3µl Identifiler plus fast PCR protocol:
Optimization of the 3µl Identifiler Plus PCR protocol began with assessing different final extension lengths to prevent the formation of -A. Similar to the other fast PCR protocols, -A was present using the 1min and 5min final extensions, but was eliminated using 10min. However, low-level NSA was observed using the 59°C annealing/extension temperature, primarily at TH01 (~169b, ~185b and occasionally at ~187b), D16S539 (~287b), vWA (~153b) and TPOX (~220b). Therefore, 61°C and 63°C were evaluated to improve primer specificity; NSA continued at a lesser extent with the use of 61°C, but was eliminated using 63°C. Since full profiles were obtained from all samples, average peak height (1015rfu) and inter-locus peak balance (average CV of LPH:TPH of 0.292) were acceptable and all PHR were >50% using 63°C, that temperature was selected, and development shifted to reducing amplification time.
Shorter initial activation times of 1min and 2min were assessed in comparison to 3min, both of which exhibited acceptable profiles, though average peak height was reduced to 874rfu and 849rfu, respectively, while average CV of LPH:TPH improved somewhat (0.278 and 0.275, respectively) for both activation times. Therefore, the 1min activation was selected for further testing. Shorter denaturation times (5sec and 10sec, compared to 15sec) were assessed next. Both data sets yielded full profiles, but the 5sec data set exhibited a decrease in average peak height (711rfu), while the 10sec data set exhibited an increase (1269rfu), compared to 15sec. Though the explanation for the peak height increase using a 10sec denaturation (compared to those obtained with 15sec) was unknown, the 10sec denaturation was selected for further study because it performed as well as or better than 15sec with regard to peak height and other profile quality criteria.
Therefore, a 10sec denaturation was next evaluated with a 50sec annealing/extension step in comparison to the 60sec annealing/extension step previously tested, using a large data set (n=88) for both in an effort to reduce skewing due to small sample size; fast profiles from these 88 samples were compared to those obtained using standard PCR. Full profiles were obtained from 93%, 91% and 94%, respectively, with an average of 96% of alleles detected from both of the fast protocols compared to 98% from standard. Fast PCR’s slightly increased rate of dropout and decreased profile completeness were likely a result of reduced peak heights obtained from fast PCR (967rfu for the 50sec data set and 675rfu for 60sec) compared to standard PCR (1303rfu). It should be noted however, that fast PCR was performed on a different thermal cycler and detected on a different 3130xl Genetic Analyzer than the standard PCR data, which could account for the differences seen in peak height. Inter-locus peak balance was acceptable from all methods, with average CVs of LPH:TPH ranging from 0.247 for standard PCR to 0.258 (50sec) and 0.261 (60sec) fast PCR protocols, which is considerably lower than that observed by Identifiler fast PCR (typically >0.300). PHR <50% were infrequent, occurring in 1% of fast PCR profiles (50sec data set only; none observed in the 60sec data set) compared to 3% of standard profiles. Overall, first pass success rates (that is, the percentage of profiles passing after the first pass/round of testing) were highest from the 50sec fast PCR data set (92%), compared to 91% for 60sec fast PCR and standard PCR (dropout and PHR <50% were the only reasons for sample failure). Thus, the 50sec annealing/extension step was selected for the optimized Identifiler Plus fast PCR protocol, which had a total run time of 49min.
3µl PowerPlex 16 HS fast PCR protocol:
Optimization of the 3µl PowerPlex 16 HS PCR protocol also began with assessing different final extension lengths to prevent the formation of -A. Similar to the other fast PCR protocols, -A was present using the 1min and 5min final extensions, but was eliminated using 10min. Furthermore, from the first round of testing with the 10min final extension step, full profiles were obtained from all samples, no signs of NSA were present, average peak heights were 1256rfu, PHR <50% (17% of samples) were limited to 0.25ng samples, but inter-locus imbalance was higher than desired (average CV of LPH:TPH was 0.438). Next, 2-step PCR cycling was evaluated using annealing/extension temperatures of 58°C, 60°C and 62°C in an effort to improve profile quality. Low-level NSA was observed using both of the lower temperatures, but not at 62°C; however, profile quality decreased using 2-step PCR, most notably via allelic dropout (11% of samples) despite increased average peak heights (1476rfu), increased occurrences of PHR <50% (28% of samples) and decreased inter-locus balance (average CV of LPH:TPH of 0.474). Therefore, 2-step was not pursued further, and development of 3-step cycling continued with an assessment of increasing ramp rates from 29% and 23% to 100% for the annealing and extensions, respectively. Compared to the use of manufacturer’s recommended ramp rates, use of 100% ramp rates exhibited allelic dropout from one sample (a 0.25ng replicate; 6% of samples), despite increased average peak heights (1580rfu), decreased occurrences of PHR <50% (11% of samples; also limited to 0.25ng) and similar inter-locus balance (average CV of LPH:TPH of 0.446); however, increased average instances of called stutter per sample and pull-up >20% accompanied these higher peak heights. Use of 100% ramp rates was further tested in conjunction with reduced annealing times (15sec compared to 30sec). Even though full profiles were obtained from all samples using the shorter annealing time, a significant reduction in average peak height was observed (890rfu) and occurrences of PHR <50% doubled (22% of samples; limited to 0.25ng), while occurrences of stutter and pull-up declined; inter-locus peak balance was not significantly affected (average CV of LPH:TPH of 0.422). Since peak heights were still at an acceptable level using 100% ramp rates and 15sec annealing, these were assessed next with shorter extension times (20sec and 15sec, compared to 30sec). Extension times less than 30sec resulted in a significant increase in allelic dropout and reduction in peak heights, such that full profiles were only obtained from 73% (15sec) and 82% (20sec) of samples. Thus, the 30sec extension was maintained.
Reduced denaturation time (10sec and 5sec, compared to 15sec) was assessed next, but as seen with a reduction in extension time, a reduction in denaturation time also resulted in significant increases in allelic dropout and decreases in peak height. Therefore, 15sec denaturation was maintained. Next, a reduction in initial activation (1min versus 2min) was assessed. With this reduction, full profiles were still obtained from all samples, while average peak height (914rfu), inter-locus balance (average CV of LPH:TPH of 0.414) and occurrences of PHR <50% (no occurrences) were not effected in a negative manner. The 1min initial activation was then tested with a 25°C final hold in comparison to 4°C, from which full profiles were obtained, a significant increase in average peak height was observed (1888rfu), accompanied by increased occurrences of pull-up (not exceeding 20% of the true allele), but not significant changes in inter- or intra-locus balance. The implementation of the 25°C final hold completed the development of the PowerPlex 16 HS fast PCR protocol, resulting in a 51min amplification protocol. It should be noted that the desired level of inter-locus peak balance (CV of LPH:TPH ? 0.350) could not be achieved.
This fast PCR protocol was tested using 89 samples, and the resulting profiles were compared to those obtained using standard PCR. Full profiles were obtained from 96% of fast profiles and 93% of standard profiles, with an average of 98% and 99% of alleles detected, respectively. Peak heights were slightly lower from fast PCR (average of 1308rfu) compared to standard (average of 1449rfu), but were still on the high end of the desired range. Inter-locus peak imbalance was higher than desired using fast and standard PCR (average CVs of LPH:TPH of 0.416 and 0.372, respectively). PHR <50% were infrequent, occurring in 2% of fast PCR profiles compared to 4% of standard profiles. Overall, first pass success rates were highest from fast PCR (94%), compared to 90% for standard PCR; dropout and PHR <50%, were the only reasons for sample failure.
Validation of Low Volume, Fast PCR Protocols Using KAPA2G
See Figures 1-3 for representative profiles from fast PCR protocols utilizing KAPA2G.
Representative Identifiler Profiles from Fast and Standard PCR.
Profiles were obtained using each of the validated Identifiler/KAPA2G fast PCR reaction volumes (A-C) and 6μl Identifiler standard PCR (D). Allele calls, allele size (bases) and peak height (rfu) are displayed for each allele.
Representative Identifiler Plus Profiles from Fast and Standard PCR.
Profiles were obtained using the validated Identifiler Plus/KAPA2G low volume, fast PCR method (A) and Identifiler Plus standard PCR (B). Allele calls, allele size (bases) and peak height (rfu) are displayed for each allele.
Representative PowerPlex 16 HS Profiles from Fast and Standard PCR.
Profiles were obtained using the validated PowerPlex 16 HS/KAPA2G low volume, fast PCR method (A) and PowerPlex 16 HS standard PCR (B). Allele calls, allele size (bases) and peak height (rfu) are displayed for each allele.
1. Determination of the optimal range of input DNA
The optimal DNA input range was determined to be 0.375-1.50ng for each of the five fast PCR protocols based on the analyses below (Figure 4).
Optimal DNA Input Ranges for Fast PCR Protocols Using KAPA2G.
An optimal range (grey) was determined for each amplification based upon the evaluated criteria (n=5 or 6 samples per DNA input;those with n=5 had a sample removed due to injection failure).
For all five fast PCR protocols, average percent alleles detected and percent full profiles increased as template amount increased, such that full profiles were obtained from nearly all samples when ?0.188ng DNA was amplified (Figure 5). Though the senstivity range (i.e., the range in which full profiles were obtained from the majority of all samples) was determined to be 0.188-3.00ng for all but 3µl Identifiler fast PCR (0.375-3.00ng), data from 0.188-3.00ng is discussed further for each of the five fast PCR methods for comparison purposes. Allele peak height and balance is summarized in Figure 6. As expected, average allele peak height increased as DNA input increased. Reproducibility of peak height was measured via average coefficient of variation per DNA input, which was ?0.350 when ?0.188ng DNA was amplified using each of the four fast PCR protocols (except for 0.188ng with a 6µl Identifiler amplification), indicating acceptable levels of reproducibility.
Sensitivity of Fast PCR Protocols Using KAPA2G.
Sensitivity is displayed as percent alleles detected and percent full profiles (n=5 or 6 per DNA input; those with n=5 had a sample removed due to injection failure).
Peak Height Summary for Fast PCR Protocols Using KAPA2G.
Average peak height, reproducibility of peak height per allele, inter-locus peak balance and intra-locus peak balance/imbalance are displayed for each DNA input (n=5 or 6 per DNA input;those with n=5 had a sample removed due to injection failure).
None of the five methods tested were able to result in the desired level of general inter-locus balance (CV of LPH:TPH ?0.350) for all DNA input amounts. Nearly all methods exhibitedCVs >0.350 at 0.188ng and 3.00ng, while PowerPlex 16 HS CVs were >0.350 for all but 3.00ng. Intra-locus balance was measured via heterozygote peakheight ratios, averaging >82% for all ?0.375ng amplifications. Instances of PHR <50% were most frequent when 0.188ng DNA was amplified, but on average occurred less than once per sample.
Various artifacts were observed above the analysis threshold, but stutter (n-4) was the most prevalent type (Figure 7). Average percent stutter ranged from 10-19% and demonstrated a slight increase for the 0.375ng samples compared to 0.750-3.00ng. This was expected given that peak heights were lower at 0.375ng compared to higher inputs; thus, any stutter peaks that met the 75rfu analysis threshold at 0.375ng were a larger percentage of the true allele peak. Instances of stutter increased as DNA input increased and tended to be much less prevalent from PowerPlex 16 HS than the Identifiler/Identifiler Plus profiles. Unacceptably high stutter peaks (>20%) occurred occassionally, but were limited to profiles obtained using 3.00ng DNA (data not shown). Other forms of stutter (n+4 and n-8) did occur above threshold on occassion, but were nearly always limited to 1.50ng and 3.00ng amplifications. Furthermore, average percent stutter for these two forms of stutter (<4% for all) was much lower than that of n-4 stutter. Unacceptably high n-8 stutter (>2 occurrences in a single profile) occurred was limited to a single 3.00ng Identifiler Plus amplification (data not shown).
Artifacts for Fast PCR Protocols.
Using KAPA2G average percent stutter and pull-up are displayed, as well as average number of detected stutter, pull-up and elevated baseline per profile (n=5 or 6 samples per DNA input;those with n=5 had a sample removed due to injection failure).
Pull-up peaks were the next most abundant artifact that was detected above threhsold, but were nearly always limited to amplification of ?1.50ng DNA and were more frequent for Identifiler Plus and PowerPlex 16 HS. Unacceptably high pull-up (>20%) was limited to 3.00ng amplifications using Identifiler (6µl) and Identifiler Plus (data not shown). A single occurrence of -A (2.7% of the true allele) was present in a 3µl Identifiler amplification using 3.00ng DNA (data not shown). Elevated baseline was limited to 1.50ng and 3.00ng and was unacceptably high (occurrences at >3 loci) at 3.00ng for all five PCR methods (data not shown). No signs of non-specific amplification were noted.
2. Stochastic threshold
Stochastic thresholds were determined for each fast PCR method and were compared to those of standard PCR (Figure 8). Fast PCR stochastic were as good as or better (85-125rfu) than those from standard PCR (120-270rfu).
Stochastic Thresholds for Fast PCR Protocols Using KAPA2G.
These were established for each amplification method by determing the point at which heterzygous loci will not be mistaken for homozygous loci due to dropout of a single allele (n=510 to 595 loci for each amplification method). ID = Identifiler, ID+ = Identifiler Plus, HS = PowerPlex 16 HS, F = fast PCR, S = standard PCR.
Precision of allele sizing was assessed based on multiple injections of positive control 9947A amplified using each of the four fast PCR methods and compared to that of standard Identifiler. All assessments indicated acceptable levels of precision per manufacture recommendations (standard deviation <0.15; figure 6).
Precision of allele sizing was assessed based on positive control 9947A amplified using the 3μl fast PCR methods that were developed for each of the three primer sets tested. All assessments indicated acceptable levels of precision per manufacture recommendations (standard deviation <0.15; figure 9).
Precision of Allele Sizing for Fast PCR Protocols using KAPA2G precision was assessed via standard deviation for each allele from 9947A positive control DNA (for each amplification method, n=9 for each of the 25 alleles for PowerPlex 16 HS and 26 alleles for Identifiler Plus/Identifiler).
As was seen previously with the comparison of various 3µl Identifiler fast PCR protocols , nearly all assessments indicated more precise allele sizing between injections compared to intra-injection precision. Furthermore, all amplification methods exhibited satisfactory precision for allele sizing.
4. Stutter assessment
For the detailed stutter analysis, n-4 stutter was the most frequent type of stutter observed and accounted for 82% (3µl PowerPlex 16 HS) to 96% (6µl Identifiler) of all stutter peaks. Percent stutter (n-4) varied more by locus than amplification method (Figure 10). Maximum observed stutter for each fast PCR method exceeded the locus specific stutter thresholds supplied by vendors for each primer set processed under their recommendations (i.e., standard PCR, using 25μl reaction volumes) (Figure 11). Unless otherwise modified by the user in the GeneMapper® ID Panel Manager, the vendor-specific thresholds will be used by GeneMapper® ID during analysis and due to the increase in percent stutter for fast PCR, more stutter peaks will be called using these fast PCR methods compared to standard. Thus, laboratories desiring to implement fast PCR (or other amplification methods with stutter thresholds different than those supplied by the vendor) should be aware of this issue.
Average Percent Stutter (n-4) for Fast PCR Protocols Using KAPA2G.
Stutter is displayed by amplification method and locus (n=1592 to 2044 stutter peaks per method). D2S1338 and D19S433 are Identifiler and Identifiler Plus loci, while Penta D and Penta E are PowerPlex 16 HS loci.ID = Identifiler, ID+ = Identifiler Plus, HS = PowerPlex 16 HS, F = fast PCR.
Figure 11: Maximum Observed Stutter (n-4) for Fast PCR Protocols Using KAPA2G Compared to Standard PCR.
Maximum observed stutter was nearly always higher than the vendor-specified, locus specific stutter thresholds for standard, full volume PCR reactions of each primer set. ID = Identifiler, ID+ = Identifiler Plus, HS = PowerPlex 16 HS, F = fast PCR.
Implementing global stutter thresholds of 20% for all loci is not an uncommon practice for reference samples . Maximum stutter limits per locus were calculated via the sum of average percent stutter plus three standard deviations (a more conservative approach adds two standard deviations ), which never exceeded 20% for any locus or amplification method. It should be noted, however, that the maximum observed percent stutter (n-4) was occasionally (0.05-0.18% of samples) above 20% for all Identifiler and Identifiler Plus fast PCR methods, ranging from 21-25%, but never exceeded 20% for PowerPlex 16 HS. Half of the profiles exhibiting n-4 stutter >20% had stutter peaks that corresponded with pull-up from another locus, 33% were from allele 25 at D18S51 and the remaining 17% were from loci with low-level peak heights (<140rfu), which are subject to stochastic effects. Thus, a global 20% n-4 stutter threshold would work well for any of the developed fast PCR methods.
Both n+4 and n-8 stutter occurred significantly less than n-4, often not occurring at some loci (data not shown). Since occurrences of n+4 and n-8 were often low for individual loci, these types of stutter were averaged across all loci. Average n+4 percent stutter ranged from 2% to 4%, whereas average n-8 percent stutter ranged from 2% to 3% (Table 7). Maximum allowable n+4 and n-8 stutter were calculated across all loci as opposed to individual loci. However, it should be noted that observed maximum n+4 exceeded calculated maximums for three fast PCR protocols (5µl Identifiler, 3µl Identifiler Plus and 3µl PowerPlex 16 HS), while observed maximum n-8 exceeded calculated maximums for all five protocols. These discrepancies could arise from the fact that all stutter peaks of the same type were grouped together since there was not enough data to calculate these values for individual loci. Given that many databasing laboratories allow a 20% n+4 stutter threshold, and the maximum observed n+4 stutter was 16.6%, it appeared reasonable to apply the same 20% global stutter threshold to n+4 stutter for all five fast PCR protocols. Many laboratories don’t provide specific allowances for n-8 stutter; therefore, based upon the data obtained from this study, global stutter thresholds for n-8 stutter were set to 12% for the Identifiler fast PCR protocols and 10% for Identifiler Plus/PowerPlex 16 HS.
|Length of 4°C Storage
||959d,e [919, 999]
||1138e [1075, 1201]
||671b [645, 697]
||1068e [1023, 1113]
||871c,d [838, 904]
||837c,d [795, 879]
||630b [606, 654]
||832c,d [812, 852]
||837c,d [811, 863]
||891d [833, 949]
||732b,c [710, 754]
||0.856 [0.850, 0.861]
||0.868 [0.865, 0.870]
||0.832 [0.830, 0.840]
||0.858 [0.851, 0.865]
||0.849 [0.839, 0.860]
||0.840 [0.829, 0.850]
||0.859 [0.853, 0.864]
||0.851 [0.845, 0.856]
||0.868 [0.863, 0.873]
||0.854 [0.848, 0.860]
||0.854 [0.844, 0.863]
||0.863 [0.853, 0.869]
|CV of LPH:TPHg
||0.370 [0.338, 0.402]
||0.319 [0.268, 0.369]
||0.383 [0.333, 0.457]
||0.350 [0.302, 0.398]
||0.357 [0.316, 0.398]
||0.370 [0.329, 0.428]
||0.356 [0.305, 0.406]
||0.359 [0.302, 0.416]
||0.388 [0.332, 0.430]
||0.347 [0.295, 0.400]
||0.354 [0.299, 0.410]
||0.385 [0.329, 0.424]
Variation Due to Storage Conditions of KAPA2G™ Fast Multiplex PCR Kit Peak height, PHR and CV of LPH:TPH averages and 95% confidence intervals are displayed for each storage condition. Peak height averages that share subscripts are not statistically different at α=0.05 according to the Tukey HSD procedure; intra- and inter-locus balance (PHR and CV of LPH:TPH) were not significantly different for any of the storage conditions using two-way ANOVA.
an=192; fn=80; gn=7
Averages in each row that share subscripts are not statistically different at α=0.05 according to the Tukey HSD procedure. Not enough data existed to calculate average percent stutter by locus for these two types of stutter.
5. Automation (Large Sample Sets)
Each of the five fast PCR protocols were tested using 86-89 buccal samples (swab cuttings or Buccal DNA CollectorTM punches) with known profiles (Table 8 for a summary of profile quality). Full profiles were obtained from ?97% of samples for each fast PCR method, with an average of ?98% alleles detected; all profiles exhibited concordant allele calls and no unexplained alleles were present. Inter-locus peak balance was assessed via the average CV of LPH:TPH and was <0.350 for all fast methods except with the PowerPlex 16 HS primer set, but it should be noted that inter-locus peak balance was also lower than desired using standard PowerPlex 16 HS (3μl amplification; data not shown). Intra-locus peak balance was assessed via average PHR and occurrences of PHR <50%. All methods exhibited average PHR between 85.8% and 86.8%, and all but 6μl Identifiler exhibited at least one profile with one or more loci exhibiting PHR <50%, though the latter differences in occurrence of profiles with PHR <50% was not found to be statistically significant for the five fast protocols (p=0.45 using Pearson’s Chi-squared test). Furthermore, if a larger sample size had been tested, 6μl Identifiler likely would have exhibited a similarly low percentage of profiles with at least one locus with a PHR <50%. Lastly, first pass success rates were ≥95% for each of the five fast PCR protocols. It should be noted that all samples exhibiting dropout with a fast PCR method also exhibited dropout when amplified using standard PCR (except for one sample amplified using 6μl Identifiler fast PCR). On the other hand, PHR <50% were generally not reproducible across any of the PCR methods.
Furthermore, it should be noted that a low level (~100rfu), unexplained artifact was observed at Amelogenin (~108b) for 1.1% of 3µl Identifiler and 5µl Identifiler fast amplifications (a single sample for both amplification volumes, figure 12). This artifact was not reproducible, and at no other time during fast PCR development or validation was this artifact observed.
Fast PCR Artifact at Amelogenin.
A low-level artifact (~108b) was identified in one 3µl Identifiler amplification (top) and one 5µl Identifiler amplification (bottom). These fast amplifications were from different samples.
6. Contamination assessment
All negative amplification controls (n=44) were free of contamination, and no signs of contamination due to the amplification process were identified in any other sample, including positive controls.
7. Lot-to-lot variation
Lot-to-lot variation of KAPA2GTM Fast Multiplex PCR Kit was assessed for three different lots of the master mix (Table 9 for a summary of profile quality). Full profiles were obtained from all samples (n=5) and positive amplification controls (n=2) using each lot. Average peak heights were significantly higher from lots 2 and 3 than lot 1, whereas no significant difference in intra-locus (average PHR and instances of PHR <50%) or inter-locus (average CV of LPH:TPH) balance were observed for the three lots. Despite lower peak heights using Lot 1, high quality, full profiles were obtained from all three lots.
8. Storage conditions
Storage conditions were assessed via 12 combinations of number of thaws and length of 4°C storage prior to use. Full profiles were obtained for all samples (n=5) and positive amplification controls (n=2). Peak heights were significantly different for many of the storage conditions (Table 10), but general trends with regard to number of thaws and length of 4°C storage were not substantiated for the majority of data subsets (Figure 13). Peak height variation due to different amplification and detection runs on the same instrument is a widely accepted concept. However, in this particular case, it was unknown whether this additional variation in peak height influenced the data such that apparent trends appeared to exist for the 3-thaw and immediate use data sets, when in fact such trends did not exist, or if additional trends existed but appeared not to. Either way, peak height values were acceptable from all data sets. Intra- and inter-locus peak balance were assessed via PHR and CV of LPH:TPH, respectively, and did not exhibit significant differences between storage conditions. Therefore, all storage conditions tested were suitable for use.
Effect of Storage Conditions on Allele Peak Height.
Average allele peak height is displayed for the various storage conditions tested (n=7 samples per data set). Consisent trends with regard to peak height were only noted for the 3 Thaws data set (decrease as length of 4°C increased) and the Immediate Amp data set (unexpected increase as number of thaws increased).