Data management and interpretation

A software tool Sphix, was developed to complement the Sequencing Bead Array (SBA) assay (Software S1). The Ion Torrent Personal Genome Machine (PGM) Torrent Suite pre-filters all sequencing data into three general categories: i) polyclonal filters, ii) low-quality filters and iii) primer-dimer (further discussed and defined in Table 1). All reads that pass thru these filters are then accessible in a FASTQ file format. Sphix handles the data processing of these files and provides algorithmic profiling of the sample. The resulting comma-separate value (CSV) files are easily integrated to other software application for further analysis and generation of graphical representation in portable document format (PDF), which could be further developed for a user-friendly experience on laptops or handheld devices (Figure S1). Sphix and training data sets (Torrent Suite reports and sequencing data files reported here) are all freely accessible (Software S1 and Data S1).

Theoretically it would be sufficient to sequence the target hybridization region (Figure 1). However a 10-bp ID trace was included for a rapid readout process and to lay the groundwork for more complex future high-throughput applications (Table S1). Future Sphix algorithms designs could then compensate for differences in reporter distributions [22] (Figure 2), and use of error-correcting barcodes [23] to decrease sequencing errors. A multidimensional ID trace strategy [24] could also be used, i.e. where a specific panel of biomarkers are used with different patient specific sets of ID traces (e.g. patient A with biomarkers X, patient B with the same biomarkers X, etc.). This would lower the overall cost per sample by screening multiple patients in the same sequencing run. Data sorting and interpretation could be nearly instantaneous when using Sphix algorithms with different ID trace information.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Figure 2. Reporter library construction and Sequencing Bead Array (SBA) assay performance.

In (a-c), the left-hand side bar-histograms detail y-axis average read counts, while the right-hand side detail y-axis read distribution percentages amongst the ten reporters. Error-bars are also included in the right-hand side graphs to illustrate the read distribution variability amongst the replicates. The upper error-bar value is the highest observed frequency, while the lower error-bar value is the lowest observed frequency. (a) Results from six individually constructed reporter libraries sequenced side-by-side without any prior selections (metadata Table S2). (b) Results from six parallel negative (water) selections showing the background noise effects (metadata Table S3). (c) Results from six quintuple HPV selections of equimolar amounts of PCR-products derived from Human Papillomavirus (HPV) plasmids HPV-16, HPV-18, HPV-33, HPV-45, and HPV-59 (metadata Table S4). (d) Read count bar-histograms (b and c) superimposed with the experimentally established threshold value (dashed line) of ~1261.5 reads, which clearly distinguishes a positive call from the background noise.

https://doi.org/10.1371/journal.pone.0076696.g002

Reporter library construction

A reporter library is defined as an equimolar set of reporters with distinct target and ID traces (Figure 1). For compatibility with the PGM instrument, the reporters constituted of ~1-micron acrylamide beads with approximately 800,000 clustered oligonucleotides [15,16]. A preferred reporter library construction strategy would be to directly conjugate synthetic oligonucleotides to the beads, thus allowing for a high quality robust manufacturing setup. Then modified nucleic-acid oligonucleotides, e.g. locked nucleic acids (LNA) [25] and peptide nucleic acids (PNA) [25], could be used for improved target hybridization. Furthermore, direct conjugation would allow for use of oligonucleotides with the target hybridization site at the 3´-end, resulting in a variety of reporter activation modes (Figure S2). Potential added benefits could allow for increased sensitivity, specificity, and dynamic range, enabling single-base resolution (e.g. SNP-calling). Direct conjugation of other types of biomolecules (e.g. antibodies) to the beads could allow for flexibility in the choice of interrogated biomarker molecule (Figure S3).

