PyroMark Q24 Advanced increases both read length and reliability of methylation analysis at positions later in the sequence. This example demonstrates 135 nucleotide dispensations and the accurate analysis of 16 different CpG positions in a single PyroMark Q24 Advanced CpG reaction. To accurately analyze all of these sites with PyroMark Q24, one would need to run 3 separate assays.
Since mutations and single nucleotide polymorphisms (SNPs) are rarely in directly neighboring positions, common Pyrosequencing chemistry might require separate assays for analysis of more than one mutation site. The new chemistry of PyroMark Q24 Advanced allows much longer runs, enabling reliable analysis of additional mutations within one run. This example shows the analysis of a 10:90 mixture of wild-type and mutated EGFR. Even after 60 dispensations, the analysis is exact.
Quantification of CpG methylation directly following a T homopolymer or between T homopolymers is especially challenging. Methylation levels are determined by the ratio of converted C nucleotides to unconverted C nucleotides following bisulfite treatment, and since the C to T conversion often leads to long T homopolymer stretches in an amplicon, this scenario occurs often. PyroMark Q24 Advanced enables accurate quantification of CpG position after or between T homopolymers.This example shows the analysis of a CpG site within a stretch of 8 T nucleotides.
Read length is a critical factor for analysis of unknown sequences, often limiting the reliable analysis to 40–80 bases. The improved chemistry and algorithm of PyroMark Q24 Advanced significantly increases the possible read length and provides higher accuracy. PyroMark Q24 (upper panel) and PyroMark Q24 Advanced (lower panel) were used for de novo sequencing using the same assay. Blue bars indicate reliably detected bases; yellow indicates bases that might have been determined correctly but should be checked by the user; red indicates unreliable readings.