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

Petra Müller,Petra Müller,Walther Parson,Walther Parson,Walther Parson,Sascha Willuweit,Ingo Bastisch,Thorsten Hadrys,Maja Sidstedt,Lutz Roewer,Francois-Xavier Laurent,Steffi Bredemeyer,Titia Sijen,Johannes Hedman,Christian Sell,Sabrina Achtruth,Maja Sidstedt,Natalie Weiler,Marc Trimborn

doi:10.1007/s00414-019-02201-2

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 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.

Highlights

With the emergence of massively parallel sequencing (MPS) technologies, molecular genetic tools have been developed to characterise the nucleotide sequence of short tandem repeat (STR) markers that have so far been analysed using fragment sizing by capillary electrophoresis (CE) [1,2,3,4,5,6,7]
Identity Single nucleotide polymorphism (SNP) results for FA10030T01A were fully concordant between runs, except for rs1294331, rs354439, and rs1382387 showing the drop-out of allele A, allele T and allele G, respectively (Table S13)
The presented results are the first MPS-data collected in a collaborative exercise performed among eight independent European laboratories using sequencing instruments of the same supplier

Summary

Introduction

With the emergence of massively parallel sequencing (MPS) technologies, molecular genetic tools have been developed to characterise the nucleotide sequence of short tandem repeat (STR) markers that have so far been analysed using fragment sizing by capillary electrophoresis (CE) [1,2,3,4,5,6,7]. Since the workflow for MPS-based sequence analysis of forensic genetic markers is more complex than for electrophoresisbased systems, we decided to reduce the complexity of this study to libraries prepared at the organising laboratory (OL) from forensically relevant samples that were shipped to the participating laboratories in the following way: In the framework of the SeqForSTRs project (EU Project ISFNr. IZ25-5793-2015-30 2017-2020, [31]) the ForenSeq DNA Signature Prep Kit [22] was used for collaborative validation experiments and population studies. The library was distributed between eight participating laboratories located in Austria, France, Germany, The Netherlands, and Sweden and were analysed on their respective MiSeq FGx sequencing platforms

Methods

Results

Conclusion