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Papers by Monika Stoljarova

Research paper thumbnail of A high volume extraction and purification method for recovering DNA from human bone

Recent studies suggested that larger volumes of digestion buffer may be better at demineralizatio... more Recent studies suggested that larger volumes of digestion buffer may be better at demineralization of pulverized bone samples . In order to handle an increased volume of crude extract, one A B S T R A C T DNA recovery, purity and overall extraction efficiency of a protocol employing a novel silica-based column, Hi-Flow 1 (Generon Ltd., Maidenhead, UK), were compared with that of a standard organic DNA extraction methodology. The quantities of DNA recovered by each method were compared by real-time PCR and quality of DNA by STR typing using the PowerPlex 1 ESI 17 Pro System (Promega Corporation, Madison, WI) on DNA from 10 human bone samples. Overall, the Hi-Flow method recovered comparable quantities of DNA ranging from 0.8 ng AE 1 to 900 ng AE 159 of DNA compared with the organic method ranging from 0.5 ng AE 0.9 to 855 ng AE 156 of DNA. Complete profiles (17/17 loci tested) were obtained for at least one of three replicates for 3/10 samples using the Hi-Flow method and from 2/10 samples with the organic method. All remaining bone samples yielded partial profiles for all replicates with both methods. Compared with a standard organic DNA isolation method, the results indicated that the Hi-Flow method provided equal or improved recovery and quality of DNA without the harmful effects of organic extraction. Moreover, larger extraction volumes (up to 20 mL) can be employed with the Hi-Flow method which enabled more bone sample to be extracted at one time.

Research paper thumbnail of Whole mitochondrial genome genetic diversity in an Estonian population sample

International Journal of Legal Medicine, 2015

Mitochondrial DNA is a useful marker for population studies, human identification, and forensic a... more Mitochondrial DNA is a useful marker for population studies, human identification, and forensic analysis. Commonly used hypervariable regions I and II (HVI/HVII) were reported to contain as little as 25 % of mitochondrial DNA variants and therefore the majority of power of discrimination of mitochondrial DNA resides in the coding region. Massively parallel sequencing technology enables entire mitochondrial genome sequencing. In this study, buccal swabs were collected from 114 unrelated Estonians and whole mitochondrial genome sequences were generated using the Illumina MiSeq system. The results are concordant with previous mtDNA control region reports of high haplogroup HV and U frequencies (47.4 and 23.7 % in this study, respectively) in the Estonian population. One sample with the Northern Asian haplogroup D was detected. The genetic diversity of the Estonian population sample was estimated to be 99.67 and 95.85 %, for mtGenome and HVI/HVII data, respectively. The random match probability for mtGenome data was 1.20 versus 4.99 % for HVI/HVII. The nucleotide mean pairwise difference was 27±11 for mtGenome and 7±3 for HVI/HVII data. These data describe the genetic diversity of the Estonian population sample and emphasize the power of discrimination of the entire mitochondrial genome over the hypervariable regions.

Research paper thumbnail of Evaluation of a novel material, Diomics X-Swab™, for collection of DNA

Forensic Science International: Genetics, 2014

Research paper thumbnail of High-quality and high-throughput massively parallel sequencing of the human mitochondrial genome using the Illumina MiSeq

Forensic Science International: Genetics, 2014

Mitochondrial DNA typing in forensic genetics has been performed traditionally using Sanger-type ... more Mitochondrial DNA typing in forensic genetics has been performed traditionally using Sanger-type sequencing. Consequently sequencing of a relatively-large target such as the mitochondrial genome (mtGenome) is laborious and time consuming. Thus, sequencing typically focuses on the control region due to its high concentration of variation. Massively parallel sequencing (MPS) has become more accessible in recent years allowing for high-throughput processing of large target areas. In this study, Nextera(®) XT DNA Sample Preparation Kit and the Illumina MiSeq™ were utilized to generate quality whole genome mitochondrial haplotypes from 283 individuals in a both cost-effective and rapid manner. Results showed that haplotypes can be generated at a high depth of coverage with limited strand bias. The distribution of variants across the mitochondrial genome was described and demonstrated greater variation within the coding region than the non-coding region. Haplotype and haplogroup diversity were described with respect to whole mtGenome and HVI/HVII. An overall increase in haplotype or genetic diversity and random match probability, as well as better haplogroup assignment demonstrates that MPS of the mtGenome using the Illumina MiSeq system is a viable and reliable methodology.

