Systematic Analysis of Stutter Percentages and Allele Peak Height and Peak Area Ratios at Heterozygous STR Loci for Forensic Casework and Database Samples

Leclair, Benoît; Frégeau, Chantal J.; Bowen, Kathy L.; Fourney, Ron M.

doi:10.1520/jfs2003312

Cited by 51 publications

(37 citation statements)

References 23 publications

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“…Use of a uniform high stutter cut-off value across all markers is a conservative approach, and in the context of HCT, will result in decreased sensitivity to identify low-level chimerism. This study also confirmed results from a previous study that markers labeled with the NED dye had lower median stutter as compared with markers labeled with the JOE or FAM dyes (7). This supports the hypothesis that the repeat structure in the markers labeled with the JOE/FAM dyes may be associated with higher stutter.…”

Section: Discussionsupporting

confidence: 90%

Systematic analysis of interference due to stutter in estimating chimerism following hematopoietic cell transplantation

Thyagarajan

Young

Floodman

et al. 2009

Clinical Laboratory Analysis

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Stutter formed during amplification of short tandem repeats (STRs) interfere with accurate engraftment monitoring following hematopoietic cell transplantation (HCT). We describe a mathematical approach to minimize the contribution of stutter when estimating chimerism following HCT. Pretransplant DNA samples from 409 donors and recipients were used to define marker-specific stutter cut-off values for all makers used in the AMPFlSTR Profiler Plus amplification kit. Mock chimerism samples (5, 20, and 50%) were used to evaluate the contribution of stutter in estimating chimerism. Three markers, vWA, D13S317, and D18S51, had overlapping stutter in the mock chimerism samples. Only D18S51 had stutter from a shared allele, whereas the other two markers had stutter from nonshared alleles. Without adjustment for stutter, D18S51 showed a 8, 6, and 4% difference from expected chimerism values (5, 20, and 50%) and after adjusting for laboratory-defined marker-specific stutter cut-offs the corresponding difference from expected chimerism values was 1, 0, and 0%. Difference from expected chimerism values in the vWa and D13S317 markers were similar before and after adjustment of stutter. Adjustment for stutter from shared alleles may improve accuracy of estimated chimerism.

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Section: Discussionsupporting

confidence: 90%

Systematic analysis of interference due to stutter in estimating chimerism following hematopoietic cell transplantation

Thyagarajan

Young

Floodman

et al. 2009

Clinical Laboratory Analysis

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“…The use of peak height imbalance thresholds other than the commonly used 70% is not novel (e.g., Moretti et al use a peak height ratio as low as 59% [8]), and the new versions of commonly used DNA analysis software allow for the incorporation of magnitude-specific peak height thresholds just like those supported by the analysis presented here. For instance, Applied Biosystems GeneMapper® ID-X allows for the use of multiple peak height thresholds based on the heights of the observed peaks.…”

Section: Discussionmentioning

confidence: 99%

“…STR typing typically involves a polymerase chain reaction (PCR) amplification step followed by size fractionation of the resulting products and fluorescent signal detection and processing. Numerous studies have shown that the signal associated with each allele of a heterozygote at a given locus is approximately equal [3][4][5][6][7][8][9]. General practice has shown that the peak height ratio, as measured by dividing the height (in relative fluorescent units (RFUs)) of the lower quantity allele by the height of the higher quantity allele "should be greater than approximately 70% in a single source sample" [3].…”

Section: Introductionmentioning

confidence: 99%

Magnitude-dependent variation in peak height balance at heterozygous STR loci

Gilder¹,

Inman

Shields

et al. 2010

Int J Legal Med

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When the smaller of two peaks at an STR locus is less than 70% the magnitude of the larger peak at that locus, the disparity is typically taken to be an indication that there is more than one contributor of template DNA to the sample being tested. An analysis of 1,763 heterozygous allele pairs suggests that a peak height imbalance threshold that varies with the magnitude of the peaks being evaluated at a locus is superior to a fixed threshold. Identifying samples that are likely to be mixtures and those that are likely to have arisen from a single source is accomplished more reliably when a statistically based, magnitude-dependent peak height imbalance threshold is used. The amelogenin locus was found to behave in a similar fashion and was also found to have no systematic bias that favored the amplification of Y or X alleles.

