Type-specific multiple sequencing primers: a novel strategy for reliable and rapid genotyping of human papillomaviruses by pyrosequencing technology

Abstract

DNA sequencing is the gold standard method for accurate microbial and viral typing. However, DNA sequencing techniques have been facing limitations in typing of human papillomaviruses when the specimen harbors multiple genotypes and yields nonspecific amplification products, resulting in nonspecific and noninterpretable sequence data. To address these limitations we have developed a type-specific multiple sequencing primer DNA-sequencing method. This new strategy is suitable for sequencing and typing of samples harboring different genotypes (co-infections with multiple genotypes) and yielding nonspecific amplifications, thus eliminating the need for nested polymerase chain reaction (PCR), stringent PCR conditions, and cloning. The new approach has also proved useful for amplicons containing low PCR yield or subdominant types, avoiding reperforming of amplifications. We have applied the multiple sequencing primer method for genotyping of clinically relevant human papillomaviruses in a clinical test panel by using a combined pool of seven type-specific sequencing primers for HPV-6, -11, -16, -18, -31, -33, and -45. Furthermore, we introduced a sequence pattern recognition approach when there was a plurality of genotypes in the sample to facilitate typing of more than one target DNA in the sample. The multiple sequencing primer method has proved to be a multifaceted approach for typing of human papillomaviruses by DNA sequencing technologies.

Figure 1

Illustrative demonstration of sequencing of amplicons containing HPV co-infections with multiple genotypes and nonspecific amplification products. a: HPV sample containing nonspecific amplification products and sequenced by the GP5+ sequencing (general) primer, resulting in sequence signals from the HPV and nonspecific products. b: Sequencing of the amplified DNA by multiple sequencing primers. Only HPV-16 will result in specific sequence signals. c: HPV sample containing co-infections of HPV-16 and HPV-18, and nonspecific amplification products, resulting in sequence signals from all present genotypes and the nonspecific amplification product by using the general primer. d: Sequencing of the same co-infected sample as described above by multiple sequencing primers. HPV-16 and HPV-18 both result in sequence signals and are genotyped easily by pattern recognition.

Figure 2

Effects of SSB on improvement of sequence signal quality. a: Pyrograms from sequencing of HPV-72 by GP5+ both in the absence and in the presence of SSB. b: Sequencing of HPV-31 in a simulated co-infection of HPV-31/40/73 sequenced by the seven-primer pool both in the absence and in the presence of SSB.

Figure 3

Ethidium bromide agarose-stained gel of HPV amplicons amplified by MY09/11 primer set resulting in nonspecific amplification products. The specific HPV band is at 450 bp. All these samples were easily genotyped by the multiple sequencing primer strategy.

Figure 4

Pyrograms of HPV-16 and HPV-18 amplicons sequenced by the seven-primer pool as reference for co-infection comparisons in Figure 5.

Figure 5

Pyrograms of co-infections of HPV-16 and HPV-18 in three different clinical samples sequenced by the seven multiple sequencing primer pool and genotyped by pattern recognition The samples contained double infections of HPV-16 and HPV-18 and were genotyped by comparing characteristic sequence signals specific for each type. The common and specific bases for each type are noted on top of each peak, characterizing each type, facilitating genotyping. Sequence peaks on positions 1, 2, 5, 6, and 9 represent HPV-16 and sequence peaks on positions 1, 2, 7, and 10 represent HPV-18. The solid and dotted arrows are for demonstration of the dominance of HPV-16 and HPV-18. The dominant type could be easily observed by comparison of single bases shown by arrows. a: HPV-16 and HPV-18 almost equal in dominance; b: HPV-18-dominant; and c: HPV-16-dominant.

Figure 6

Genotyping of a challenging clinical sample containing dominant co-infections or nonspecific amplification products by using general primer resulting in nonspecific sequence signals (a) and resulting in specific sequencing signals (genotyping accurately HPV-33) (b). c: Pyrogram of HPV-33 from plasmid as a reference for comparison with b.