The 3130 Genetic Analyzer and 3730 DNA Analyzer generated more variability than the other instruments (Supplemental Fig. 9). The maximum standard deviation of any allele was 0.16 bases, observed at FGA with the largest alleles (44.2–50.2), on both instruments. The 0.5-base bin window set by the bin file is greater than three standards deviations of either 0.1 or 0.16 bases, the largest sizing variations observed. Sizing variability increased with locus and allele size. Those loci with the largest sizes; FGA, Penta D, DYS391, TPOX, and Penta E, had alleles with the greatest standard

deviations. Figure options Download full-size image Download high-quality image (170 K) Download as PowerPoint slide Figure options Download full-size image Download high-quality image (168 K) Download as PowerPoint slide Amplification of repeat selleck chemical sequences by DNA polymerases often produces slippage products

one or more repeat units shorter or larger than the true sequence length [15] and [16]. Because the level of stutter products as a percentage of the full-length allele products remains roughly constant, filters can be constructed to remove allele calls on PLX3397 concentration stutter position peaks below that stutter percentage. To calculate the average observed stutter for each locus, 116 unrelated genomic DNAs were amplified with the PowerPlex® Fusion System for 30 cycles. Samples were detected using an Applied Biosystems® 3500xl Genetic Analyzer using a 1.2 kV 18 s or 1.2 kV 12 s injection. A peak height ratio of the stutter peak height to the allele peak height was calculated. To ensure accurate calculation of the true stutter ratio, allele peak heights greater than 30,000 RFU and less than 175 RFU were removed from the data set. Stutter peaks that resided between two true alleles two repeats apart (e.g., 8, 10) were removed as well. Peaks in this position are often inflated Sitaxentan due to the additive effect of minus and plus stutter peaks migrating at the same size. The stutter filter for the GeneMapper®ID and ID-X files is set as the mean stutter ratio at each

locus plus three standard deviations. The GeneMapper® ID-X stutter file includes filters for plus stutter for the trinucleotide repeat locus D22S1045 and the n − 2 peak seen with D1S1656. The highest stutter percentages were seen with D12S391 and D1S1656, and the stutter ratio increased with increasing repeat number. The stutter data and summary are presented in Supplemental Tables 2 and 3. Figure options Download full-size image Download high-quality image (385 K) Download as PowerPoint slide Laboratories commonly reduce reaction volume for cost-saving purposes. Although recent STR system improvements have allowed the use of a variety of solid support substrates containing inhibitory chemicals, amplification reactions using these materials with reduced reaction volumes can be negatively affected. Results with reduced reaction volumes of 12.5 μl and 6.