Dataset Information

Evaluation of single nucleotide polymorphism typing with invader on PCR amplicons and its automation.

ABSTRACT: Large-scale pharmacogenetics and complex disease association studies will require typing of thousands of single-nucleotide polymorphisms (SNPs) in thousands of individuals. Such projects would benefit from a genotyping system with accuracy >99% and a failure rate <5% on a simple, reliable, and flexible platform. However, such a system is not yet available for routine laboratory use. We have evaluated a modification of the previously reported Invader SNP-typing chemistry for use in a genotyping laboratory and tested its automation. The Invader technology uses a Flap Endonuclease for allele discrimination and a universal fluorescence resonance energy transfer (FRET) reporter system. Three hundred and eighty-four individuals were genotyped across a panel of 36 SNPs and one insertion/deletion polymorphism with Invader assays using PCR product as template, a total of 14,208 genotypes. An average failure rate of 2.3% was recorded, mostly associated with PCR failure, and the typing was 99.2% accurate when compared with genotypes generated with established techniques. An average signal-to-noise ratio (9:1) was obtained. The high degree of discrimination for single base changes, coupled with homogeneous format, has allowed us to deploy liquid handling robots in a 384-well microtitre plate format and an automated end-point capture of fluorescent signal. Simple semiautomated data interpretation allows the generation of approximately 25,000 genotypes per person per week, which is 10-fold greater than gel-based SNP typing and microsatellite typing in our laboratory. Savings on labor costs are considerable. We conclude that Invader chemistry using PCR products as template represents a useful technology for typing large numbers of SNPs rapidly and efficiently.

SUBMITTER: Mein CA

PROVIDER: S-EPMC311429 | biostudies-literature | 2000 Mar

REPOSITORIES: biostudies-literature

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Evaluation of single nucleotide polymorphism typing with invader on PCR amplicons and its automation.

Mein C A CA Barratt B J BJ Dunn M G MG Siegmund T T Smith A N AN Esposito L L Nutland S S Stevens H E HE Wilson A J AJ Phillips M S MS Jarvis N N Law S S de Arruda M M Todd J A JA

Genome research 20000301 3

Large-scale pharmacogenetics and complex disease association studies will require typing of thousands of single-nucleotide polymorphisms (SNPs) in thousands of individuals. Such projects would benefit from a genotyping system with accuracy >99% and a failure rate <5% on a simple, reliable, and flexible platform. However, such a system is not yet available for routine laboratory use. We have evaluated a modification of the previously reported Invader SNP-typing chemistry for use in a genotyping l ...[more]

PMID: 10720574

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Project description:Highly specific amplification of complex DNA pools without bias or template-independent products (TIPs) remains a challenge. We have developed a procedure using phi29 DNA polymerase and trehalose and optimized control of amplification to create micrograms of specific amplicons without TIPs from down to sub-femtograms of DNA. The amplicons from 5 ng and 0.5 ng DNA, which were from originally good quality of gDNA (05-050), or partially degraded gDNA (04-018), were validate with Illumina HumanHap550-Duo Genotyping Beadchip. As seen in (Suppl. Table 5a), the call rates (97.30% to 99.07%) and accuracy or concordance ( > 99.85% for the SNPs called in both amplicon and natural reference) for 5 ng derived amplicons with both Wpa and Gv2 were close to each other and close to native gDNA (call rate: 98.3% to 99.75%). These call rates were better than a recent report (amplicon 95.9% vs. un-amplified 98.5%), in which the early kit Repli-g 625S was applied, and re-genotyping was performed when the performance was low and duplicate samples were filtered for the highest call rate. The genotyping accuracy of Wpa was actually in the same range as the variation in technical replicates with similar SNP typing arrays (99.87% and 99.88%, replicated Affymetrix array, or between Affymetrix and Illumina arrays). Importantly, the genotyping concordance for amplicons generated from 0.5 ng with Wpa (99.88% and 99.69%) were also close to the technical replicates. In this case, the call rates of Wpa were slightlyreduced compared to that with 5 ng input, but the call rate for the partially degraded sample 04-018, was modestly improved over Gv2 (92.06 % vs. 90.53%). Wpa data also showed some amplification non-uniformity among different locations, resulting in some “artificial CNVs” similar to Gv2 (exampled as in Suppl. Fig. 5 and Suppl. Table 6), with the outputs obtained by taking unamplified gDNAs as their reference. This imbalance however was consistent and reproducible for each method but different between Wpa and Gv2. These artificial CNVs can be efficiently cancelled if pair-wise amplified test and reference are compared, as observed in CGH result (Fig. 4 and Suppl. Fig. 4), also supported by others {Pugh 2008}. It is interesting to note that the representation of chromosomal terminal sequences was greatly improved with Wpa compared with Gv2 (Fig. 5), and that some of these regions were significantly under-amplified or even lost with Gv2 (Suppl. Fig. 5 and Suppl. Table 6, 7), as also independently reported recently {Pugh 2008}. This occurred especially in the terminal 3 to 5 Mb and sometimes extended to 10 Mb in many chromosome termini, and was particularly serious when low levels or degraded DNA was as input. An analysis for 5 Mb termini is shown (Suppl. Table 5b calculated all involved SNPs as a cohort. Fig. 5 and Suppl. Tables 6 and 7 were the result for each chromosome terminus). Importantly, the SNP typing was also greatly improved, outstandingly exemplified by the amplicons of 0.5 ng input for the partially degraded 04-018, with Wpa versus Gv2 call rate of 91.9% vs. 84.45% and accuracy of 99.57% vs. 98.62%. The result also showed that these terminal regions underrepresentation in Gv2 was not absolutely associated with the distance-to-end, but possibly was a sequence related issue. Keywords: Whole-pool amplification, whole genome SNP typing

