Highlights
- •non-LUS stutter is common and detectable above threshold in sequence data.
- •non-LUS and LUS stutter products occurred in a mutually exclusive manner.
- •Accounting for non-LUS stutter will improve sequenced-based mixture analysis.
- •A LUS-only stutter correction will not accurately account for non-LUS stutter.
- •n-0 stutter products were observed.
Abstract
Forensic DNA analysis is among the most well-recognized and well-developed forensic
disciplines. The field’s use of DNA markers known as short tandem repeats (STRs) offer
a robust means of discriminating individuals while also introducing challenges to
the analysis. One of these challenges, stutter, is the result of a non-biological
artifact introduced during PCR. The formation and amplification of these stutter products
can occur at rates as high as 15–20% of the parent allele. The challenge inherent
in this process is differentiating stutter artifacts from true alleles, particularly
in the presence of a minor contributor. Traditionally, DNA profiles are obtained using
capillary electrophoresis (CE), where amplified DNA fragments are separated by size,
not sequence, and the identification of stutter is performed on a locus-specific level.
The use of CE-based fragment data rather than sequence-based data, has limited the
community’s understanding of the precise behavior of stutter. Massively parallel sequencing
(MPS) data provides an opportunity to better characterize stutter, permitting a more
accurate means of detecting both size- or longest uninterrupted stretch (LUS)-based
stutter but also allele and motif-specific stutter characteristics. This study sheds
light on the value of characterizing motif- and allele-specific stutter, including
non-LUS stutter, when using MPS methods. Analysis and characterization of stutter
sequences was performed using data generated from 539 samples amplified with the ForenSeq
and PowerSeq 46GY library preparation kit and sequenced on the Illumina MiSeq FGx.
Assessment of non-LUS stutter begins with calculating stutter rates for all potential
stutter products at a given locus (and allele), additionally, the occurrence of these
discrete stutter products were quantified. Results show that although the LUS sequence
stutters at a higher rate than non-LUS motifs, the non-LUS stutter products do occur
at detectable levels and potentially influence sequence-based mixture analysis. The
data indicate that the stutter from one motif or allele can be distinguished from
another motif or allele based on their unique stutter rates; however, the number of
stutter products from each motif or allele may similarly make up the overall pool
of stutter products. Motif- and allele-specific stutter models provide the most comprehensive
analysis of sequence stutter rates and provide the ability to differentiate stutter
sequences more accurately from true allele stutter. This information provides a foundation
for including the characterization of non-LUS stutter products when analyzing DNA
profiles, specifically mixtures with potential low-level contributors.
Keywords
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Article info
Publication history
Published online: April 16, 2022
Accepted:
April 11,
2022
Received in revised form:
March 16,
2022
Received:
January 14,
2022
Identification
Copyright
© 2022 Elsevier B.V. All rights reserved.