Research paper thumbnail of A high volume extraction and purification method for recovering DNA from human bone

Forensic Science International: Genetics, 2014

Recent studies suggested that larger volumes of digestion buffer may be better at demineralizatio... more Recent studies suggested that larger volumes of digestion buffer may be better at demineralization of pulverized bone samples . In order to handle an increased volume of crude extract, one A B S T R A C T DNA recovery, purity and overall extraction efficiency of a protocol employing a novel silica-based column, Hi-Flow 1 (Generon Ltd., Maidenhead, UK), were compared with that of a standard organic DNA extraction methodology. The quantities of DNA recovered by each method were compared by real-time PCR and quality of DNA by STR typing using the PowerPlex 1 ESI 17 Pro System (Promega Corporation, Madison, WI) on DNA from 10 human bone samples. Overall, the Hi-Flow method recovered comparable quantities of DNA ranging from 0.8 ng AE 1 to 900 ng AE 159 of DNA compared with the organic method ranging from 0.5 ng AE 0.9 to 855 ng AE 156 of DNA. Complete profiles (17/17 loci tested) were obtained for at least one of three replicates for 3/10 samples using the Hi-Flow method and from 2/10 samples with the organic method. All remaining bone samples yielded partial profiles for all replicates with both methods. Compared with a standard organic DNA isolation method, the results indicated that the Hi-Flow method provided equal or improved recovery and quality of DNA without the harmful effects of organic extraction. Moreover, larger extraction volumes (up to 20 mL) can be employed with the Hi-Flow method which enabled more bone sample to be extracted at one time.

Research paper thumbnail of High sensitivity multiplex short tandem repeat loci analyses with massively parallel sequencing

Forensic Science International: Genetics, 2015

STR typing in forensic genetics has been performed traditionally using capillary electrophoresis ... more STR typing in forensic genetics has been performed traditionally using capillary electrophoresis (CE). However, CE-based method has some limitations: a small number of STR loci can be used; stutter products, dye artifacts and low level alleles. Massively parallel sequencing (MPS) has been considered a viable technology in recent years allowing high-throughput coverage at a relatively affordable price. Some of the CE-based limitations may be overcome with the application of MPS. In this study, a prototype multiplex STR System (Promega) was amplified and prepared using the TruSeq DNA LT Sample Preparation Kit (Illumina) in 24 samples. Results showed that the MinElute PCR Purification Kit (Qiagen) was a better size selection method compared with recommended diluted bead mixtures. The library input sensitivity study showed that a wide range of amplicon product (6-200ng) could be used for library preparation without apparent differences in the STR profile. PCR sensitivity study indicated that 62pg may be minimum input amount for generating complete profiles. Reliability study results on 24 different individuals showed that high depth of coverage (DoC) and balanced heterozygote allele coverage ratios (ACRs) could be obtained with 250pg of input DNA, and 62pg could generate complete or nearly complete profiles. These studies indicate that this STR multiplex system and the Illumina MiSeq can generate reliable STR profiles at a sensitivity level that competes with current widely used CE-based method.

Research paper thumbnail of A high volume extraction and purification method for recovering DNA from human bone

Recent studies suggested that larger volumes of digestion buffer may be better at demineralizatio... more Recent studies suggested that larger volumes of digestion buffer may be better at demineralization of pulverized bone samples . In order to handle an increased volume of crude extract, one A B S T R A C T DNA recovery, purity and overall extraction efficiency of a protocol employing a novel silica-based column, Hi-Flow 1 (Generon Ltd., Maidenhead, UK), were compared with that of a standard organic DNA extraction methodology. The quantities of DNA recovered by each method were compared by real-time PCR and quality of DNA by STR typing using the PowerPlex 1 ESI 17 Pro System (Promega Corporation, Madison, WI) on DNA from 10 human bone samples. Overall, the Hi-Flow method recovered comparable quantities of DNA ranging from 0.8 ng AE 1 to 900 ng AE 159 of DNA compared with the organic method ranging from 0.5 ng AE 0.9 to 855 ng AE 156 of DNA. Complete profiles (17/17 loci tested) were obtained for at least one of three replicates for 3/10 samples using the Hi-Flow method and from 2/10 samples with the organic method. All remaining bone samples yielded partial profiles for all replicates with both methods. Compared with a standard organic DNA isolation method, the results indicated that the Hi-Flow method provided equal or improved recovery and quality of DNA without the harmful effects of organic extraction. Moreover, larger extraction volumes (up to 20 mL) can be employed with the Hi-Flow method which enabled more bone sample to be extracted at one time.

Research paper thumbnail of Whole mitochondrial genome genetic diversity in an Estonian population sample

International Journal of Legal Medicine, 2015

Mitochondrial DNA is a useful marker for population studies, human identification, and forensic a... more Mitochondrial DNA is a useful marker for population studies, human identification, and forensic analysis. Commonly used hypervariable regions I and II (HVI/HVII) were reported to contain as little as 25 % of mitochondrial DNA variants and therefore the majority of power of discrimination of mitochondrial DNA resides in the coding region. Massively parallel sequencing technology enables entire mitochondrial genome sequencing. In this study, buccal swabs were collected from 114 unrelated Estonians and whole mitochondrial genome sequences were generated using the Illumina MiSeq system. The results are concordant with previous mtDNA control region reports of high haplogroup HV and U frequencies (47.4 and 23.7 % in this study, respectively) in the Estonian population. One sample with the Northern Asian haplogroup D was detected. The genetic diversity of the Estonian population sample was estimated to be 99.67 and 95.85 %, for mtGenome and HVI/HVII data, respectively. The random match probability for mtGenome data was 1.20 versus 4.99 % for HVI/HVII. The nucleotide mean pairwise difference was 27±11 for mtGenome and 7±3 for HVI/HVII data. These data describe the genetic diversity of the Estonian population sample and emphasize the power of discrimination of the entire mitochondrial genome over the hypervariable regions.

