ISO 17025 validation of a next-generation sequencing assay for relationship testing
Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › fagfællebedømt
The HID-Ion AmpliSeq™ Identity Panel is a next-generation sequencing assay with 90 autosomal and 34 Y-chromosome SNPs that are amplified in one PCR step and subsequently sequenced using the Ion Personal Genome Machine (Ion PGM™) System. This assay was validated for relationship testing in our ISO 17025 accredited laboratory in 2015. Here, the essential parts of the validation report submitted to the Danish Accreditation Fund are presented. A total of 100 unrelated Danes were typed in duplicates and the locus balance, heterozygote balance (Hb) and noise levels were analysed in detail. Two loci were disregarded for casework because genotyping was uncertain. Hb for rs7520386 was skewed and high levels of noise were observed in rs576261. Three general acceptance criteria for analysis of single-source samples were defined: (i) sequencing depth > 200 reads, (ii) noise level < 3% and (iii) Hb > 0.3. A Python script named SNPonPGM was developed to assist the analyst by highlighting loci that do not fulfil the general acceptance criteria. Furthermore, SNPonPGM has functions that reduce the hands-on time of the reporting officer to a few minutes per case. Mixtures with DNA from two individuals in a 1:24 ratio were readily identified using the three criteria and the SNPonPGM script.
|Status||Udgivet - nov. 2016|