Primer Designer Tool - Thermo Fisher Scientific

APPLICATION NOTEPrimer Designer Tool Primer Designer Tool A comprehensive solution for NGS data verification of humanexome DNA sequences by optimized Sanger sequencingExome or whole genomesequencing by next-generationsequencing (NGS) technologiestypically reveals a large numberof variations from a referencegenome. Many of these variationscould underlie variations inphenotypic traits, increasedrisk of disease susceptibility,or reveal somatic mutationsthat are potentially oncogenic.Moreover, these variants mayoccur in genomic regions thatare challenging to sequencewith high accuracy by NGS orare underrepresented by readnumbers. In these situations itis recommended to follow up byverification using an orthogonalmethod such as traditionalfluorescent Sanger sequencingand automated capillaryelectrophoresis. To facilitate thedesign process and workflow forNGS data verification of humanexome sequences we havedeveloped a complete solutionconsisting of a comprehensivepanel of over 300,000 predesigned PCR primer pairscovering over 95% of human exons.These PCR primers are designedto be compatible for downstreamSanger sequencing either withtraditional BigDye Terminator (BDT) sequencing kits v1.1 and v3.1 orthe workflow-optimized BigDye Direct cycle sequencing kit (BDD). Theprimer designs can be accessed through a user-friendly web portal. Theportal also enables direct ordering of custom-synthesized primer pairs.In this application note we demonstrate the utility and convenience ofthis offering, called Primer Designer tool, to the scientific communityby showing the process and workflow from NGS-delivered variant call toSanger-verified result. Primer Designer tool makes primer selection for re-sequencing easyNGS users may want to verify the finding of a SNP or other variant inthe human exome if an actionable decision depends on the accuracyof the result. To that end, the locus of interest has to be re-analyzedwith an orthologous method such as PCR-based Sanger sequencing.Designing PCR primers for a locus of interest can be both tediousand challenging. The researcher has to navigate into reference genesequence files or other DNA sequence resource sites, find the targetlocus, ascertain that the primers are highly specific for the locus and

NGS confirmation by Sanger/CE sequencingCurrent bottleneckThe process may repeat if the primer design failsNGS reveals SNP or variantQuestion: is result true or false?PrimerdesignSNPcheckOrder primers fromoligo vendorRun CEsequencingPrimer Designer solutionNGS analysisNeed to verify?Primer Design Toolall-in-one functionalityRun CEsequencing User-friendly online tool Complete primer design for human exome coverage ( 300,000 primer pairs) Fully automated online primer ordering All primers are 100% quality checked by mass spectroscopy Direct access for ION usersFigure 1: NGS data verification workflow can be tedious and time-consuming (upper pane) or streamlined and convenient using the new PrimerDesigner tool (lower pane). SNP of interest, and that primer-dimers and undesiredsecondary structures are avoided. Finally, the designhas to be transferred into an ordering sheet of anoligo vendor and then it is a matter of good designand experimental skills to proceed with PCR andcycle sequencing to obtain the DNA sequence from acapillary electrophoresis (CE) trace.To simplify this process, we have developed the PrimerDesigner tool, which integrates the gene and primerselection, design, ordering and synthesis process with agreater than 95% chance of obtaining a successful andaccurate sequencing result in a user-friendly workflow(Figure 1). The Primer Designer tool comprises a virtual designcollection of over 300,000 primer pairs that cover 95.9%of human coding exons and 74.9% of human noncodingexons. Complete sequence coverage is achieved for 94%of coding exons and 68% of noncoding target exons.Sophisticated bioinformatics rules and filters wereapplied to help ensure high target specificity and toavoid SNP locations as well as to minimize the formationof primer-dimers. Designing the Primer Designer toolThe basis for the design collection was the entire humanhg19 reference genome refGene.txt.gz). The totalnumber of RefSeq genes was 23,645 and the numberof transcripts was 40,344. The total number of exonswas 227,706 which added up to a total of approximately70 million bp. The size range for exons was between2 and 91,670 bp; intron sizes ranged between 2 and1,043,912 bp (Figure 2). Great care was taken to avoidcommon SNPs in the primer designs. Over 11 millionSNPs from dbsnp135 snp135Common.txt.gz) wereconsidered. The typical exon in the human genome can be capturedwith one amplicon; however, a high number of exonsexceeded the intended target length of 500 bp. Sincemany of these are noncoding, preference was given onlyto coding exons (Figure 3).To accommodate long exons into “manageable”amplicons of approximately 500 bp, two or more

