Applied biosystems 3500xl genetic analyzer

Genomic DNA was isolated using the Nucleo® Spin Tissue Kit (Macherey-Nagel, Düren, Germany) in accordance to the manufacturer’s instructions. The PowerPlex® 16 HS System was used for DNA typing [33 (link)]. In the system the following loci are co-amplified: D18S51, D21S11, TH01, D3S1358, Penta E, FGA, TPOX, D8S1179, vWA and Amelogenin, CSF1PO, D16S539, D7S820, D13S317, D5S818 and Penta D. In addition, a size standard was included as well as Taq DNA polymerase in the master mix. All 16 loci were amplified simultaneously from 100 pg of genomic DNA in a single tube and analyzed in a single injection. Automatic assignment of genotypes using the GeneMapper® ID and ID-X software were applied after separation of the PCR products on an Applied Biosystems 3500xL Genetic Analyzer (ThermoFisher Scientific, Bonn, Germany).

Genomic DNA was extracted from the cell cultures and the tumor samples from the patients using a DNeasy Blood & Tissue Kit (Qiagen, Valencia, CA, USA). The Gene Print PowerPlex 16HS System (Promega, Madison, WI, USA) was used to amplify the extracted DNA. The amplified fragments were detected using an Applied Biosystems 3500xL Genetic Analyzer (Thermo Fisher Scientific).

The DNA markers targeted in this study (N263, N565 and N571) were previously mined from genomic data obtained from over 25 Papaver somniferum L. individuals. The markers contain microsatellite sequences for use in potential downstream genetic identification assays (Supplementary Information: Table 13). The marker sequences were queried using the NCBI BLAST database^{33 (link)}. MegaBLAST^{34 (link)} results did not return any hits, while Blastn results yielded hits with a maximum query coverage of 26% and no e-value under 0.088, with the majority over 1.0. These results indicate that these sequences are unique. In addition, the primerBLAST query results did not return any target sequences within the nr database, therefore specificity for the Papaver somniferum L. targets are expected^{35 (link)}. Note, the poppy-specific primer sequences used in this study are available from the corresponding author on reasonable request.
Capillary electrophoresis was used to further characterize the DNA markers. 2.0 µL of each DNA extract was amplified using the parameters discussed in previous sections; 1 µL of each amplicon was combined with 9 µL of a formamide-GS600LIZ size standard master mix and analyzed on an Applied Biosystems 3500xL Genetic Analyzer (Thermo Fisher Scientific Inc.). Capillary electrophoresis was performed using an injection voltage of 1.6 kV and a 10 s injection time.

The second cohort included twenty-eight BM samples at diagnosis; they were enrolled for targeted bisulfite sequencing. MethPrimer software, (http://www.urogene.org/cgi-bin/methprimer/methprimer.cgi), was used for designing a specific set of primer pairs (M13-tailed PCR and LGALS12 primers) shown in Table 3 that binds only to bisulfite-modified DNA. This analysis was focused on a genomic region rich in CpG islands (by using DBCAT software, http://dbcat.cgm.ntu.edu.tw), containing 11 CpGs at nt −445 to nt −213 upstream of exon 1 in the predicted promoter region of LGALS12. This region also includes binding site for the transcription factor well known as SP1 that binds to GC-rich motives of many promoters (Katzenmaier et al., 2017 (link)).
PCR reaction was carried out on Applied Biosystems™ Veriti™ 96-Well Fast Thermal Cycler (Thermo Fisher Scientific). Sequencing was carried out using BigDye™ Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions after the cycle sequencing. Products were purified using The BigDye® XTerminator™ Purification Kit (Thermo Fisher Scientific). Applied Biosystems™ 3500 XL Genetic Analyzer (Thermo Fisher Scientific) carried out targeted automatic bisulfite sequencing reaction and sequencing data analysis.

Multiplex amplification of Y-STRs was performed on an Applied Biosystem® Veriti™ 96‐well thermal cycler (ThermoFisher Scientific) by using the Yfiler™ Plus PCR Amplification Kit (ThermoFisher Scientific) according to the manufacturer's protocol utilising 1 ng of genomic DNA. The amplified DNAs were then electrophoresed on the 24-capillary Applied Biosystems® 3500xL Genetic Analyzer (ThermoFisher Scientific) and the fragment analysis was performed using GeneMapper® ID-X software v.1.4 (ThermoFisher Scientific).

Amplification of rpoB gene in faecal samples was performed using KOD FX Neo DNA polymerase (TOYOBO) with primers shown in Supplementary Table 1. PCR products were cloned into pGEM-T vector (Promega, A3600). DNA was extracted using FavorPrep^TM Plasmid DNA Extraction Mini Kit (Favorgen, FAPDE001-1) and subjected to sequencing using Applied Biosystems 3500xL Genetic Analyzer (Thermo Fisher Scientific). Sequencing data of rpoB gene was compared with rpoB gene of P. asaccharolytica (CP002689.1) and P. uenonis (NZ_BAJM01000009) using A plasmid Editor (version 2).