Prism 3130xl genetic analyzer

In order to evaluate maternal contamination, sequence coverage over the genome, artifact formation and events of allelic dropout, WGA products were diluted at a concentration of 40 ng/µL to perform a quantitative fluorescent PCR by Elucigene QST*R kit—a highly multiplexed single tube assay comprising a total of 16 markers for the detection of the 3 most common autosomal aneuploidies in chromosomes 13, 18 and 21—and QST*R-XY kit—a 12-plex single tube assay for determination of sex chromosomal aneuploidies, including Klinefelter and Turner syndromes—(Elucigene Diagnostics—Citylabs, Nelson Street, Manchester, United Kingdom) [20 (link)]. This 12-plex QF-PCR enables the identification of the Amelogenin marker, which amplifies non-polymorphic sequences on the X (104 bp) and Y (110 bp) chromosomes, and of the non-polymorphic Y-specific SRY marker, which allows also gender determination. The QF-PCR products were separated in an ABI PRISM 3130 xl Genetic Analyzer and fragment analysis was performed with Gene Mapper 4.0 software (Applied Biosystems by Life Technologies LTD, Warrington, UK).

DNA sequencing was carried out using the Sanger method with an Applied Biosystems (Foster City, CA, USA) automatic DNA sequencer (ABI PRISM 3130xl Genetic Analyzer) and an Applied Biosystems BigDye (ver. 3.1) kit. Blast search (Altschul et al., 1990 (link)) was carried out using the NCBI nucleotide database “16S rRNA sequences (Bacteria and Archaea)” with the program selection optimized for “Highly similar sequences (megablast).”

DNA was extracted from whole individuals using the Qiagen DNeasy Blood & Tissue Kit following the manufacturer's protocol for animal tissue. We developed two multiplex reactions to amplify ten microsatellite loci in P. elegans. Seven of the microsatellite loci were identified from a draft transcriptome of P. elegans (Heikkinen, Kesäniemi, & Knott, 2017), and primers were designed to amplify these loci using WebSat software (Martins, Lucas, Neves, & Bertioli, 2009). Three of the loci (Pe6, Pe7, and Pe19) were described previously (Kesäniemi, Boström et al., 2012) (see Table 1). Multiplex PCR reactions of 10 μl were performed containing 1x Qiagen Multiplex PCR Master Mix, 0.2 μmol/L of each primer, and 1 μl DNA template (diluted 1:20). The PCR had an initial activation step of 15 min at 95°C followed by 30 cycles of 30 s at 94°C, 90 s at 60°C, and 60 s at 72°C, and a final extension for 30 min at 60°C. Fragments were separated using an ABI PRISM 3130xl Genetic analyzer with Gene Scan™ 500 LIZ™ size standard (Applied Biosystems) in our own laboratory. The results were analyzed with GeneMapper^® v.5 Software (Applied Biosystems).