However, since direct conjugation was not yet available with PGM compatible acrylamide beads. The proof-of-concept setup was based on clonal amplification of synthetic oligonucleotide templates via emulsion PCR. Emulsion PCR technology relies on an enzyme producing the DNA, with increased risk of beads with suboptimal DNA copy number, and other introduced artifacts. For added compatibility, the general priming site was placed at the 3´-end of all reporters (Figure 1). Three different template resources were investigated: i) a synthetic single-stranded DNA oligonucleotide with a linker sequence complementary to the attached bead primer (the positive strand), ii) its reverse-complement strand (the negative strand), and iii) complementary strands annealed to form a double-stranded DNA template. All template variants were able to generate reporter libraries containing the correct sequence composition; however the double-stranded templates were used for this assay to achieve a more even reporter distribution. The synthetic oligonucleotide templates used for emulsion PCR were, on average, ~115-bp in length (Table S1). These ultramers add complexity in terms of costs and inherent need for extensive purification. Variants with a shorter linker region (average size 85-bp) were also investigated and successfully implemented (data not shown), making the longer versions optional depending on library complexity and quality parameters desired. In-house oligonucleotide synthesis was employed without extensive purification protocols; while sufficient for an initial proof-of-concept, the lower quality grade accounts for variability. This phenomenon is clearly reflected in the library reporter read distribution (Figure 2), and thus the assay could be further optimized by more extensive purification of the oligonucleotide templates, and inclusion of internal normalization standards. Since the reporter oligonucleotide templates have many common regions (Table S1), one could employ a ligation assembly strategy of smaller oligonucleotides to increase oligonucleotide quality and lower production cost [26,27], or produce longer oligonucleotides via a PCR derived strategy [28].

A model 10-plex SBA assay was designed to genotype ten high-risk HPV genotypes (HPV-16, HPV-18, HPV-33, HPV-35, HPV-39, HPV-45, HPV-52, HPV-56, HPV-58, and HPV-59) commonly associated with cervical cancer progression [21]. For each targeted genotype, unique oligonucleotide templates with distinct target hybridization sites and a corresponding ID trace were used to generate libraries of reporters (Table S1). To investigate construction reproducibility, identical reporter libraries were generated from six separate emulsion PCRs and sequenced individually to examine library variability (Figure 2a, Table 1a, and Table S2). The emulsion PCR reaction generated approximately 100-µl of reporter library with an estimated average bead density of ~5.93 x10⁵ beads/μl. A fixed reporter library volume of 10-µl was used per SBA reaction (total ~5.93 x10⁶ beads). However the average reporter population in a sequencing run was only 6.87 x10⁵, resulting in a loading efficiency of only ~10% and, accordingly loss of resolution. In an ideal case for a 10-plex library, each reporter type would constitute 10% of all the called reporters. The observed average reporter distribution range was 6.63%-12.48%, attributable to differences in sequence composition, template concentrations, template quality, and handling. A variety of oligonucleotide template concentrations were explored; however, it proved difficult to lower the polyclonality factor, which is an inherent problem with the emulsion PCR method. Once produced, the library was refrigerated and stored for up to one month. Reporter libraries sequenced directly after production and those sequenced within a one-month time frame demonstrated little or no degradation in quality, which could be tracked via fluorescence measurements of the bead-clustered oligonucleotides and via sequencing runs (data not shown).

SBA assay validation and performance

The SBA assay methodology was divided into two events; i) target hybridization to activate the reporters, and ii) selection of activated reporters. The SBA assay experimental settings were optimized with synthetic targets and PCR products derived from plasmids with cloned HPV DNA fragments (henceforth referred to as HPV plasmids). Target hybridization temperature profiles were explored ranging in temperature from 37-50°C in 5 minute to 24-hour protocols. Annealing times less than one hour resulted in reproducibility difficulties, and thus a 1-hour protocol was chosen, as little or no improvements were observed with longer protocols. A higher annealing temperature also resulted in lowered observed background noise. Reporter selection was explored using both a manual approach (with a handheld magnetic-rack), and an automated robotic system; the automated variant offered uniform reproducibility and ease-of-use, and was therefore used in all detailed experiments. Target-to-bead ratios were explored by combing a fixed amount of reporter library beads (~5.93 x10⁶) with 1-picomolar target molecules in variable reaction volumes of 5-40-µl, (equivalent of ~1.48 x10⁵ -1.19 x10⁶ beads/μl). The higher bead densities rendered high background signals, while a less dense reaction volume produced a stronger signal-to-noise ratio. Activated reporters clumping and falsely co-selecting unwanted neighboring reporters, and or general steric hindrance due to high concentration of beads might have caused this. However bead density calculations were based on fluorescent measurements and estimations (Table 1 and Table S2), and should not be considered absolute values. Also optical effects caused by the beads was not accounted for in the fluorescent measurements.

