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Inter-laboratory study on standardized MPS libraries : evaluation of performance, concordance, and sensitivity using mixtures and degraded DNA

Müller, Petra ; Sell, Christian ; Hadrys, Thorsten ; Hedman, Johannes LU ; Bredemeyer, Steffi ; Laurent, Francois Xavier ; Roewer, Lutz ; Achtruth, Sabrina ; Sidstedt, Maja LU and Sijen, Titia , et al. (2020) In International Journal of Legal Medicine 134(1). p.185-198
Abstract

We present results from an inter-laboratory massively parallel sequencing (MPS) study in the framework of the SeqForSTRs project to evaluate forensically relevant parameters, such as performance, concordance, and sensitivity, using a standardized sequencing library including reference material, mixtures, and ancient DNA samples. The standardized library was prepared using the ForenSeq DNA Signature Prep Kit (primer mix A). The library was shared between eight European laboratories located in Austria, France, Germany, The Netherlands, and Sweden to perform MPS on their particular MiSeq FGx sequencers. Despite variation in performance between sequencing runs, all laboratories obtained quality metrics that fell within the manufacturer’s... (More)

We present results from an inter-laboratory massively parallel sequencing (MPS) study in the framework of the SeqForSTRs project to evaluate forensically relevant parameters, such as performance, concordance, and sensitivity, using a standardized sequencing library including reference material, mixtures, and ancient DNA samples. The standardized library was prepared using the ForenSeq DNA Signature Prep Kit (primer mix A). The library was shared between eight European laboratories located in Austria, France, Germany, The Netherlands, and Sweden to perform MPS on their particular MiSeq FGx sequencers. Despite variation in performance between sequencing runs, all laboratories obtained quality metrics that fell within the manufacturer’s recommended ranges. Furthermore, differences in locus coverage did not inevitably adversely affect heterozygous balance. Inter-laboratory concordance showed 100% concordant genotypes for the included autosomal and Y-STRs, and still, X-STR concordance exceeded 83%. The exclusive reasons for X-STR discordances were drop-outs at DXS10103. Sensitivity experiments demonstrated that correct allele calling varied between sequencing instruments in particular for lower DNA amounts (≤ 125 pg). The analysis of compromised DNA samples showed the drop-out of one sample (FA10013B01A) while for the remaining three degraded DNA samples MPS was able to successfully type ≥ 87% of all aSTRs, ≥ 78% of all Y-STRs, ≥ 68% of all X-STRs, and ≥ 92% of all iSNPs demonstrating that MPS is a promising tool for human identity testing, which in return, has to undergo rigorous in-house validation before it can be implemented into forensic routine casework.

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published
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keywords
Collaborative study, ForenSeq DNA Signature Prep Kit, Inter-laboratory study, Massively parallel sequencing, Short tandem repeats
in
International Journal of Legal Medicine
volume
134
issue
1
pages
14 pages
publisher
Springer
external identifiers
  • scopus:85075393562
  • pmid:31745634
ISSN
0937-9827
DOI
10.1007/s00414-019-02201-2
language
English
LU publication?
yes
id
6a292f31-1e71-42b5-a779-3f6e27ce0869
date added to LUP
2019-12-11 10:55:46
date last changed
2020-05-26 05:36:50
@article{6a292f31-1e71-42b5-a779-3f6e27ce0869,
  abstract     = {<p>We present results from an inter-laboratory massively parallel sequencing (MPS) study in the framework of the SeqForSTRs project to evaluate forensically relevant parameters, such as performance, concordance, and sensitivity, using a standardized sequencing library including reference material, mixtures, and ancient DNA samples. The standardized library was prepared using the ForenSeq DNA Signature Prep Kit (primer mix A). The library was shared between eight European laboratories located in Austria, France, Germany, The Netherlands, and Sweden to perform MPS on their particular MiSeq FGx sequencers. Despite variation in performance between sequencing runs, all laboratories obtained quality metrics that fell within the manufacturer’s recommended ranges. Furthermore, differences in locus coverage did not inevitably adversely affect heterozygous balance. Inter-laboratory concordance showed 100% concordant genotypes for the included autosomal and Y-STRs, and still, X-STR concordance exceeded 83%. The exclusive reasons for X-STR discordances were drop-outs at DXS10103. Sensitivity experiments demonstrated that correct allele calling varied between sequencing instruments in particular for lower DNA amounts (≤ 125 pg). The analysis of compromised DNA samples showed the drop-out of one sample (FA10013B01A) while for the remaining three degraded DNA samples MPS was able to successfully type ≥ 87% of all aSTRs, ≥ 78% of all Y-STRs, ≥ 68% of all X-STRs, and ≥ 92% of all iSNPs demonstrating that MPS is a promising tool for human identity testing, which in return, has to undergo rigorous in-house validation before it can be implemented into forensic routine casework.</p>},
  author       = {Müller, Petra and Sell, Christian and Hadrys, Thorsten and Hedman, Johannes and Bredemeyer, Steffi and Laurent, Francois Xavier and Roewer, Lutz and Achtruth, Sabrina and Sidstedt, Maja and Sijen, Titia and Trimborn, Marc and Weiler, Natalie and Willuweit, Sascha and Bastisch, Ingo and Parson, Walther},
  issn         = {0937-9827},
  language     = {eng},
  number       = {1},
  pages        = {185--198},
  publisher    = {Springer},
  series       = {International Journal of Legal Medicine},
  title        = {Inter-laboratory study on standardized MPS libraries : evaluation of performance, concordance, and sensitivity using mixtures and degraded DNA},
  url          = {http://dx.doi.org/10.1007/s00414-019-02201-2},
  doi          = {10.1007/s00414-019-02201-2},
  volume       = {134},
  year         = {2020},
}