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“…Armonk, NY: IBM Corp.) were used to illustrate differences of peak balance (also peak height ratio or heterozygote balance). Peak balance was calculated for each heterozygous combination (at least five individuals per allele combination) according to Method #2, developed by Leclair et al (2004), and defined as the ratio of peak height of the longer allele over that of the shorter allele. For comparison of allele sizes standard deviation and range were calculated for each marker using Microsoft Excel (2010).…”

Section: Discussionmentioning

confidence: 99%

Interlaboratory comparison of fig (Ficus carica L.) microsatellite genotyping data and determination of reference alleles

Hladnik

Jakše

Khadari

et al. 2018

AAS

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Microsatellites have been identified as the marker of choice in plant genotyping projects. However, due to length discrepancies obtained between different laboratories for the same allele, interlaboratory comparison of fingerprinting results is often a difficult task. The objectives of this study were to compare genotyping results of two laboratories, to evaluate genetic parameters of microsatellite markers and to determine reference allele sizes for fig cultivars from the Istrian peninsula. Genotyping results of ninety fig (Ficus carica L.) accessions were comparable between the laboratories despite differences observed when comparing electropherograms of different capillary electrophoresis systems. Differences in lengths of the same alleles were detected due to different PCR methods and laboratory equipment, but the distances between alleles of the same locus were preserved. However, locus FSYC01 exhibited one allele dropout which led to misidentification of 28 heterozygotes as homozygote individuals suggesting this locus as unreliable. Allele dropout was assigned to the tail PCR technology or to a touchdown PCR protocol. Genotypes of twenty-four reference cultivars from the Istrian peninsula were confirmed by both laboratories. These results will contribute to the usage of markers with greater reliability, discrimination power and consequently, to more reliable standardization with other fig genotyping projects.Key words: microsatellite marker; reference genotype; interlaboratory comparison; Ficus carica L. IZVLEČEK MEDLABORATORIJSKA PRIMERJAVA REZULTATOV GENOTIPIZACIJE FIGE Z MIKROSATELITSKIMI MARKERJI (Ficus carica L.) IN DOLOČITEV REFERENČNIH ALELOVMikrosateliti so se izkazali kot zelo uporabni markerji pri genetskih raziskavah rastlin. Zaradi odstopanj dolžin enakih alelov v različnih laboratorijih, je primerjava rezultatov med laboratoriji pogosto težavna. Namen raziskave je bil primerjati rezultate genotipizacije dveh laboratorijev, ovrednotiti genetske parametre mikrosatelitskih markerjev in določiti dolžine referenčnih alelov za sorte fig istrskega polotoka. Rezultati genotipizacije devetdesetih vzorcev fige (Ficus carica L.) so bili primerljivi med laboratorijema, kljub razlikam, ki smo jih opazili pri primerjavi elektroferogramov različnih sistemov kapilarnih elektroforez. Razlike med dolžinami enakih alelov med laboratorijema so bile odkrite zaradi različnih metod PCR in analitske opreme, vendar pa so bile razlike med aleli istega lokusa ohranjene. Pri lokusu FSYC01 smo ugotovili izpad alela, kar je privedlo do napačne identifikacije; namesto 28 heterozigotov smo posameznike določili kot homozigote. Ugotovljena lastnost nakazuje na nezanesljivost lokusa FSYC01. Izpad alela smo pripisali uporabi ekonomične metode PCR ali uporabi protokola PCR s postopnim nižanjem temperature prileganja začetnih oligonukleotidov. Genotipi štiriindvajsetih referenčnih sort istrskega polotoka so bili potrjeni v obeh laboratorijih. Rezultati raziskave bodo prispevali k uporabi bolj zanesljivih mikrosatelitskih m...

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Systematic Analysis of Stutter Percentages and Allele Peak Height and Peak Area Ratios at Heterozygous STR Loci for Forensic Casework and Database Samples

Cited by 51 publications

References 23 publications

Systematic analysis of interference due to stutter in estimating chimerism following hematopoietic cell transplantation

Systematic analysis of interference due to stutter in estimating chimerism following hematopoietic cell transplantation

Magnitude-dependent variation in peak height balance at heterozygous STR loci

Interlaboratory comparison of fig (Ficus carica L.) microsatellite genotyping data and determination of reference alleles

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