Background noise is caused by unspecific aggregation between reporters and magnetic selection beads. To confidently distinguish a positive call, a minimum detectable value is needed. A threshold was therefore experimentally determined by performing six replicate selections on a negative (pure water), processed with the same conditions as the genomic samples (Figure 2b, Table 1b, and Table S3). Next-Generation Sequencing (NGS) read count data follows a Poisson or negative binominal distribution (when overdispersion persists) [29]. When read counts are below ~100 (e.g. individual reporter read counts, Figure 2), even a small increase in counts reduces the impact of shot noise, while at higher counts shot noise effects are lower. However since the proof-of-concept SBA assay operates at higher read count a level, a simplifying assumption that the background read count noise follows a normal distribution was made. The statistical three-sigma rule (the mean + 3x the standard deviation) was applied to the background noise data set, to establish a minimum threshold that would account for variability. This assumption is only applicable for sufficient number of background reads (i.e. mean reporter read counts > 100 reads), and would need to be statistically adjusted for future SBA embodiments with lowered background noise. There is also an uneven distribution of reporter reads in the background noise. In theory, reporter specific thresholds could be used to account for differences. However, a conservative threshold value of ~1261.5 was used, based on the reporter for HPV-56 with the highest background read count. Henceforth: reporter reads > threshold = positive call, and reporter read counts ≤ threshold = background noise. The threshold is specific to this library set, and for other applications similar thresholds need to be established.

To determine a lower detection limit of the SBA assay, target-to-bead ratios were explored using a fixed bead density, while varying the amount of targets from 1-picomolar to 1-femtomolar molecules. Assay capability was investigated employing a two-target detection model in which two synthetic targets, T-45 and T-59 (corresponding to biomarker sequences HPV-45, and HPV-59, respectively) were present at various concentrations; T-45 was present at a fixed amount of 1-picomolar molecules, while T-59 ranged from 1-picomolar down to 1-femtomolar molecules through a ten-fold dilution series. A lower detection limit of 10-femtomolar was observed (data not shown). However with an estimated bead-loading efficiency of ~10%, only a subset of the reporters are actually read. This fraction is dependent on total reporter population in the set, and could be further optimized with improved loading schemes, and chips supporting more wells. Target-to-reporter ratio is essential and will affect the capabilities of the assay. Any given reporter requires a minimum finite number of targets in order to be activated and selected. Targets in abundance will saturate the reporters, which could allow for improved ranges by adjusting amounts of total reporters used in the assay.

PCR products derived from HPV plasmids of the ten targeted HPV genotypes were individually detected with the SBA assay, and results were validated by amplicon Pyrosequencing [26,30]. Patients may carry multiple HPV infections and, though rare, have been shown to carry as many as five genotypes [30]. Therefore, the SBA assay was further deployed to screen pools of PCR products derived from different HPV plasmids. Equimolar pooling of PCR products from the five high-risk genotypes HPV-16, HPV-18, HPV-33, HPV-45, and HPV-59 (1/5 component each) was used as a target source to simulate a quintuple infection. The experiment was repeated six times to also investigate reproducibility of the assay, and in all cases all five HPV subtypes were successfully detected above the threshold (Figure 2c and d, Table 1c, and Table S4).

