Abstract
Skeletal remains recovered from missing persons’ cases are often exposed to harsh environmental conditions resulting in the DNA being damaged, degraded, and/or the samples containing PCR inhibitors. In this study, the efficacy of common extraction methods was evaluated to remove high levels of PCR inhibitors commonly encountered with human remains, and their downstream compatibility with the two leading sequencing chemistries and platforms for human identification purposes. Blood, hair, and bone samples were spiked with high levels of inhibitors commonly identified in each particular substrate in order to test the efficiency of various DNA extraction methods prior to sequencing. Samples were extracted using three commercial extraction kits (DNA IQ™, DNA Investigator, and PrepFiler® BTA), organic (blood and hair only), and two total demineralization protocols (bone only)). Massively parallel sequencing (MPS) was performed using two different systems: Precision ID chemistry and a custom AmpliSeq™ STR and iiSNP panel on the Ion S5™ System and the ForenSeq DNA Signature Prep Kit on the MiSeq FGx™. The overall results showed that all DNA extraction methods were efficient and are fully compatible with both MPS systems. Key performance indicators such as STR and SNP reportable alleles, read depth, and heterozygote balance were comparable for each extraction method. In samples where CE-based STRs yielded partial profiles (bone), MPS-based STRs generated more complete or full profiles. Moreover, MPS panels contain more STR loci than current CE-based STR kits and also include SNPs, which can further increase the power of discrimination obtained from these samples, making MPS a desirable choice for the forensic analysis of such challenging samples.
| Original language | English |
|---|---|
| Pages (from-to) | 51-58 |
| Number of pages | 8 |
| Journal | International journal of legal medicine |
| Volume | 133 |
| Issue number | 1 |
| DOIs | |
| State | Published - 14 Jan 2019 |
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Keywords
- DNA extraction
- Ion S5™
- Massively parallel sequencing
- MiSeq FGx™
- Missing persons
- PCR inhibitors
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}
Assessment of impact of DNA extraction methods on analysis of human remain samples on massively parallel sequencing success. / Zeng, Xiangpei; Elwick, Kyleen; Mayes, Carrie; Takahashi, Maiko; King, Jonathan L.; Gangitano, David; Budowle, Bruce; Hughes-Stamm, Sheree.
In: International journal of legal medicine, Vol. 133, No. 1, 14.01.2019, p. 51-58.Research output: Contribution to journal › Article › Research › peer-review
TY - JOUR
T1 - Assessment of impact of DNA extraction methods on analysis of human remain samples on massively parallel sequencing success
AU - Zeng, Xiangpei
AU - Elwick, Kyleen
AU - Mayes, Carrie
AU - Takahashi, Maiko
AU - King, Jonathan L.
AU - Gangitano, David
AU - Budowle, Bruce
AU - Hughes-Stamm, Sheree
PY - 2019/1/14
Y1 - 2019/1/14
N2 - Skeletal remains recovered from missing persons’ cases are often exposed to harsh environmental conditions resulting in the DNA being damaged, degraded, and/or the samples containing PCR inhibitors. In this study, the efficacy of common extraction methods was evaluated to remove high levels of PCR inhibitors commonly encountered with human remains, and their downstream compatibility with the two leading sequencing chemistries and platforms for human identification purposes. Blood, hair, and bone samples were spiked with high levels of inhibitors commonly identified in each particular substrate in order to test the efficiency of various DNA extraction methods prior to sequencing. Samples were extracted using three commercial extraction kits (DNA IQ™, DNA Investigator, and PrepFiler® BTA), organic (blood and hair only), and two total demineralization protocols (bone only)). Massively parallel sequencing (MPS) was performed using two different systems: Precision ID chemistry and a custom AmpliSeq™ STR and iiSNP panel on the Ion S5™ System and the ForenSeq DNA Signature Prep Kit on the MiSeq FGx™. The overall results showed that all DNA extraction methods were efficient and are fully compatible with both MPS systems. Key performance indicators such as STR and SNP reportable alleles, read depth, and heterozygote balance were comparable for each extraction method. In samples where CE-based STRs yielded partial profiles (bone), MPS-based STRs generated more complete or full profiles. Moreover, MPS panels contain more STR loci than current CE-based STR kits and also include SNPs, which can further increase the power of discrimination obtained from these samples, making MPS a desirable choice for the forensic analysis of such challenging samples.
AB - Skeletal remains recovered from missing persons’ cases are often exposed to harsh environmental conditions resulting in the DNA being damaged, degraded, and/or the samples containing PCR inhibitors. In this study, the efficacy of common extraction methods was evaluated to remove high levels of PCR inhibitors commonly encountered with human remains, and their downstream compatibility with the two leading sequencing chemistries and platforms for human identification purposes. Blood, hair, and bone samples were spiked with high levels of inhibitors commonly identified in each particular substrate in order to test the efficiency of various DNA extraction methods prior to sequencing. Samples were extracted using three commercial extraction kits (DNA IQ™, DNA Investigator, and PrepFiler® BTA), organic (blood and hair only), and two total demineralization protocols (bone only)). Massively parallel sequencing (MPS) was performed using two different systems: Precision ID chemistry and a custom AmpliSeq™ STR and iiSNP panel on the Ion S5™ System and the ForenSeq DNA Signature Prep Kit on the MiSeq FGx™. The overall results showed that all DNA extraction methods were efficient and are fully compatible with both MPS systems. Key performance indicators such as STR and SNP reportable alleles, read depth, and heterozygote balance were comparable for each extraction method. In samples where CE-based STRs yielded partial profiles (bone), MPS-based STRs generated more complete or full profiles. Moreover, MPS panels contain more STR loci than current CE-based STR kits and also include SNPs, which can further increase the power of discrimination obtained from these samples, making MPS a desirable choice for the forensic analysis of such challenging samples.
KW - DNA extraction
KW - Ion S5™
KW - Massively parallel sequencing
KW - MiSeq FGx™
KW - Missing persons
KW - PCR inhibitors
UR - http://www.scopus.com/inward/record.url?scp=85055560372&partnerID=8YFLogxK
U2 - 10.1007/s00414-018-1955-9
DO - 10.1007/s00414-018-1955-9
M3 - Article
VL - 133
SP - 51
EP - 58
JO - International Journal of Legal Medicine
JF - International Journal of Legal Medicine
SN - 0937-9827
IS - 1
ER -