle Aspirates, Real-time PCR data
DNA and RNA were isolated from various biopsy or aspirate samples. Nucleic acid functionality was tested by real-time PCR yielding good Ct values.
Figure 5b. Core Needle Biopsies and Fine Needle Aspirates, Sequencing data
Isolated nucleic acids were then sequenced with the Oncomine Focus assays on the PGM. High sequencing metrics were achieved for DNA and RNA such as mean read lengths, % uniformity, % no strand bias, % end to end reads and % reads on target.
Figure 6. Manual vs. Automated Extractions
Equivalent yields were achieved by manual extracting versus automating purifications on the Duo or Flex purification systems.
CONCLUSIONS In summary, we have a robust workflow that allow for the extraction of DNA and RNA from the same FFPE sample. Samples can be processed manually or with automation on the KingFisher purification systems with concordant variant detection. From tiny amounts of material such as CNB’s or FNA’s to bigger tissues such as tumor resections, samples generated quality sequencing data meeting all of our core sequencing metrics.
TRADEMARKS/LICENSING For Research Use Only. Not for use in diagnostic procedures. ©2016 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. TaqMan is a registered trademark of Roche Molecular Systems, Inc., used under permission and license. Bioanalyzer is a trademark of Agilent Technologies, Inc.