Differences in i) oligonucleotide template quality, ii) emulsion PCR construct yield, iii) sample source quality, iv) chip-loading, v) batches of kits and chips, and vi) handling, contributed to library variability between sequencing runs (Table 1 and Tables S2-S4). Reporter selections slightly increase polyclonal and significantly increase the low quality filtering, while the percentage of uncalled reads decrease (Table 1c). This phenomenon is also true for the background noise profile with significantly higher degree of low-quality filtering albeit lowered polyclonal filtering (Table 1b). Selection of beads with a multitude of target hybridization match and mismatch sites, may cause the increase in polyclonal filtering. In both cases (Table 1b and c) the total amount of beads to be loaded on the sequencing chip is lower after the selection when comparing with sequencing of a native reporter library (Table 1a). This also results in higher total read-count variability, with an increase in the standard deviation of the reporter population. This might further favor certain background noise effect with sup-optimal beads interfering with the process. However since the PGM Torrent Suite filtering algorithms is company proprietary knowledge, it is difficult to further speculate on the causal effects. It could be advantageous for future work to alter or completed remove these filters as a main PGM screening objective is to ensure long high-quality reads, while the SBA assay requires significantly lower read-lengths with less emphasis on read quality, which could be further compensated by use of error-correcting barcodes [23]. When the bar-histograms are viewed with y-axis read distributions of called reporters percentages (reporter read count/called reporters) the profile variation is significantly lower (Figure 2a-c right-hand graphs), which indicates potential for a very robust assay once handling can be automated. Optimizing target-to-bead ratio, reaction salt conditions and exploring alternative activation strategies (Figures S2 and S3), could further enhance overall assay performance and utility. Future improvements in sequencing sensors could allow for nanoparticle-sized reporters with lowered demand on numbers of clustered oligonucleotides. Such improvements would greatly increase target surface-area display and possibly target binding capacities, thus allowing for sensitivity near the nanoparticle bio-barcode assay attomolar range [12], thus circumventing need for prior nucleic acid amplification.

The Ion PGM Ion 314 chip was used throughout this work to achieve cost benefits. For situations that call for expanded reporter libraries, such as parallel sample interrogations, and increased dynamic range, sequencing chips with extended features should be explored. At present, the Ion PGM [15,16] is compatible with the Ion 314 chip having ~1.2 M wells, the Ion 316 chip having ~6.3 M wells, and the Ion 318 chip having ~11.3 M wells. The Ion Proton [15,16] uses sub-micron beads, allowing for decreased well-size and an even higher throughput with the PI chip having ~165 M wells, the PII chip having ~660 M wells, and the upcoming PIII chip having ~1.2 B wells (http://www.lifetechnologies.com. Accessed 2013 Aug 24).

Genotyping of clinical samples

Our main objective was to screen samples with unknown viral loads and profiles using PCR products derived from authentic clinical samples. Standardized nested PCR using general HPV primer sets PGMY09/11 and GP5+/6+ [31] was performed on 20 cervical tumor samples (extracted genomic DNA). As a comparator, amplicons were directly sequenced by Pyrosequencing [26,30]. The presence of HPV was confirmed in 19 of the 20 samples, and genotype results achieved by the two methods were in agreement, with the exception for samples OM-2215 and OM-2258 (Figure 3, Figure S1 and, and Table S5). A co-infection of HPV-16 and HPV-18 in OM-2215, and a co-infection of HPV-16 and HPV-45 in OM-2258 were detected with the SBA assay, whereas amplicon Pyrosequencing only detected the dominant genotype in each sample (HPV-18 and HPV-45 respectively). This was due to a higher sensitivity and dynamic range for the SBA assay. To confirm these findings, separate PCR screens were performed employing genotype-specific primers for HPV-16, HPV-18 and HPV-45, which in all cases generated the correct products with accurate sequence information. For sample OM-1299, neither method was able to detect any HPV genotype. Therefore ten additional separate PCR reactions were made using all ten possible genotype-specific primers [26,30], which did not either result in any genotype match rendering it a true negative with current assay conditions. Among the remaining 17 samples, ten were positive for HPV-16, four for HPV-18, one for HPV-45, and two for HPV-59.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Figure 3. Sequencing Bead Array (SBA) assay screens of 20 cervical tumor samples.

