PMC

SNPs which can be used to differentiate between the defined groups must be perfectly conserved within each group (green column in the group’s consensus graph) and must differ between the groups (orange or red column in the global consensus graph at the top). These positions can be automatically identified and are marked by red columns in the alignment.

Zoomed in view of an alignment where groups have been defined. The number of sequences which need to be displayed in order to capture the essential differences between the groups is significantly reduced (the five shown groups contain 94 sequences), but drilling down to the single sequence level is still easily possible, as visible in group “advC”. Bases which are identical to the consensus sequence (or reference sequence, which can be chosen manually) are gray, differing bases are colored based on the selected coloring scheme – “BioEdit” in this case.

In multiplex pyrosequencing, several primers are used simultaneously in the sequencing reaction so that their signals overlap. A1: In this example, primer 1 (upper part) reads the sequence TTAACCT and primer 1 (middle part) reads the sequence CGCCGTC. Since the signals overlap, the fingerprint (lower part) represents the sequence TTCAAGCCCCGTTC. It is important to note that in this fingerprint, it is not possible to tell which base was read by which primer. A2: The T→C mutation after primer 1 and the C→T mutation after primer 2 are used as targets for differentiating between two species. However, they cancel each other out, causing the fingerprints for A1 and A2 to be identical. B: Moving primer 1 one base to the left alleviates this problem: the fingerprints for B1 and B2 are now different. This demonstrates the importance of correct pyrosequencing primer positioning relative to all utilized SNPs.

Display of an alignment with two pyrosequencing primers, one of which is visible on the screen (green annotation), and four PCR primers, one of which is also visible (blue annotation). For each primer, the melting temperature is displayed at the 5′ end and the length is displayed at the 3′ end. A line connects the forward PCR primer with its reverse counterpart (not visible, offscreen). The product size is shown in the middle of the connecting line, and the red color warns of a high difference in predicted melting temperature. In subfigure A, the predicted pyrograms from the two pyrosequencing primers are shown for each group – with just 5 cycles of the pyrosequencing machine, a unique pyrogram can be obtained for each of the groups. In subfigure B, the pyrosequencing primer has been moved one base to the left, thus preventing sequencing of one SNP. This leads to the predicted pyrograms for advE and advB being identical.