PCR Primer Design Tool for Sanger Sequencing Confirmation
1
Michael Wenz, Stephan Berosik, Achim Karger, Pius Brzoska, Fangqi Hu, Xiaoqing You. Thermo Fisher Scientific, 180 Oyster Point Blvd., South San Francisco, CA 94080 RESULTS A New PCR Primer Design Tool for Sanger Sequencing Confirmation Thermo Fisher Scientific • 5791 Van Allen Way • Carlsbad, CA 92008 • lifetechnologies.com ABSTRACT High data quality and accuracy are recognized characteristics of Sanger re- sequencing projects and are primary reasons that next generation sequencing projects compliment their results by capillary electrophoresis data validation. We have developed an on-line tool called Primer Designer™ to streamline the NGS-to- Sanger sequencing workflow by taking the laborious task of PCR primer design out of the hands of the researcher by providing pre-designed assays for the human exome. The primer design tool has been created to enable scientists using next generation sequencing to quickly confirm variants discovered in their work by providing the means to quickly search, order and receive suitable pre-designed PCR primers for Sanger sequencing. Using the Primer Designer™ tool to design M13-tailed and non-tailed PCR primers for Sanger sequencing we will demonstrate validation of 28-variants across 24- amplicons and 19-genes using the BDD, BDTv1.1 and BDTv3.1 sequencing chemistries on the 3500xl Genetic Analyzer capillary electrophoresis platform. INTRODUCTION The Applied Biosystems® Primer Designer™ tool enables NGS scientists to quickly and accurately validate variants by Sanger sequencing. An essential part of successful Sanger sequencing is the quality of the design, synthesis and purification of the gene specific PCR primers. By navigating to www.lifetechnologies.com and entering the search terms “Sanger Primers” or “Primer Designer™ Tool” researchers can now save valuable time by choosing from a collection of >300,000 human exome amplicon targets and view their associated primer pair sequences for free. The pre-designed primers for PCR and Sanger sequencing can be ordered directly from Life Technologies with or without M13 tails, HPLC or desalt-purification only. With the Primer Designer™ Tool it is possible to confirm novel SNPs by complimentary Sanger sequencing within days. MATERIALS AND METHODS The Primer Designer™ Tool was used to identify primers for 192-gene targets or amplicons from the “Top 60” genes covered in the Ion TargetSeq™ Exome Kit. Of these 192-targets, 24-amplicon targets were selected for discussion here based on 28-SNPs variants found using NGS that could be subject to confirmation by Sanger sequencing. Four sets of 192-amplicon target primers each were ordered through Life Technologies including M13-tailed/desalted, M13-tailed HPLC purification, non- tailed/desalted and non-tailed/HPLC purified. The Primer Designer™ Tool primer design is such that each primer pair, regardless of location within the human exome, has a target T m of 63°C +/- 3°C to support universal PCR conditions which was used for all 192-amplicons. All data were generated on the Applied Biosystems® 3500xl Genetic Analyzer and Veriti® thermal cycler. The Primer Designer™ tool primers generated amplicons of no greater than 600-bp using starting input such as target information (Gene, SNP ID, RefSeq, or FASTA sequence), chromosome position, or a .vcf file from NGS. The following will demonstrate how NGS SNP validation was achieved. Materials PN AmpliTaq Gold ® 360 PCR Mix 4398881 BigDye ® Direct PCR/Sequencing Kit 4458688 BigDye ® Terminator v1.1 Sequencing Kit 4337451 BigDye ® Terminator v3.1 Sequencing Kit 4337456 BigDye XTerminator ® Purification Kit 4376484 UP DN/Rnase Free Water 10977-023 DNA Buffer (10mM Tris/0.1mM EDTA, pH 8.0) T0223 Primers ordered through the Life Technologies web site were delivered dried down at ambient temperature and were re-suspended to a 100 μM working solution in a DNA re- suspension buffer; primers were then pooled to an optimal concentration based upon PCR needs; 2.4 μM each for common PCR reactions or 0.8 μM primer for BigDye® Direct PCR. BigDye® Terminator v1.1, v3.1 