The Applied Biosystems™ NGM Detect™ PCR Amplification Kit – As promising as promised?

Abstract

We describe a paternity case with three genetic incompatibilities between a three-year-old boy and his putative father.STR analysis of 2 out of 25 markers revealed the absence of paternal alleles and presence of two maternal alleles at D2S441 and D2S1338 loci in the child. The rest 23 STR markers served to confirm paternity. In addition, we analyzed Y-STRs and determined the same haplotype in the child and his putative father.With massive parallel sequencing on HID Ion GeneStudio S5 System using Precision ID GlobalFiler NGS STR Panel v2 (Applied Biosystems) we confirmed the presence of two alleles of maternal origin at D2S441, D2S1338 loci and identified two maternal alleles at additional locus D2S1776 located on chromosome 2 in the child.Finally, we confirm paternity. Three loci 'exclusion' was due to maternal uniparental disomy of chromosome 2 in the child.

A systematic approach to improve downstream single‐cell analysis for the DEPArray™ technology

“…When using low amounts of DNA, the IQC peaks within the NGMDetect kit tend to pull up peaks in adjacent color channels and distort the 6‐FAM channel scale, as the peaks are several folds higher than the sample peaks. As this was described to be one of the drawbacks by Burch et al [50], we were able to detect increased punctures but no serious disadvantages in allele designation, especially as scales can be adjusted manually. To date, Promega kits do not contain additional quality markers, which has been outlined to change in the future with the development of their 8‐dye chemistry reagents (PowerPlex® 35GY System and PowerPlex® 18E System [15],).…”

“…When using low amounts of DNA, the IQC peaks within the NGMDetect kit tend to pull up peaks in adjacent color channels and distort the 6-FAM channel scale, as the peaks are several folds higher than the sample peaks. As this was described to be one of the drawbacks by Burch et al [50], we were able to detect increased punctures but no serious disadvantages in allele designation, especially as scales can be adjusted manually. To date, Promega kits do not contain additional quality markers, which has been outlined to change in the future with the development of increased sensitivity (1) the commonly applied PCR cycles and analytical threshold associated with the manufacturer's recommended protocol would require optimization that cannot be fully achieved by reduced amplification volume in all kits and (2) insufficient assay performance (i.e., no LoD of 1 copy) makes a kit-to-kit evaluation at a single-cell level difficult.…”

“…The lower occurrence of artifacts detected with the Fusion 6C kit might be reflective of the lower average peak heights observed as suggested by Tay et al [40] for their use of the QIAGEN Investigator® 24plex QS kit. The suitable performance of NGMDetect might be due to the kit being improved by shortening amplicons with the intended use for challenging samples from which full genetic profiles could not be obtained with other kits supplied by Life Technologies [50]. In any case, artifacts are sporadic snapshots of a given amplification process and not reproducible, particularly when low DNA amounts are used.…”

“…The suitable performance of NGMDetect might be due to the kit being improved by shortening amplicons with the intended use for challenging samples from which full genetic profiles could not be obtained with other kits supplied by Life Technologies [50]. In any case, artifacts are sporadic snapshots of a given amplification process and not reproducible, particularly when low DNA amounts are used.…”

Beyond simple kinship and identification: aDNA analyses from a 17th-19th century crypt in Germany

“…DNA extracts were amplified using the AmpFLSTR™ NGM SElect™ [47] and the NGM Detect™ [48] PCR amplification kits (both from Thermo Fisher Scientific), according to the manufacturers' instructions. 30 amplification cycles were run with both STR kits.…”