amplicons were designed that overlap for 50 bp so thatdouble coverage by forward and reverse sequence wasguaranteed (Figure 4).The Primer Designer tool web portalThe Primer Designer tool is accessible and free for useon the Life Technologies website (Figure 5):www.lifetechnologies.com/primerdesigner For the complimentary display of primer sequencesa log-in into a user account at Life Technologies.comis required. Although the ordering and purchase ofa primer pair as oligonucleotides from this tool arenot obligatory, orders are fulfilled promptly if thisoption is selected. Customers ordering from thistool will benefit from fast turnaround time—in USand Canada desalted primers typically ship within 2business days, and HPLC-purified primers typicallyship within 6 business days from receipt of order—and economical pricing. Each primer order is qualitycontrolled by mass spectroscopy and shipped witha detailed certificate of analysis. Synthesis is doneon a 50 nmol scale for HPLC-purified primers,which allows for 100 PCR and sequencing reactions.Desalted primers are synthesized on a 25 nmol scalethat allows for many more reactions as the yield istypically 3–5 times higher.To find the locus of interest the target information fieldcan be queried for a gene symbol like CFTR or KRAS, aSNP rs identification number, a COSMIC mutation ID,a RefSeq or FASTA sequence file or simply a string ofnucleotides (minimum 50).Alternatively, chromosomal coordinates can be usedor an NGS-derived variant call formatted file (.vcf) canbe directly uploaded. Most conveniently, users of theIon PGM or Proton sequencing platforms benefit fromhaving a direct link from variant to a correspondingprimer pair design within the Variant Caller Export Toolin the Ion Torrent Reporter suite. %10%5%5%0%0%Target sequence sizeTarget sequence sizeFigure 2: Exon size distribution (in bp) of the hg19 reference %0% Amplicon sizeAmplicon size00100100200300400200 Size300(bp) 400Size (bp)500500600600Figure 3: Size distribution (in bp) of the Primer Designer humanexome collection. Clicking on “Search” will launch the tool and a list ofprimer pairs will be returned that match the searchcriteria.When multiple primer pairs are available it is advisableto use the “View Primer on Map” feature which showsthe location of the amplicon generated by the primerpair on a simplified genomic map (Figure 6). By clickingon an exon in the interactive map (depicted as a gray boxor bar) the exon number is indicated.100 200 300 400 500 600 700 800 900 1000100 200 300 400 500 600 700 800 900 1000Size (bp)Size (bp)0050 bp paddingIntron50 bp paddingExonAmplicon 1Amplicon 250 bp overlapFigure 4: Overlapping amplicons to cover long exons.Intron

Figure 5: Primer Designer tool. The user-friendly interface takes only seconds to find your primers. Figure 6: Overview map of primer pairs.

Primer Ordering OptionsThe Primer Designer tool providesthe capability for fully-automatedonline primer order placement fromyour user account. The following options are available(Figure 7): Primer 5 -tail option: non-tailedvs. M13-tailed Purification: desalted vs. HPLC Primer pair vs. single primerDespite the slightly higher cost,we recommend the use of M13tailed PCR primers since a variantof interest might be located closeto either end of a pre-designedassay. With M13-primed sequencingthe “blind spot” or PCR-specificpriming region of approximately20 nucleotides at the 5 end istypically well resolved, leadinginto the first base of the amplicontarget sequence. A visual depictionof this is illustrated in Figure 8.The BigDye Direct sequencingkit contains specially engineeredM13 sequencing primers whichexhibit even better 5 resolutionusing POP‑7 compared to BigDye KitBigDye Direct CycleSequencing Kit(BDD) Figure 7: The primer option window.Terminator v3.1 and POP‑7 sequencing. An overview of the sequencingchemistry options are provided in Table 1. We also strongly recommendsequencing both the forward and reverse strands of an amplicon; this isnot only good laboratory practice because it validates a sequence by havingdouble coverage from either side, but it may be instrumental to delineate thebreak points of heterozygous insertion / deletions or strong stops induced bydifficult sequences such as extensive homopolymers. Which BigDye sequencing kit to use?The PCR primers designed by the Primer Designer tool can be configured tobe compatible with any of the existing BigDye Terminator sequencing kits.The features of these kits are listed in Table 1. BenefitsNotes and considerations Easy-to-use single-plate workflow from PCR to Sangersequencing—no sample transfer—just add reagents fromfirst to last steps Kit includes PCR and sequencing reagents priced andformulated ready to go—no need to dilute Time-to-result (PCR to DNA sequence) as fast as 4 hoursfor short amplicons ( 400 bp) Superior 5 resolution on fast POP-7 polymer (equalsmore data) Excellent data quality Primers for PCR must be ordered M13-tailed (forwardand reverse) in the Primer Designer tool—desaltedpurification is acceptable to yield very good data Fast time-to-result: quick delivery of desalted primersmeans data sooner (no waiting for HPLC purification) HPLC-purified M13-specific primers for sequencing arealready included in the kit Reactions with very high GC content ( 70%) DNA requiresupplemental GC enhancer reagent (Cat. No. 4398848) Widely-used sequencing reagent Good 5 resolution when used in Sanger sequencing withPOP-6 polymer Non-tailed or M13-tailed primers can be used Can be used as sequencing reagent and fragment-analysisreagent (SEQ FA) when used with dye set E5 5X dilution buffer is included with kit Sanger-sequencing run time for POP-6 is slower thanPOP-7 M13 sequencing primers are not included in the kit but canbe ordered HPLC-purified in Primer Designer tool; HPLCpurification requires extra time Established standard sequencing reagent Non-tailed or M13-tailed primers can be used Uniform high data quality possible for most sequencecompositions including homopolymeric sequences 5X dilution buffer is included with kit Resolution at 5 end—first 25 bases after primer aretypically masked or trimmed when run with POP-7 M13 sequencing primers are not included in the kit but canbe ordered HPLC-purified in Primer Designer tool. HPLCpurification requires extra time BigDye Terminatorv1.1 Sequencing Kit(BDT v1.1) BigDye Terminatorv3.1 Sequencing Kit(BDT v3.1) Table 1: Overview of benefits and considerations for selection of BigDye sequencing kits.