and Direct sequencing chemistries were all performed across a subset of the 192-designed primers and across all of the 24- amplicon/28-SNP NGS confirmation targets. The Primer Designer™ search is started by entering either specific target information, a chromosome region of interest or by pointing to a NGS .vcf file. A search of the >300,000 PCR pre- designed primer pairs will return a list of primer pair options to choose from. By clicking on the “View Primer on Map” option nearest to the primer set of interest a map of the target exome within the gene of interest can be displayed. Click the “View Details” option beneath the primer pair of interest to reveal additional information including the primer chromosome location, Gene, Cosmic, and SNP detail related to the designed PCR primer set, view both forward and reverse primer sequences for download or for placement of an oligo synthesis order. Three step process to confirm NGS results by Sanger Sequencing using the VR Toolkit ( ww.apps.lifetechnologies.com/vrtoolkit ) 1) Build a reference sequence based on the designed primers 2) Run Variant Reporter® Software locally and load the reference file created in step #1 3) Use the “Variant Confirmation” application in the VR Tool kit. Step 1:Input the Primer ID that corresponds to the PCR primer pair ID that was generated by the Primer Designer™ Tool. A reference file (.vrr) is generated by the VR Toolkit that can be imported by Variant Reporter® Software. Step 2: Use Variant Reporter software to identifying SNPs contained within the Sanger sequencing data. The reference file generated in step #1 is used for analysis and SNP identification. The example shows data generated by the Primer Designer™ Tool for the XPC gene; a control gDNA (bottom two traces) does not produce the SNP while the specimen of interest does. The variant report is exported from Variant Reporter® for step #3 below. Step 3: Generate a Variant Confirmation Table by selecting: • the NGS .vcf file, • reference file (step #1) and • Variant Reporter® result (step #2) and clicking on “Generate Report” in the VR Toolkit. NGS confirmation is indicated when the NGS(.vcf) and VR (.txt) results match. Sanger sequencing results using BigDye® Direct and BigDye® Terminator sequencing chemistries confirm 27/28-SNPs identified using NGS. In addition to the BigDye® chemistries, each primer set was synthesized with and without M13-tails as well as with standard desalting and HPLC purification methods where each version generated the same Sanger sequencing result. Note that in some instances, high-lighted in orange, a definitive sequencing answer is found in only one sequencing direction due to additional sequence content such as homopolymers or heterozygous insertion/deletions obscuring the sequencing results of the opposite strand. In choosing the sequencing chemistry it is important to consider the proximity of the SNP of interest to the PCR priming sites as the BigDye® Terminator v3.1 and POP-7™ combination may miss variants adjacent to the 5’-region due to a 25-30-bp compression. Tailed PCR and the use of M13 fwd/rev sequencing primers affords sequencing through the PCR specific priming region as the sequencing priming site is M13 sequence specific; this is beneficial if a SNP of interest is near the 5’-gene specific (PCR) priming region where there is a risk of missing the SNP if 5’-sequencing resolution is sub-par for the first 5-10-bp. The BigDye® Direct PCR/Sequencing kit is only compatible with M13-tailed PCR primers. The BigDye® Direct/POP-7™ and BigDye® Terminator v1.1/POP-6™ chemistry and 3500xl Genetic Analyzer run module combinations provide comparable 5’ sequencing resolution although the 3500xl POP-6™ sequencing run module used to capture at least 600-bp is significantly longer than the POP-7™ module. CE Verification Results for 24-amplicons/28-SNPs CONCLUSIONS We demonstrated the successful use of the Primer Designer™ Tool and a next generation sequencing data confirmation by Sanger sequencing analysis workflow using 24-genes containing 28 SNPs. The Primer Designer™ Tool generated PCR primer designs for 