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Performance and concordance of the ForenSeq™ system for autosomal and Y chromosome short tandem repeat sequencing of reference-type specimens.
Forensic Sci Int Genet. 2017 05; 28:1-9.FS

Abstract

Though the utility of next-generation sequencing (NGS) technologies for forensic short tandem repeat (STR) typing has been evident for several years, commercially available assays and software solutions developed specifically to meet forensic needs have only recently become available. One of these, the ForenSeq™ DNA Signature Prep Kit (Illumina, Inc.) sequences 27 autosomal STR (aSTR) and 24 Y chromosome STR (Y-STR) loci (concurrent with additional nuclear markers) per multiplexed sample, with automated secondary and tertiary analyses of the data accomplished via the associated ForenSeq™ Universal Analysis Software (UAS). In this study we investigated the performance of the ForenSeq system for aSTR and Y-STR typing by examination of 151 sample libraries developed from high quality DNAs amplified at the target 1ng template. Utilizing PCR Primer Mix B, greater than 99.5% of aSTR loci and 97.0% of Y-STR loci were recovered when 42 or fewer sample libraries were pooled for sequencing. A direct comparison of UAS developed fragment length results to capillary electrophoresis (CE) based data identified only two allele call discrepancies when no UAS quality flag was triggered. Review of the ForenSeq data indicated that most samples with total sequence read counts exceeding 40,000 could be interpreted to develop nearly complete aSTR genotypes or Y-STR haplotypes. However, markers D22S1045 and DYS392 produced poor or inconsistent results even when sample read counts were greater than 85,000. Excluding these two loci, analyst-interpreted aSTR and Y-STR ForenSeq profiles were 99.96% and 100% concordant, respectively, with CE data. In addition to demonstrating concordance on par with other CE kit to kit comparisons, the results from this study will assist laboratories seeking to develop workflows for high volume processing and analysis of aSTRs and Y-STRs from reference-type specimens using the ForenSeq system.

Authors+Show Affiliations

DNA Support Unit, Federal Bureau of Investigation Laboratory, 2501 Investigation Parkway, Quantico, VA 22135, USA; Counterterrorism and Forensic Science Research Unit, Visiting Scientist Program, Federal Bureau of Investigation Laboratory, 2501 Investigation Parkway, Quantico, VA 22135, USA. Electronic address: Rebecca.Just@ic.fbi.gov.DNA Support Unit, Federal Bureau of Investigation Laboratory, 2501 Investigation Parkway, Quantico, VA 22135, USA.DNA Support Unit, Federal Bureau of Investigation Laboratory, 2501 Investigation Parkway, Quantico, VA 22135, USA.DNA Support Unit, Federal Bureau of Investigation Laboratory, 2501 Investigation Parkway, Quantico, VA 22135, USA.

Pub Type(s)

Evaluation Study
Journal Article

Language

eng

PubMed ID

28126691

Citation

Just, Rebecca S., et al. "Performance and Concordance of the ForenSeq™ System for Autosomal and Y Chromosome Short Tandem Repeat Sequencing of Reference-type Specimens." Forensic Science International. Genetics, vol. 28, 2017, pp. 1-9.
Just RS, Moreno LI, Smerick JB, et al. Performance and concordance of the ForenSeq™ system for autosomal and Y chromosome short tandem repeat sequencing of reference-type specimens. Forensic Sci Int Genet. 2017;28:1-9.
Just, R. S., Moreno, L. I., Smerick, J. B., & Irwin, J. A. (2017). Performance and concordance of the ForenSeq™ system for autosomal and Y chromosome short tandem repeat sequencing of reference-type specimens. Forensic Science International. Genetics, 28, 1-9. https://doi.org/10.1016/j.fsigen.2017.01.001
Just RS, et al. Performance and Concordance of the ForenSeq™ System for Autosomal and Y Chromosome Short Tandem Repeat Sequencing of Reference-type Specimens. Forensic Sci Int Genet. 2017;28:1-9. PubMed PMID: 28126691.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Performance and concordance of the ForenSeq™ system for autosomal and Y chromosome short tandem repeat sequencing of reference-type specimens. AU - Just,Rebecca S, AU - Moreno,Lilliana I, AU - Smerick,Jill B, AU - Irwin,Jodi A, Y1 - 2017/01/09/ PY - 2016/09/23/received PY - 2016/12/30/revised PY - 2017/01/01/accepted PY - 2017/1/28/pubmed PY - 2017/7/8/medline PY - 2017/1/28/entrez KW - Concordance KW - ForenSeq KW - Massively parallel sequencing (MPS) KW - Next generation sequencing (NGS) KW - Short tandem repeat (STR) SP - 1 EP - 9 JF - Forensic science international. Genetics JO - Forensic Sci Int Genet VL - 28 N2 - Though the utility of next-generation sequencing (NGS) technologies for forensic short tandem repeat (STR) typing has been evident for several years, commercially available assays and software solutions developed specifically to meet forensic needs have only recently become available. One of these, the ForenSeq™ DNA Signature Prep Kit (Illumina, Inc.) sequences 27 autosomal STR (aSTR) and 24 Y chromosome STR (Y-STR) loci (concurrent with additional nuclear markers) per multiplexed sample, with automated secondary and tertiary analyses of the data accomplished via the associated ForenSeq™ Universal Analysis Software (UAS). In this study we investigated the performance of the ForenSeq system for aSTR and Y-STR typing by examination of 151 sample libraries developed from high quality DNAs amplified at the target 1ng template. Utilizing PCR Primer Mix B, greater than 99.5% of aSTR loci and 97.0% of Y-STR loci were recovered when 42 or fewer sample libraries were pooled for sequencing. A direct comparison of UAS developed fragment length results to capillary electrophoresis (CE) based data identified only two allele call discrepancies when no UAS quality flag was triggered. Review of the ForenSeq data indicated that most samples with total sequence read counts exceeding 40,000 could be interpreted to develop nearly complete aSTR genotypes or Y-STR haplotypes. However, markers D22S1045 and DYS392 produced poor or inconsistent results even when sample read counts were greater than 85,000. Excluding these two loci, analyst-interpreted aSTR and Y-STR ForenSeq profiles were 99.96% and 100% concordant, respectively, with CE data. In addition to demonstrating concordance on par with other CE kit to kit comparisons, the results from this study will assist laboratories seeking to develop workflows for high volume processing and analysis of aSTRs and Y-STRs from reference-type specimens using the ForenSeq system. SN - 1878-0326 UR - https://www.unboundmedicine.com/medline/citation/28126691/Performance_and_concordance_of_the_ForenSeq™_system_for_autosomal_and_Y_chromosome_short_tandem_repeat_sequencing_of_reference_type_specimens_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S1872-4973(17)30001-7 DB - PRIME DP - Unbound Medicine ER -