Bubble chart representation of the SBA assay screens of 20 cervical tumor samples (extracted genomic DNA). The data is based on number of called reporters (metadata in Figure S1 and Table S5). Results were called as follows: ten single infections for Human Papillomavirus (HPV) genotype HPV-16, four single infections for HPV-18, two single infections for HPV-59, one single infection for HPV-45, one dual co-infection for HPV-16, and HPV-18, one dual co-infection for HPV-16, and HPV-45, and one genotype negative sample. OM-1299 was negative as no reporter read count was above the background noise threshold.

https://doi.org/10.1371/journal.pone.0076696.g003

The general nested primer set PGMY09/11 and GP5+/6+ [31], is described capable of amplifying a spectrum of approximately 30 different genotypes. Future embodiments of the HPV genotyping SBA assay will aim to explore multiple amplification regions and expand the SBA reporter set to cover the vast majority of classified HPV genotypes [19,20], as the true strength of the technology lies in its multiplexing capabilities. Since only a limited set of HPV genotypes were interrogated using the SBA assay, the negative result for OM-1299 may be due to the presence of an un-interrogated HPV genotype. Other possible reasons for why the sample was negative despite its tumor origin may be; i) a true negative that does not carry anymore HPV virus due to prior cleared infections, ii) the genomic DNA extract did not captured any HPV DNA, iii) the HPV DNA was integrated into the host genome without the incorporation of L1 gene for which the PCR targets, vi) the PCR primer sets might have been ineffective to capture the HPV DNA due to genetic sequence variations or similar, or v) it may also reflect that the OM-1299 case (http://www.oncomatrix.com. Accessed 2013 Aug 24) was derived from an invasive adenocarcinoma, of which some types do not contain any HPV DNA [32].

Data management and interpretation

The software tool Sphix, was written in PERL (http://www.perl.org. Accessed 2013 Aug 24). Experimental data generated by the Ion Personal Genome Machine (PGM) software, the Torrent Suite 3.42 (Life Technologies, Carlsbad, CA), was manually retrieved from the Torrent Server, including FASTQ sequence data and sequencing run metrics of importance. Sphix performs barcode de-multiplexing by matching a given set of 10-bp ID traces with the sequence at the very beginning (5'-end) of each read, followed by binning and digital quantification of reporters. Only perfect matches were scored, counted and added to the results matrix. Based on the results, a CSV file is generated from which graphical bar-histogram representation can be generated using various third party software applications (e.g. Microsoft Excel), revealing the genotype profile of the sample. Sphix and training data sets are available online (Software S1 and Data S1).

The PGM dispenses a predefined custom flow order of nucleotides, wherein 4-flows make one cycle. The sequencing workflow requires that an initial 4-bp key sequence is used for read indexing for filtering and quality assessments. A theoretical minimum PGM dispensation order to cover the 4-bp key followed by a 10-bp ID trace from the predefined set of 153 ID traces (Table S1), was determined to be 10 cycles (i.e. 40 flows), corresponding to an estimated sequencing run time of 10 minutes. However, to cover reads that were shorter than expected, we chose to use 100 flows, corresponding to a sequencing runtime of ~37 minutes.

Reporter library construction

A 10-plex HPV reporter library was constructed for multiplex genotyping of PCR products derived from the nested PCR product of general PCR primer-sets PGMY09/11 and GP5+/6+ [31]. The PCR generates an ~150-bp amplicon containing genotype specific sequence variations within the region. By aligning all the possible combinations, genotype unique sites of 20-25-bp could be found within the primer-flanking region. The GenBank sequences for respective HPV genotype (HPV-16; HQ644299, HPV-18; GQ180792, HPV-33; HQ537707, HPV-35; JX129488, HPV-39; M62849, HPV-45; EF202156, HPV-52; HQ537739, HPV-56; EF177181, HPV-58; HQ537768, and HPV-59; EU918767) were aligned to find genotype-specific hybridization candidate sites. Multiple candidates were chosen for each of the ten interrogated genotypes. The following design criteria was setup to filter the candidates: i) 20-25-bp of length, ii) melting temperatures (T_m) of 52°C < Tm < 61°C, and iii) GC contents of 45% < GC < 60%. Sequences with hairpins or more than 5 single repeat were also eliminated. The online software application OligoAnalyzer 3.1 (Integrated DNA Technologies, Coralville, IA) was used to calculate all the criteria parameters. To ensure relative uniqueness, all sequences were compared to one another using megaBLAST (v 2.2.10) to eliminate any with an 80% match in 20 bases. The alignment and filtering resulted in one unique hybridization site for each of the ten interrogated HPV genotypes (Table S1).