192- amplicon gene targets that performed well using universal PCR conditions as well as with the BigDye® Terminator sequencing chemistries. In addition to the Primer-Designer™ Tool a data analysis workflow for the comparison the NGS data to the Sanger sequencing results using the VR Tool Kit was demonstrated. The combination of these free tools enables efficient and effective confirmation of NGS variants. TRADEMARKS/LICENSING For Research Use Only. Not for use in diagnostic procedures. AmpliTaq Gold is a registered trademark of Roche Molecular Systems, Inc. The authors declare a conflict of interest. BigDye Direct BigDye Terminator v1.1 BigDye Terminatorv3.1 CE Genotype CE Genotype CE Genotype NGS/CE Confirmed NOTCH2 247286 A A/T A/T A/T A/T YES PDE4DIP 247326 C C/T C/T C/T C/T YES PDE4DIP 247326 G G/A G/A G/A G/A YES EXT2 328600 T T/C T/C T/C T/C YES CCND2 115094 C C/G C/G C/G C/G YES ARID2 336029 T T/C T/C T/C T/C YES ALK 192315 A A/T A/T A/T A/T YES MSH6 359011 A A/G A/G A/G A/G YES MSH6 359024 T C C C C YES FN1 205929 C C/T C/T C/T C/T YES BCR 362193 G G/A G/A-fwd G/A-fwd G/A-fwd YES BCR 362194 C C/T C/T C/T C/T YES BCR 362197 G G/A G/A G/A G/A YES MN1 221970 A A/G A/A A/A A/A NO CYP2D6 225801 G G>T G>T G>T G>T YES CYP2D6 225801 G G>A G>A G>A G>A YES CYP2D6 225801 T T>G T>G T>G T>G YES CYP2D6 225801 G G>C G>C G>C G>C YES FANCD2 363573 G G/T G/T G/T G/T YES XPC 227180 G G/A G/A G/A G/A YES FOXP1 235541 A A/T A/T A/T A/T YES EPHB1 240070 G G/A G/A G/A G/A YES FBXW7 254918 G G/A G/A G/A G/A YES MTRR 257547 A A/G A/G-rev* A/G-rev* A/G-rev* YES TNFAIP3 281646 C C/T C-f/indel-r C-f/indel-r C-f/indel-r YES TNFAIP3 281648 A A/C A/C A/C A/C YES SYNE1 282848 A A/G A/G A/G A/G YES SYNE1 282961 T T/C T/C T/C T/C YES Gene Primer#/ Assay Reference NGS Genotype
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Transcript of PCR Primer Design Tool for Sanger Sequencing Confirmation
Michael Wenz, Stephan Berosik, Achim Karger, Pius Brzoska, Fangqi Hu, Xiaoqing You.
Thermo Fisher Scientific, 180 Oyster Point Blvd., South San Francisco, CA 94080
RESULTS
A New PCR Primer Design Tool for Sanger Sequencing Confirmation
Thermo Fisher Scientific • 5791 Van Allen Way • Carlsbad, CA 92008 • lifetechnologies.com
ABSTRACT High data quality and accuracy are recognized characteristics of Sanger re-sequencing projects and are primary reasons that next generation sequencing projects compliment their results by capillary electrophoresis data validation. We have developed an on-line tool called Primer Designer™ to streamline the NGS-to-Sanger sequencing workflow by taking the laborious task of PCR primer design out of the hands of the researcher by providing pre-designed assays for the human exome. The primer design tool has been created to enable scientists using next generation sequencing to quickly confirm variants discovered in their work by providing the means to quickly search, order and receive suitable pre-designed PCR primers for Sanger sequencing. Using the Primer Designer™ tool to design M13-tailed and non-tailed PCR primers for Sanger sequencing we will demonstrate validation of 28-variants across 24-amplicons and 19-genes using the BDD, BDTv1.1 and BDTv3.1 sequencing chemistries on the 3500xl Genetic Analyzer capillary electrophoresis platform.
INTRODUCTION The Applied Biosystems® Primer Designer™ tool enables NGS scientists to quickly and accurately validate variants by Sanger sequencing. An essential part of successful Sanger sequencing is the quality of the design, synthesis and purification of the gene specific PCR primers. By navigating to www.lifetechnologies.com and entering the search terms “Sanger Primers” or “Primer Designer™ Tool” researchers can now save valuable time by choosing from a collection of >300,000 human exome amplicon targets and view their associated primer pair sequences for free. The pre-designed primers for PCR and Sanger sequencing can be ordered directly from Life Technologies with or without M13 tails, HPLC or desalt-purification only. With the Primer Designer™ Tool it is possible to confirm novel SNPs by complimentary Sanger sequencing within days.