Research paper thumbnail of Evaluation of a novel material, Diomics X-Swab™, for collection of DNA

Forensic Science International: Genetics, 2014

Research paper thumbnail of High-quality and high-throughput massively parallel sequencing of the human mitochondrial genome using the Illumina MiSeq

Forensic Science International: Genetics, 2014

Mitochondrial DNA typing in forensic genetics has been performed traditionally using Sanger-type ... more Mitochondrial DNA typing in forensic genetics has been performed traditionally using Sanger-type sequencing. Consequently sequencing of a relatively-large target such as the mitochondrial genome (mtGenome) is laborious and time consuming. Thus, sequencing typically focuses on the control region due to its high concentration of variation. Massively parallel sequencing (MPS) has become more accessible in recent years allowing for high-throughput processing of large target areas. In this study, Nextera(®) XT DNA Sample Preparation Kit and the Illumina MiSeq™ were utilized to generate quality whole genome mitochondrial haplotypes from 283 individuals in a both cost-effective and rapid manner. Results showed that haplotypes can be generated at a high depth of coverage with limited strand bias. The distribution of variants across the mitochondrial genome was described and demonstrated greater variation within the coding region than the non-coding region. Haplotype and haplogroup diversity were described with respect to whole mtGenome and HVI/HVII. An overall increase in haplotype or genetic diversity and random match probability, as well as better haplogroup assignment demonstrates that MPS of the mtGenome using the Illumina MiSeq system is a viable and reliable methodology.

Research paper thumbnail of A high volume extraction and purification method for recovering DNA from human bone

Forensic Science International: Genetics, 2014

Recent studies suggested that larger volumes of digestion buffer may be better at demineralizatio... more Recent studies suggested that larger volumes of digestion buffer may be better at demineralization of pulverized bone samples . In order to handle an increased volume of crude extract, one A B S T R A C T DNA recovery, purity and overall extraction efficiency of a protocol employing a novel silica-based column, Hi-Flow 1 (Generon Ltd., Maidenhead, UK), were compared with that of a standard organic DNA extraction methodology. The quantities of DNA recovered by each method were compared by real-time PCR and quality of DNA by STR typing using the PowerPlex 1 ESI 17 Pro System (Promega Corporation, Madison, WI) on DNA from 10 human bone samples. Overall, the Hi-Flow method recovered comparable quantities of DNA ranging from 0.8 ng AE 1 to 900 ng AE 159 of DNA compared with the organic method ranging from 0.5 ng AE 0.9 to 855 ng AE 156 of DNA. Complete profiles (17/17 loci tested) were obtained for at least one of three replicates for 3/10 samples using the Hi-Flow method and from 2/10 samples with the organic method. All remaining bone samples yielded partial profiles for all replicates with both methods. Compared with a standard organic DNA isolation method, the results indicated that the Hi-Flow method provided equal or improved recovery and quality of DNA without the harmful effects of organic extraction. Moreover, larger extraction volumes (up to 20 mL) can be employed with the Hi-Flow method which enabled more bone sample to be extracted at one time.

Research paper thumbnail of High sensitivity multiplex short tandem repeat loci analyses with massively parallel sequencing

Forensic Science International: Genetics, 2015

STR typing in forensic genetics has been performed traditionally using capillary electrophoresis ... more STR typing in forensic genetics has been performed traditionally using capillary electrophoresis (CE). However, CE-based method has some limitations: a small number of STR loci can be used; stutter products, dye artifacts and low level alleles. Massively parallel sequencing (MPS) has been considered a viable technology in recent years allowing high-throughput coverage at a relatively affordable price. Some of the CE-based limitations may be overcome with the application of MPS. In this study, a prototype multiplex STR System (Promega) was amplified and prepared using the TruSeq DNA LT Sample Preparation Kit (Illumina) in 24 samples. Results showed that the MinElute PCR Purification Kit (Qiagen) was a better size selection method compared with recommended diluted bead mixtures. The library input sensitivity study showed that a wide range of amplicon product (6-200ng) could be used for library preparation without apparent differences in the STR profile. PCR sensitivity study indicated that 62pg may be minimum input amount for generating complete profiles. Reliability study results on 24 different individuals showed that high depth of coverage (DoC) and balanced heterozygote allele coverage ratios (ACRs) could be obtained with 250pg of input DNA, and 62pg could generate complete or nearly complete profiles. These studies indicate that this STR multiplex system and the Illumina MiSeq can generate reliable STR profiles at a sensitivity level that competes with current widely used CE-based method.