The SBA oligonucleotides [100 µM] were synthesized in house (Stanford Genome Technology Center, Palo Alto, CA), and subsequently pooled with corresponding reverse complement strand in 1X STE-buffer [10 mM Tris (pH 7.6), 50 mM NaCl, and 0.1 mM EDTA]. Duplex templates were annealed in the GeneAmp PCR system 9700 (Life Technologies, Carlsbad, CA) at 95°C for 2 minutes followed by slow cooling until room temperature was reached. Following the hybridization step, all template duplexes were pooled at equimolar concentrations, and further diluted to 32 pM with ultraPURE H₂O (Life Technologies, Carlsbad, CA). The diluted pool later served as the reporter library template for emulsion PCR. The emulsion PCR was performed using the Ion One Touch DL System with the Ion OneTouch 200 Template Kit v2 DL (Life Technologies, Carlsbad, CA), using ~2-micron acrylamide beads [15,16], in order to be compatible with the Ion PGM instrument (Life Technologies, Carlsbad, CA). The automated Ion OneTouch ES (Life Technologies, Carlsbad, CA) emulsion PCR library enrichment and cleanup procedure yields approximately 100-µl of reporter library stock in 1X Wash/Storage Buffer [PBS, supplemented with 0.2% Tween-20 (pH 7.7)].

For each SBA assay reaction, a fixed amount of 10-µl of reporter library was used. Bead density estimates of the constructed reporter libraries, were based on fluorescently based quantitative measurements using a single-stranded DNA assay on the Qubit 2.0 Fluorometer (Life Technologies, Carlsbad, CA). Each constructed reporter library was measured in three replicates of 3-µl, with three independent measurements. Furthermore the fluorescent measurements provided means for tracking degradation of the bead-clustered oligonucleotides for storage purposes, which was notable post one-month time frame. Quality control was also performed by sequencing the native reporter libraries with the Ion PGM instrument when using the Ion 314 chips with the Ion PGM 200 Sequencing Kit (Life Technologies, Carlsbad, CA) according to the manufacturer’s instructions.

SBA assay validation and performance

Cloned HPV-plasmids for HPV-16, HPV-18, and HPV-45 were kindly provided by Dr. E.M. de Villiers (DKFZ, Heidelberg, Germany); HPV-33 and HPV-39 from Dr. M. Favre (Institute Pasteur, Paris, France); HPV-35 and HPV-56 DNA from Dr. A. Lorincz (Digene Corporation, Gaithersburg, MD, USA), HPV-52 DNA from Dr. W. Lancaster (Wayne State University School of Medicine, Detroit, MI, USA), and HPV-58 and HPV-59 DNA from Dr. T. Matsukura (National Institute of Health, Tokyo, Japan). The HPV plasmid concentrations were normalized at 100-ng/ml using an ND-100 Spectrophotometer (Thermo, Fisher Scientific, Waltham, MA). PCR amplification was performed using the nested PCR with general primers PGMY09/11 and GP5+/6+ [31] according to a previously described protocol [36], with an initial plasmid DNA concentration of 100-ng/ml, or ultraPURE H₂O for background noise selections (Figure 2b, Table 1b, and Table S3). The GP6+ primer carried a 5'-end biotin molecule for cleaning and selection purposes. A magnetic workstation, the Magnatrix 1200 (NorDiag, Biotrin International Ltd., Dublin, Ireland) was employed for automating the lab work. Running custom-made scripts, the robot generated single-stranded templates of the PCR amplicons. M-270 beads (Life Technologies, Carlsbad, CA) were used for immobilizing the DNA followed by magnetic collection and washing, initially in 1X TE-buffer [10 mM Tris–HCl (pH 7.5), 0.1 mM EDTA] followed by thorough washes with purified H₂O [18.2 MΩ.cm at 25 °C) (EMD Millipore, Billerica, MA] and NaOH [0.1 M] with constant mixing. Finally, the single-stranded products were released by heating to 80°C for 1 second in purified H₂O [37].

