1. A Novel Method for Multiplex Genotyping in a Single Reactor Using GTPlex-PyroSeq 
Myungsok Oh, Benjamin Douglass Hoehn, Youngho Moon, Taejeong Oh, Youngbok Ko, Sungwhan An 
The Journal of Molecular Diagnostics 
Volume 14, Issue 4, Pages (July 2012)
DOI: /j.jmoldx
Copyright © 2012 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

2. Figure 1
Background for ID sequence based genotyping by pyrosequencing. A: Using individual nucleotides as ID marks (IDms) can identify up to four type-specific sequences in a single reactor. The IDm is inserted between the sequencing primer site and the type-specific sequence. When used in combination with a specified dispensation order, unique peaks can then be identified for each of the genotypes identified by the specific IDms. B: Using a single-nucleotide IDm is limited because IDms are affected by the subsequent type-specific sequence, generating undesired multiple peaks (genotype 1) or double peaks (genotype 2), confusing identification. For this reason, genotyping using a single IDm has significant limitations. C: To ensure the IDm is unaffected by the subsequent type-specific sequence, an additional nucleotide is inserted to separate the IDm and the subsequent type-specific sequence. This nucleotide is called a sign post (SP). As a result, the IDm is unaffected until the SP is reached in the dispensation order. The combination of the IDm and the SP is now called the ID sequence. D: In the case when the first nucleotide in the type-specific sequence is identical to the SP, or multiple repeats of the SP, the height of the peak will proportionally increase. To overcome this problem, an additional nucleotide can be added to the ID sequence (IDm and the SP) to separate it from the type-specific sequence. This nucleotide is called the end mark, and the ID sequence is now composed of an IDm, an SP, and an Em.
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2012 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

3. Figure 2
The relationship between the number of SPs and ID sequences. A: To identify more than three type-specific sequences, it is possible to extend that number by adding additional SPs. Two additional sequences can be distinguished for each subsequent addition of an SP beyond the first (SP1). So the maximum number of IDms that can be used for a set number of SPs can be determined using the formula 2N + 1, when N equals the number of SPs. B: An example of five genotype ID sequences generated using two SPs and related dispensation order is shown: three cases where the IDms are before SP1 and two cases where the IDms fall between SP1 and SP2. IDms are bolded and underlined.
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2012 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

4. Figure 3
Schematic diagram of ID sequence and dispensation order generation. A: Reciprocity allows for either the design of the ID sequence to dictate the dispensation order or the ID sequence may be designed using a predetermined dispensation order. In this case, the dispensation order (ATGC)n is used to design the ID sequences. In the dispensation order, three IDms may be located ahead of SP1, and two IDms may be located between each subsequent SP. Sequencing seven genotypes requires three SP according to the formula (2N + 1). Applying these rules, seven type-specific ID sequences can be generated with the selected dispensation order (the dispensation order does not include the Em). Expected pyrograms can be observed adjacent to each ID sequence. B: Using the ID sequences and dispensation order generated in A, four pyrograms are depicted showing the expected results for single or multiple genotypes. Genotype IDs are bolded and underlined, and correspond to the ID sequences in A; genotypes are numbered in descending order.
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2012 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

5. Figure 4
The GTPlex system demonstrates high fidelity in HPV genotyping. A: Schematic diagram of the unique ID sequences for 15 high-risk HPV genotypes and the dispensation order generated using the GTPlex system. SPs (black bars) and IDms (white bars) with genotype are indicated. B: Pyrosequencing results from HPV L1 DNA corresponding to 15 high-risk types genotypes using the GTPlex system. IDs are bolded and underlined. C: Multiple genotyping assays using type-specific HPV L1 PCR products in a single reaction mixture. IDs are bolded and underlined, with the genotype indicated above the peak.
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2012 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

6. Figure 5
Testing of human cervical cancer cell lines using the GTPlex HPV genotyping system. A: A flowchart that outlines in a stepwise manner the GTPlex system from sample collection to pyrosequencing. B: HPV genotyping using genomic DNA from known cervical cancer cell lines. The ID sequences and dispensation order are the same as those shown in Figure 4A. C33A cells were used as a negative control. Sequence IDs are bolded and labeled.
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2012 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions