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Using real-time PCR as strategy to evaluate performance of PCR and Sanger sequencing reactions
Forensic Science International: Genetics Supplement Series xxx (xxxx) xxx–xxx
Contents lists available at ScienceDirect
Forensic Science International: Genetics Supplement Series journal homepage: www.elsevier.com/locate/fsigss
Using real-time PCR as strategy to evaluate performance of PCR and Sanger sequencing reactions B. Freire-Paspuela, G. Burgosb,⁎ a b
Laboratorios de Investigación, Universidad de las Américas (UDLA), Quito, Ecuador Escuela de Medicina, Facultad de Ciencias de la Salud, Universidad de las Américas (UDLA), Quito, Ecuador
A R T I C LE I N FO
A B S T R A C T
Keywords: Sanger sequencing Real-time PCR mtDNA
Sanger sequencing is an indispensable technique for mitochondrial ancestry studies. However, it is a time consuming process that requires several resources to obtain high quality electropherograms. To optimize the efficiency of DNA analysis, we used real-time PCR (qPCR) to evaluate Sanger sequencing reaction performance before capillary electrophoresis. qPCR was performed to amplify Mitochondrial Control Region (CR). We determined that final PCR product quantification can be done using qPCR final fluorescence instead of visualization in agarose gel. Real-time sequencing reactions were done using BigDye Terminator v3.1 and SYBR Safe (Invitrogen); in addition, a dissociation curve step from 65 °C to 95 °C was added at the end of the thermal cycler protocol. The sequencing qPCR products were purified and run in a 3130 Genetic Analyzer (Applied Biosystems). The results demonstrated that fluorescence at 65 °C during the dissociation curve was directly related with DNA quantity in the sequencing qPCR reaction. Furthermore, samples that presented higher fluorescence at 65 °C generated better quality electropherograms. We suggest that electropherogram quality prediction before capillary electrophoresis is possible using sequencing qPCR. In this way, it is possible to increase workflow efficiency in laboratory procedures and identify errors during samples processing.
1. Introduction Sanger sequencing is an indispensable technique in mitochondrial ancestry studies and consist of several steps that are susceptible to manipulation errors; however, problems during sample processing cannot be detected until capillary electrophoresis, representing a waste of resources and time. Previous studies have used real-time PCR (qPCR) and high-resolution melting analysis of mtDNA to identify specific polymorphisms [1]. In this study, we propose a methodology using qPCR to evaluate Sanger sequencing reactions performance, making it possible to increase laboratory work efficiency and detect deficient sequencing reactions before capillary electrophoresis. 2. Material and methods DNA of fifteen donors was purified using a phenol-chloroform protocol. Mitochondrial Control Region (CR) were amplified by qPCR using L15900 and H639 primers [2]. qPCR reactions were performed in a final volume of 15 μL using 0.45U GoTaq DNA Polymerase, 1X colorless GoTaq Flexi buffer (Promega), 1.2 μM MgCl2, 0.3 mM dNTPs, 0.2 μM of each primer, 2X SYBR Safe (Invitrogen) and 7.5 ng DNA.
⁎
qPCR reactions were performed in a CFX96 C1000 Touch Thermal Cycler (Bio-Rad) under the following conditions: 95 °C for 2 min, then 40 cycles of 30 s at 95 °C, 30 s at 58 °C, 90 s at 72 °C, a final elongation of 5 min at 72 °C and a final dissociation curve step from 65 °C to 95 °C. To evaluate qPCR product quantity, band intensity in a 2% (w/v) agarose gel stained with SYBR Safe was compared to final fluorescence during qPCR reaction. After confirming amplification, the qPCR products were purified using Agencourt AMPure XP (Beckman Coulter) and magnetic Agencourt SPRIPlate 96 Super Magnet Plate as described by Burgos et al. (2017) [3]. Purified qPCR product of one sample was quantified with Nanodrop spectrophotometer (Thermo Scientific) and diluted in three serial concentrations to subsequently perform a trend line during the sequencing qPCR. Sequencing qPCR reactions of the serial dilutions and the rest of the samples were performed in a final volume of 12 μL using 1 μL BigDye Terminator v3.1 Ready Reaction Mix (Applied Biosystems), 0.75X sequencing buffer, 0.5 μM L15900 primer, 2X SYBR Safe and 2 μL of purified qPCR product. Thermal cycler conditions followed the manufacturer’s specifications and a dissociation step from 65 °C to 95 °C was included at the end of the protocol. A trend line with the three previously purified qPCR dilutions was done to compare DNA
Corresponding author. E-mail address: [email protected] (G. Burgos).
https://doi.org/10.1016/j.fsigss.2019.10.079 Received 16 September 2019; Accepted 7 October 2019 1875-1768/ © 2019 Elsevier B.V. All rights reserved.
Please cite this article as: B. Freire-Paspuel and G. Burgos, Forensic Science International: Genetics Supplement Series, https://doi.org/10.1016/j.fsigss.2019.10.079
Forensic Science International: Genetics Supplement Series xxx (xxxx) xxx–xxx
B. Freire-Paspuel and G. Burgos
Fig. 1. (A) Dissociation curve of sequencing qPCR. (B) Trend line obtained comparing the DNA quantity in the sequencing qPCR and the fluorescence at 65 °C. (C) Comparison between DCF65 and the electropherogram (%HQ) of 15 samples.
quantity to fluorescence at 65 °C in the dissociation curve. Sequencing qPCR products were purified using Agencourt CleanSEQ (Beckman Coulter) as described by Burgos et al. (2017) [3] and run in a ABI3130 genetic analyzer. Electropherogram quality was determined using Geneious 11.1.5 and consisted of the percentage of untrimmed high quality bases in the sequence (%HQ). Finally, fluorescence at 65 °C during the dissociation curve (DCF65) was compared to the electropherograms %HQ to set a fluorescence threshold that could be used to identify possible bad quality reactions before capillary electrophoresis.
in the sequencing qPCR reaction. 4. Conclusions The results showed that mitochondrial CR amplification by realtime PCR is useful for evaluating PCR products that will be subsequently sequenced. Therefore, qPCR could be used as an alternative to PCR product visualization in an agarose gel. In addition, sequencing qPCR can be used for sample-evaluation before final purification and to predict electropherograms quality before capillary electrophoresis. In this way, it is possible to increase workflow efficiency in laboratory procedures and identify errors during samples processing.
3. Results and discussion 3.1. Mitochondrial control region qPCR
Role of funding The mitochondrial control region qPCR reaction generated a product of 1308bp (Fig. S1A) and final fluorescence of 2000RFU (Fig. S1B), indicating that final fluorescence in the amplification curve was related with the band intensity in the agarose gel. Therefore, SYBR Safe stained qPCR could be used as an alternative to PCR product visualization in an agarose gel. Only amplified products with final fluorescence of more than 1500RFU were used for sequencing qPCR.
Financial support was provided by Grant MED.GB.18.06 from Dirección General de Investigación, Universidad de Las Américas, Quito, Ecuador. Declaration of Competing Interest None.
3.2. Sequencing qPCR Appendix A. Supplementary data During the dissociation curve of sequencing qPCR, the DNA serial dilutions had fluorescence values from 2960 to 3220RFU at 65 °C (DCF65) (Fig. 1A). These results generated a linear trend line with a minimal variation of R = 0.9969 (Fig. 1B). In conclusion, we saw a direct relation between DCF65 and the DNA quantity in the sequencing qPCR reaction. The electropherograms of the fifteen samples presented values from 0 to 70%HQ and DCF65 from 2600 to 3360RFU. The trend line generated with these values indicated a direct correlation between DCF65 and electropherogram %HQ (Fig. 1C). Electropherogram with high %HQ presented a uniform signal, less background and higher peaks. For instance, reactions with values lower than 3000RFU generated low quality electropherograms (less than 30%HQ). Therefore, a DCF65 threshold was set at 3000RFU to obtain high quality electropherograms and samples with a lower DCF65 value were discarded. This makes it possible to identify samples that could generate low quality electropherograms due to problems during purification or lack of DNA
Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.fsigss.2019.10.079. References [1] A. dos Santos, I. Soares, T. de Almeida, A. de Souza, R. Grazinoli, A. Mencalha, Highresolution melting (HRM) of hypervariable mitochondrial DNA regions for forensic science, J. Forensic Sci. 63 (2018) 536–540, https://doi.org/10.1111/1556-4029. 13552. [2] P. Cavalcanti, F. Carvalho, E. Carvalho, D. Silva, A mini-primer set in a multiplex PCR fashion covering the MTDNA control region from submerged skeletal remains, Forensic Sci. Int. Genet. Suppl. Ser. (2017), https://doi.org/10.1016/j.fsigss.2017. 09.194 1875–1768. [3] G. Burgos, B. Freire-Paspuel, T. Restrepo, M. Camargo, A. Ibarra, O. Palacio, M.E. Sánchez, E. Tejera, Magnetic beads: an alternative method to enzymatic purification for SNaPshot reactions, Forensic Sci. Int. Genet. Suppl. Ser. 6 (2017) e368–e369, https://doi.org/10.1016/j.fsigss.2017.09.107.
2
Contents lists available at ScienceDirect
Forensic Science International: Genetics Supplement Series journal homepage: www.elsevier.com/locate/fsigss
Using real-time PCR as strategy to evaluate performance of PCR and Sanger sequencing reactions B. Freire-Paspuela, G. Burgosb,⁎ a b
Laboratorios de Investigación, Universidad de las Américas (UDLA), Quito, Ecuador Escuela de Medicina, Facultad de Ciencias de la Salud, Universidad de las Américas (UDLA), Quito, Ecuador
A R T I C LE I N FO
A B S T R A C T
Keywords: Sanger sequencing Real-time PCR mtDNA
Sanger sequencing is an indispensable technique for mitochondrial ancestry studies. However, it is a time consuming process that requires several resources to obtain high quality electropherograms. To optimize the efficiency of DNA analysis, we used real-time PCR (qPCR) to evaluate Sanger sequencing reaction performance before capillary electrophoresis. qPCR was performed to amplify Mitochondrial Control Region (CR). We determined that final PCR product quantification can be done using qPCR final fluorescence instead of visualization in agarose gel. Real-time sequencing reactions were done using BigDye Terminator v3.1 and SYBR Safe (Invitrogen); in addition, a dissociation curve step from 65 °C to 95 °C was added at the end of the thermal cycler protocol. The sequencing qPCR products were purified and run in a 3130 Genetic Analyzer (Applied Biosystems). The results demonstrated that fluorescence at 65 °C during the dissociation curve was directly related with DNA quantity in the sequencing qPCR reaction. Furthermore, samples that presented higher fluorescence at 65 °C generated better quality electropherograms. We suggest that electropherogram quality prediction before capillary electrophoresis is possible using sequencing qPCR. In this way, it is possible to increase workflow efficiency in laboratory procedures and identify errors during samples processing.
1. Introduction Sanger sequencing is an indispensable technique in mitochondrial ancestry studies and consist of several steps that are susceptible to manipulation errors; however, problems during sample processing cannot be detected until capillary electrophoresis, representing a waste of resources and time. Previous studies have used real-time PCR (qPCR) and high-resolution melting analysis of mtDNA to identify specific polymorphisms [1]. In this study, we propose a methodology using qPCR to evaluate Sanger sequencing reactions performance, making it possible to increase laboratory work efficiency and detect deficient sequencing reactions before capillary electrophoresis. 2. Material and methods DNA of fifteen donors was purified using a phenol-chloroform protocol. Mitochondrial Control Region (CR) were amplified by qPCR using L15900 and H639 primers [2]. qPCR reactions were performed in a final volume of 15 μL using 0.45U GoTaq DNA Polymerase, 1X colorless GoTaq Flexi buffer (Promega), 1.2 μM MgCl2, 0.3 mM dNTPs, 0.2 μM of each primer, 2X SYBR Safe (Invitrogen) and 7.5 ng DNA.
⁎
qPCR reactions were performed in a CFX96 C1000 Touch Thermal Cycler (Bio-Rad) under the following conditions: 95 °C for 2 min, then 40 cycles of 30 s at 95 °C, 30 s at 58 °C, 90 s at 72 °C, a final elongation of 5 min at 72 °C and a final dissociation curve step from 65 °C to 95 °C. To evaluate qPCR product quantity, band intensity in a 2% (w/v) agarose gel stained with SYBR Safe was compared to final fluorescence during qPCR reaction. After confirming amplification, the qPCR products were purified using Agencourt AMPure XP (Beckman Coulter) and magnetic Agencourt SPRIPlate 96 Super Magnet Plate as described by Burgos et al. (2017) [3]. Purified qPCR product of one sample was quantified with Nanodrop spectrophotometer (Thermo Scientific) and diluted in three serial concentrations to subsequently perform a trend line during the sequencing qPCR. Sequencing qPCR reactions of the serial dilutions and the rest of the samples were performed in a final volume of 12 μL using 1 μL BigDye Terminator v3.1 Ready Reaction Mix (Applied Biosystems), 0.75X sequencing buffer, 0.5 μM L15900 primer, 2X SYBR Safe and 2 μL of purified qPCR product. Thermal cycler conditions followed the manufacturer’s specifications and a dissociation step from 65 °C to 95 °C was included at the end of the protocol. A trend line with the three previously purified qPCR dilutions was done to compare DNA
Corresponding author. E-mail address: [email protected] (G. Burgos).
https://doi.org/10.1016/j.fsigss.2019.10.079 Received 16 September 2019; Accepted 7 October 2019 1875-1768/ © 2019 Elsevier B.V. All rights reserved.
Please cite this article as: B. Freire-Paspuel and G. Burgos, Forensic Science International: Genetics Supplement Series, https://doi.org/10.1016/j.fsigss.2019.10.079
Forensic Science International: Genetics Supplement Series xxx (xxxx) xxx–xxx
B. Freire-Paspuel and G. Burgos
Fig. 1. (A) Dissociation curve of sequencing qPCR. (B) Trend line obtained comparing the DNA quantity in the sequencing qPCR and the fluorescence at 65 °C. (C) Comparison between DCF65 and the electropherogram (%HQ) of 15 samples.
quantity to fluorescence at 65 °C in the dissociation curve. Sequencing qPCR products were purified using Agencourt CleanSEQ (Beckman Coulter) as described by Burgos et al. (2017) [3] and run in a ABI3130 genetic analyzer. Electropherogram quality was determined using Geneious 11.1.5 and consisted of the percentage of untrimmed high quality bases in the sequence (%HQ). Finally, fluorescence at 65 °C during the dissociation curve (DCF65) was compared to the electropherograms %HQ to set a fluorescence threshold that could be used to identify possible bad quality reactions before capillary electrophoresis.
in the sequencing qPCR reaction. 4. Conclusions The results showed that mitochondrial CR amplification by realtime PCR is useful for evaluating PCR products that will be subsequently sequenced. Therefore, qPCR could be used as an alternative to PCR product visualization in an agarose gel. In addition, sequencing qPCR can be used for sample-evaluation before final purification and to predict electropherograms quality before capillary electrophoresis. In this way, it is possible to increase workflow efficiency in laboratory procedures and identify errors during samples processing.
3. Results and discussion 3.1. Mitochondrial control region qPCR
Role of funding The mitochondrial control region qPCR reaction generated a product of 1308bp (Fig. S1A) and final fluorescence of 2000RFU (Fig. S1B), indicating that final fluorescence in the amplification curve was related with the band intensity in the agarose gel. Therefore, SYBR Safe stained qPCR could be used as an alternative to PCR product visualization in an agarose gel. Only amplified products with final fluorescence of more than 1500RFU were used for sequencing qPCR.
Financial support was provided by Grant MED.GB.18.06 from Dirección General de Investigación, Universidad de Las Américas, Quito, Ecuador. Declaration of Competing Interest None.
3.2. Sequencing qPCR Appendix A. Supplementary data During the dissociation curve of sequencing qPCR, the DNA serial dilutions had fluorescence values from 2960 to 3220RFU at 65 °C (DCF65) (Fig. 1A). These results generated a linear trend line with a minimal variation of R = 0.9969 (Fig. 1B). In conclusion, we saw a direct relation between DCF65 and the DNA quantity in the sequencing qPCR reaction. The electropherograms of the fifteen samples presented values from 0 to 70%HQ and DCF65 from 2600 to 3360RFU. The trend line generated with these values indicated a direct correlation between DCF65 and electropherogram %HQ (Fig. 1C). Electropherogram with high %HQ presented a uniform signal, less background and higher peaks. For instance, reactions with values lower than 3000RFU generated low quality electropherograms (less than 30%HQ). Therefore, a DCF65 threshold was set at 3000RFU to obtain high quality electropherograms and samples with a lower DCF65 value were discarded. This makes it possible to identify samples that could generate low quality electropherograms due to problems during purification or lack of DNA
Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.fsigss.2019.10.079. References [1] A. dos Santos, I. Soares, T. de Almeida, A. de Souza, R. Grazinoli, A. Mencalha, Highresolution melting (HRM) of hypervariable mitochondrial DNA regions for forensic science, J. Forensic Sci. 63 (2018) 536–540, https://doi.org/10.1111/1556-4029. 13552. [2] P. Cavalcanti, F. Carvalho, E. Carvalho, D. Silva, A mini-primer set in a multiplex PCR fashion covering the MTDNA control region from submerged skeletal remains, Forensic Sci. Int. Genet. Suppl. Ser. (2017), https://doi.org/10.1016/j.fsigss.2017. 09.194 1875–1768. [3] G. Burgos, B. Freire-Paspuel, T. Restrepo, M. Camargo, A. Ibarra, O. Palacio, M.E. Sánchez, E. Tejera, Magnetic beads: an alternative method to enzymatic purification for SNaPshot reactions, Forensic Sci. Int. Genet. Suppl. Ser. 6 (2017) e368–e369, https://doi.org/10.1016/j.fsigss.2017.09.107.
2