MATERIALS AND METHODS The Primer Designer™ Tool was used to identify primers for 192-gene targets or amplicons from the “Top 60” genes covered in the Ion TargetSeq™ Exome Kit. Of these 192-targets, 24-amplicon targets were selected for discussion here based on 28-SNPs variants found using NGS that could be subject to confirmation by Sanger sequencing. Four sets of 192-amplicon target primers each were ordered through Life Technologies including M13-tailed/desalted, M13-tailed HPLC purification, non-tailed/desalted and non-tailed/HPLC purified. The Primer Designer™ Tool primer design is such that each primer pair, regardless of location within the human exome, has a target Tm of 63°C +/- 3°C to support universal PCR conditions which was used for all 192-amplicons. All data were generated on the Applied Biosystems® 3500xl Genetic Analyzer and Veriti® thermal cycler. The Primer Designer™ tool primers generated amplicons of no greater than 600-bp using starting input such as target information (Gene, SNP ID, RefSeq, or FASTA sequence), chromosome position, or a .vcf file from NGS. The following will demonstrate how NGS SNP validation was achieved.
Materials PN
AmpliTaq Gold® 360 PCR Mix 4398881
BigDye® Direct PCR/Sequencing Kit 4458688
BigDye® Terminator v1.1 Sequencing Kit 4337451
BigDye® Terminator v3.1 Sequencing Kit 4337456
BigDye XTerminator® Purification Kit 4376484
UP DN/Rnase Free Water 10977-023
DNA Buffer (10mM Tris/0.1mM EDTA, pH 8.0) T0223
Primers ordered through the Life Technologies web site were delivered dried down at ambient temperature and were re-suspended to a 100 µM working solution in a DNA re-suspension buffer; primers were then pooled to an optimal concentration based upon PCR needs; 2.4 µM each for common PCR reactions or 0.8 µM primer for BigDye® Direct PCR. BigDye® Terminator v1.1, v3.1 and Direct sequencing chemistries were all performed across a subset of the 192-designed primers and across all of the 24-amplicon/28-SNP NGS confirmation targets.
The Primer Designer™ search is started by entering either specific target information, a chromosome region of interest or by pointing to a NGS .vcf file.
A search of the >300,000 PCR pre-designed primer pairs will return a list of primer pair options to choose from. By clicking on the “View Primer on Map” option nearest to the primer set of interest a map of the target exome within the gene of interest can be displayed.
Click the “View Details” option beneath the primer pair of interest to reveal additional information including the primer chromosome location, Gene, Cosmic, and SNP detail related to the designed PCR primer set, view both forward and reverse primer sequences for download or for placement of an oligo synthesis order.
Three step process to confirm NGS results by Sanger Sequencing using the VR Toolkit ( ww.apps.lifetechnologies.com/vrtoolkit )
1) Build a reference sequence based on the designed primers
2) Run Variant Reporter® Software locally and load the reference file created in step #1
3) Use the “Variant Confirmation” application in the VR Tool kit.
Step 1:Input the Primer ID that corresponds to the PCR primer pair ID that was generated by the Primer Designer™ Tool. A reference file (.vrr) is generated by the VR Toolkit that can be imported by Variant Reporter® Software.
Step 2: Use Variant Reporter software to identifying SNPs contained within the Sanger sequencing data. The reference file generated in step #1 is used for analysis and SNP identification. The example shows data generated by the Primer Designer™ Tool for the XPC gene; a control gDNA (bottom two traces) does not produce the SNP while the specimen of interest does. The variant report is exported from Variant Reporter® for step #3 below.
Step 3: Generate a Variant Confirmation Table by selecting: • the NGS .vcf file, • reference file (step #1) and • Variant Reporter® result (step #2) and clicking on “Generate Report” in the VR Toolkit. NGS confirmation is indicated when the NGS(.vcf) and VR (.txt) results match.
