Multiplex Pyrosequencing

Abstract

We describe here the development of a new and simple single-tube multiplex Pyrosequencing assay. Genomic DNA or cDNA was employed to PCR amplify region(s) using biotinylated and normal primer(s). Subsequent to capture of PCR products on streptavidin-coated beads, single-stranded DNA separation and hybridization of multiple sequencing primers, Pyrosequencing was performed. The obtained pyrogram resulted in a unique pattern in which the intensity of the signal determined the number of incorporated nucleotide(s). Here, we demonstrate the use of this multiplex Pyrosequencing for single nucleotide polymorphisms genotyping and microbial typing.

Figure 1

Schematic presentation of the principle of multiplex Pyrosequencing on a single template (left) and on multiple templates (right). PCR was performed with one of the primers biotinylated. Subsequent to immobilization of the product on streptavidin-coated magnetic beads and single-stranded elution, several sequencing primers were hybridized to the template(s) and Pyrosequencing was performed. A unique pattern was obtained, determining the exact sequence of different nucleotide positions.

Figure 2

Schematic demonstration of the expected patterns obtained from multiplex analyses of two SNPs residing on a DNA template (A). Raw data in the pyrogram demonstrate two of the four possible expected patterns (B). The correct sequences are shown above each pyrogram and the order of dispensation is demonstrated below the pyrograms.

Figure 3

Pyrogram demonstrating raw data obtained from multiplex Pyrosequencing using multiple templates obtained from multiplex PCR. Gel electrophoresis separation resolves two templates obtained from multiplex PCR (A). The left-hand lane represents the DNA marker, which is a 100 bp DNA ladder. Raw pyrogram data from each DNA template using a single primer are demonstrated in (B) and using multiple primers are shown in (C). Theoretical pattern expected from each nucleotide addition is demonstrated in the top panel of (C). The order of nucleotide dispensation is demonstrated below the pyrograms.

Figure 3

Pyrogram demonstrating raw data obtained from multiplex Pyrosequencing using multiple templates obtained from multiplex PCR. Gel electrophoresis separation resolves two templates obtained from multiplex PCR (A). The left-hand lane represents the DNA marker, which is a 100 bp DNA ladder. Raw pyrogram data from each DNA template using a single primer are demonstrated in (B) and using multiple primers are shown in (C). Theoretical pattern expected from each nucleotide addition is demonstrated in the top panel of (C). The order of nucleotide dispensation is demonstrated below the pyrograms.

Figure 4

Sequence alignment of seven different 5′ UTRs of HCV sequences. PCR-INR-HCV and PCR-INF-HCV demonstrate the hybridization positions of nested PCR primers. HCV-seq-F1, HCV-seq-F2 and HCV-seq-F3 show positions of the sequencing primers. Pyrosequencing of highlighted nucleotides result in a specific pattern of nucleotide incorporation.

Figure 5

Theoretical pattern (left) and pyrogram (right) obtained from six different subtypes using Multiplex Pyrosequencing. The height of the y-axis is determined by the number of incorporated nucleotides and the x-axis is determined by time. Nucleotides were added according to the specified order at 1 min intervals. The added nucleotide is indicated below the pyrogram.