Project description:Highly specific amplification of complex DNA pools without bias or template-independent products (TIPs) remains a challenge. We have developed a procedure using phi29 DNA polymerase and trehalose and optimized control of amplification to create micrograms of specific amplicons without TIPs from down to sub-femtograms of DNA. The amplicons from 5 ng and 0.5 ng DNA, which were from originally good quality of gDNA (05-050), or partially degraded gDNA (04-018), were validate with Illumina HumanHap550-Duo Genotyping Beadchip. As seen in (Suppl. Table 5a), the call rates (97.30% to 99.07%) and accuracy or concordance ( > 99.85% for the SNPs called in both amplicon and natural reference) for 5 ng derived amplicons with both Wpa and Gv2 were close to each other and close to native gDNA (call rate: 98.3% to 99.75%). These call rates were better than a recent report (amplicon 95.9% vs. un-amplified 98.5%), in which the early kit Repli-g 625S was applied, and re-genotyping was performed when the performance was low and duplicate samples were filtered for the highest call rate. The genotyping accuracy of Wpa was actually in the same range as the variation in technical replicates with similar SNP typing arrays (99.87% and 99.88%, replicated Affymetrix array, or between Affymetrix and Illumina arrays). Importantly, the genotyping concordance for amplicons generated from 0.5 ng with Wpa (99.88% and 99.69%) were also close to the technical replicates. In this case, the call rates of Wpa were slightlyreduced compared to that with 5 ng input, but the call rate for the partially degraded sample 04-018, was modestly improved over Gv2 (92.06 % vs. 90.53%). Wpa data also showed some amplification non-uniformity among different locations, resulting in some âartificial CNVsâ similar to Gv2 (exampled as in Suppl. Fig. 5 and Suppl. Table 6), with the outputs obtained by taking unamplified gDNAs as their reference. This imbalance however was consistent and reproducible for each method but different between Wpa and Gv2. These artificial CNVs can be efficiently cancelled if pair-wise amplified test and reference are compared, as observed in CGH result (Fig. 4 and Suppl. Fig. 4), also supported by others {Pugh 2008}. It is interesting to note that the representation of chromosomal terminal sequences was greatly improved with Wpa compared with Gv2 (Fig. 5), and that some of these regions were significantly under-amplified or even lost with Gv2 (Suppl. Fig. 5 and Suppl. Table 6, 7), as also independently reported recently {Pugh 2008}. This occurred especially in the terminal 3 to 5 Mb and sometimes extended to 10 Mb in many chromosome termini, and was particularly serious when low levels or degraded DNA was as input. An analysis for 5 Mb termini is shown (Suppl. Table 5b calculated all involved SNPs as a cohort. Fig. 5 and Suppl. Tables 6 and 7 were the result for each chromosome terminus). Importantly, the SNP typing was also greatly improved, outstandingly exemplified by the amplicons of 0.5 ng input for the partially degraded 04-018, with Wpa versus Gv2 call rate of 91.9% vs. 84.45% and accuracy of 99.57% vs. 98.62%. The result also showed that these terminal regions underrepresentation in Gv2 was not absolutely associated with the distance-to-end, but possibly was a sequence related issue. Keywords: Whole-pool amplification, whole genome SNP typing The overall goal of the part of study was a validation of the quality of the amplicons from different amounts (5ng and 0.5 ng) of original starting gDNA, good quality (sample 05-050) or partially degraded gDNA (sample 04-018), with our new procedure Wpa, and with native gDNA as control, in terms of the call rate and accuracy (allele bias) in addition to the uniformity of the sequence amplified (sequence representation or sequence bias). Amplified or native genomic DNA isolated from patients was in-parallel analyzed/genotyped with the same experimental platform, of which the native genomic DNAs were used as the standard controls. For the sequence representation, the two alleles of the SNPsâ signal of a panel of multiple native DNAsâ signal provided by the experimental platform (Illumina) was used as the reference, so that an abstract signal for sequence representation of each SNP and for all SNPs was obtained.

Dataset Information

Evaluation of single nucleotide polymorphism typing with invader on PCR amplicons and its automation.

Publications

Evaluation of single nucleotide polymorphism typing with invader on PCR amplicons and its automation.

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