Sanger sequencing results using BigDye® Direct and BigDye® Terminator sequencing chemistries confirm 27/28-SNPs identified using NGS. In addition to the BigDye® chemistries, each primer set was synthesized with and without M13-tails as well as with standard desalting and HPLC purification methods where each version generated the same Sanger sequencing result. Note that in some instances, high-lighted in orange, a definitive sequencing answer is found in only one sequencing direction due to additional sequence content such as homopolymers or heterozygous insertion/deletions obscuring the sequencing results of the opposite strand. In choosing the sequencing chemistry it is important to consider the proximity of the SNP of interest to the PCR priming sites as the BigDye® Terminator v3.1 and POP-7™ combination may miss variants adjacent to the 5’-region due to a 25-30-bp compression. Tailed PCR and the use of M13 fwd/rev sequencing primers affords sequencing through the PCR specific priming region as the sequencing priming site is M13 sequence specific; this is beneficial if a SNP of interest is near the 5’-gene specific (PCR) priming region where there is a risk of missing the SNP if 5’-sequencing resolution is sub-par for the first 5-10-bp. The BigDye® Direct PCR/Sequencing kit is only compatible with M13-tailed PCR primers. The BigDye® Direct/POP-7™ and BigDye® Terminator v1.1/POP-6™ chemistry and 3500xl Genetic Analyzer run module combinations provide comparable 5’ sequencing resolution although the 3500xl POP-6™ sequencing run module used to capture at least 600-bp is significantly longer than the POP-7™ module.
CE Verification Results for 24-amplicons/28-SNPs
CONCLUSIONS We demonstrated the successful use of the Primer Designer™ Tool and a next generation sequencing data confirmation by Sanger sequencing analysis workflow using 24-genes containing 28 SNPs. The Primer Designer™ Tool generated PCR primer designs for 192-amplicon gene targets that performed well using universal PCR conditions as well as with the BigDye® Terminator sequencing chemistries. In addition to the Primer-Designer™ Tool a data analysis workflow for the comparison the NGS data to the Sanger sequencing results using the VR Tool Kit was demonstrated. The combination of these free tools enables efficient and effective confirmation of NGS variants.
TRADEMARKS/LICENSING For Research Use Only. Not for use in diagnostic procedures. AmpliTaq Gold is a registered trademark of Roche Molecular Systems, Inc. The authors declare a conflict of interest.
BigDye Direct BigDye Terminator v1.1 BigDye Terminatorv3.1
CE Genotype CE Genotype CE GenotypeNGS/CE
ConfirmedNOTCH2 247286 A A/T A/T A/T A/T YESPDE4DIP 247326 C C/T C/T C/T C/T YESPDE4DIP 247326 G G/A G/A G/A G/A YESEXT2 328600 T T/C T/C T/C T/C YESCCND2 115094 C C/G C/G C/G C/G YESARID2 336029 T T/C T/C T/C T/C YESALK 192315 A A/T A/T A/T A/T YESMSH6 359011 A A/G A/G A/G A/G YESMSH6 359024 T C C C C YESFN1 205929 C C/T C/T C/T C/T YESBCR 362193 G G/A G/A-fwd G/A-fwd G/A-fwd YESBCR 362194 C C/T C/T C/T C/T YESBCR 362197 G G/A G/A G/A G/A YESMN1 221970 A A/G A/A A/A A/A NO
CYP2D6 225801 G G>T G>T G>T G>T YESCYP2D6 225801 G G>A G>A G>A G>A YESCYP2D6 225801 T T>G T>G T>G T>G YESCYP2D6 225801 G G>C G>C G>C G>C YESFANCD2 363573 G G/T G/T G/T G/T YESXPC 227180 G G/A G/A G/A G/A YESFOXP1 235541 A A/T A/T A/T A/T YESEPHB1 240070 G G/A G/A G/A G/A YESFBXW7 254918 G G/A G/A G/A G/A YESMTRR 257547 A A/G A/G-rev* A/G-rev* A/G-rev* YESTNFAIP3 281646 C C/T C-f/indel-r C-f/indel-r C-f/indel-r YESTNFAIP3 281648 A A/C A/C A/C A/C YESSYNE1 282848 A A/G A/G A/G A/G YESSYNE1 282961 T T/C T/C T/C T/C YES
GenePrimer#/ Assay Reference
NGS Genotype
