A blank control was done with template without sequencing primer. blOligo reactions contained 4 pmol of the blocking oligonucleotide with a dideoxy residue at its 3′ end. Sequencing primers and blOligos used are listed in (Table 1).TdT Treatment
For TdT (E.C. 22.214.171.124) treatment, Dynabeads with single-stranded templates were incubated in 5 µL of One-Phor-All-Buffer PLUS (Amersham Biosciences) containing 0.5 mM ddCTP and 2.5 U TdT in a total volume of 50 µL at 37°C for 30 min. Afterwards, they were washed in 100 µL washing buffer and treated by Pyrosequencing as described above.Results and Discussion
As outlined in (Figure 1), self-priming of ssDNA template may falsify the outcome of Pyrosequencing. (Figure 2) and 3 give two experimental examples for the self-priming problem. In (Figure 2), Pyrosequencing is performed on single-stranded templates obtained from the reverse transcription PCR (RT-PCR) of CARD 15 gene transcripts to analyze allele-specific expression. SNP rs2067085 was used as a marker for the allelic imbalance in heterozygotes reported previously using another SNP (12). Theoretical histograms for homozygotes and heterozygotes are clearly informative and distinguishable ((Figure 2)A). However, using cDNA of a CC homozygotic individual (all individuals were genotyped using genomic DNA and Sanger sequencing), ghost peaks appear at positions informative for the G allele, although there is no G allele present ((Figure 2)B). For the heterozygotic individual, the SNP determining peaks appear too high, indicating an imbalance in allelic expression (Figure (2)B, right column). The same problems were also detectable for cDNA, with templates obtained from cloned PCR products. Ghost peaks also appeared at the SNP determining positions and falsified the results in a similar way ((Figure 2)C).
A blank control experiment in which only the template was used in the Pyrosequencing reaction (i.e., without sequencing primer) indicated that self-priming of the template's 3′ end results in the occurrence of signals at positions of the informative bases ((Figure 2)D). Although the Pyrograms differ slightly for the three genotypes, ghost peaks are consistent at positions of the bases informative for SNP rs2067085. Beside the examples presented, in our hands, such blank control experiments reveal a 3′-end self-priming for nearly all inspected sequences and are an indispensable control when setting up Pyrosequencing analyses.
If the CARD15 blOligo was added during Pyrosequencing, no signals at all were obtained with either template ((Figure 2)E). Consequently, if the blOligo was used together with the sequencing primer, the Pyrogram gave the clear-cut expected result ((Figure 2)G). With the same targets, we tested the second possible method of locking the 3′ end of the single-stranded template; in this case, by attaching a ddCMP using TdT. As with the use of the blOligo, the Pyrosequencing reactions with the TdT-modified templates obtained from the cDNA and cloned PCR fragments indicated no self-priming in the blank control ((Figure 2)F) and gave the expected result in the analysis ((Figure 2)H).
Aside from SNP typing, quantitative data analysis is the main advantage of Pyrosequencing. The quantification is done by the calculation of relative peak heights. Informative bases are compared with a base common to all templates (calibration to an adenine should be avoided because the A-peaks often appear too high).
(Table 2) summarizes the quantitative analysis of allele-specific expression of CARD15 transcripts in four individuals. By standard Pyrosequencing, the results indicate an allelic imbalance in all samples with the G allele being overexpressed. However, in every case, the total amount of the allele-specific bases is conflicting with the amount of a common base. They sum to values larger than 1, which indicates the presence of background signals ((Table 2), right column). Both the use of blOligo and TdT treatment shift the results significantly so that Σ G + C becomes closer to 1. If we accept a maximum deviation from 1.0 of 10%, which is obtained in all cases of TdT treatment, an allelic imbalance is seen for only two of the samples and, furthermore, with a favored C allele expression.Table 2. Analysis of Allele-Specific Expression of CARD15 in Leucocytes of Four Individuals
Values were calculated through the analysis of single nucleotide polymorphism (SNP) rs2067085 on caspase recruitment domain family, member 15 (CARD15) cDNA by Pyrosequencing. For each individual, Pyrosequencing was performed using the standard procedure and after locking the 3′ ends of PCR products with the CARD15 blOligo (blocking oligonucleotide). Bold numbers indicate optimal Pyrosequencing performance after deoxynucleotidyl transferase (TdT) treatment.