The SBA assay was executed by i) target hybridization to activate and immobilize the reporters, and ii) selection of activated reporters. A 10-µl aliquot of merged reporter library stock was further diluted to a total of 30-µl in 1X Annealing Buffer [PBS, supplemented with 0.2% Tween-20 (pH 7.7)]. Target DNA was added in 10-µl aliquots (using only 10-µl of the 50-µl PCR products) in 1X Annealing Buffer to make the total reaction volume to 40-µl. The mixture was sonicated at maximum intensity for 10 seconds using the desktop water bath and sonicator B2500A-DTH (VWR, Radnor, PA). Sonication ensures maximum mixture and target exposure. Target hybridization was accomplished using an initial heating at 95°C for 2 minutes with a GeneAmp PCR system 9700, then immediately relocated to a Thermomixer R & MTP Microblock (Eppendorf, Hamburg, Germany) programmed at 70°C, followed by slow cooling to 45°C with constant shaking at 1300 rpm to ensure the reporters avoided sedimentation for maximum target exposure. The activated reporters were then incubated at room temperature for 5 minutes with continuous mixing with 13-µl streptavidin coated paramagnetic MyOne C1-beads (Life Technologies, Carlsbad, CA) in 1X Annealing Buffer Magnetic selection was performed manually with 1X Wash/Storage Buffer and 1X Elution buffer [0.1 M NaOH supplemented with 0.1% Tween-20] while using a Dynal Invitrogen bead separation magnetic rack (Life Technologies, Carlsbad, CA). The final product was further washed and concentrated with 1X Annealing Buffer to yield a total volume of 3-µl ready for sequencing primer annealing and sequencing readout with Ion PGM instrument using the Ion 314 chips with the Ion PGM 200 Sequencing Kit according to manufacturer instructions. The Sequencing runs were programmed at a constant of 100 flows for all reactions to ensure full-length ID traces. In addition to the manual magnetic selection process, the process was also compatible with the Ion OneTouch ES protocol (Life Technologies, Carlsbad, CA).

Genotyping of clinical samples

The 20 cervical tumor samples (extracted genomic DNA) were obtained commercially (Oncomatrix, Inc., San Marcos, CA) from different females with accession codes OM-1078, OM-1272, OM-1299, OM-1301, OM-1452, OM-1464, OM-1530, OM-1569, OM-1668, OM-1741, OM-1751, OM-1848, OM-1854, OM-1967, OM-1980, OM-2006, OM-2059, OM-2215, OM-2257, and OM-2258. The samples were PCR amplified using a nested PCR with general primers PGMY09/11 and GP5+/6+ [31,36], with an initial sample concentration of 40-ng/ml genomic DNA, which yielded 50-µl PCR product. The full 50-µl PCR sample volume was used for all OM-samples, except: OM-1301, OM-1569, OM-1668, OM-1751, OM-1848, and OM-1854, for which only 10-µl PCR sample volume was used due to initial high DNA target concentration. The Magnatrix 1200 was programmed to generate single-stranded templates of the PCR amplicons.

In the cases of OM-2215 and OM-2258, amplicon Pyrosequencing [26,30] detected only a single (dominant) genotype, while the SBA detected two genotypes in each sample (HPV-16 and HPV-18 in OM-2215, and HPV-16 and HPV-45 in OM-2258). To confirm these findings, secondary PCR amplifications were performed using the genotype specific Multiple Sequencing Primer (MSP) MSP-16, MSP-18 and MSP-45 in separate reactions together with GP6+ [30], followed by Pyrosequencing using MSP-16, MSP-18 and MSP-45 as sequencing primers. In the case of OM-1299, where none of the methods manage to confirm a genotype, secondary PCR amplifications were performed using all the ten included genotype specific Multiple Sequencing Primer (MSP) MSP-16, MSP-18, MSP-33, MSP-35, MSP-39, MSP-45, MSP-52, MSP-56, MSP-58, and MSP-59 in separate reactions together with GP6+ [30], followed by Pyrosequencing using the included MSP sequencing primers individually.