Abi prism 3730 genetic analyser

Molecular analysis was carried out on up to 20 miracidia per child from the 2010 collections depending on the infection intensity of the child. We replicated the identical methodologies of the original 2005 and 2006 surveys in order that the data were directly comparable [13 (link)], since the original miracidial samples were no longer available for re-analysis. In brief, DNA preparation was carried out on the Whatman cards as per the manufacturers protocol (Whatman FTA cards®). PCR was carried out using the previously published multiplex assay using seven pairs of microsatellite primers [15 (link)], namely SMD28, SMDA28, SDM25, SMD89, CA11-1, SMS9-1 and SMU31768. Forward primers were labelled using 6-FAM, PET, VIC and NED dyes. Microsatellite sizing was performed on the same ABI Prism 3730 Genetic Analyser with a LIZ-500 size standard (Applied Biosystems, Cheshire, UK) as the 2005 and 2006 samples.

Genomic DNA was extracted from the voucher specimens using the Sangon Fungus Genomic DNA Extraction kit (Sangon Biotech Co. Ltd., Shanghai, China), according to the manufacturer’s instructions. Primer pairs ITS5/ITS4 (White et al. 1990 (link)), LR0R/LR5 (Vilgalys and Hester 1990 (link)), and atp6-2/atp6-3 (Kretzer and Bruns 1999 (link)) were used for amplifying ITS, nrLSU and atp6, respectively. PCR reactions was performed in a total volume of 25 μl containing 0.5 μl template DNA, 11 μl distilled water, 0.5 μl of each primer and 12.5 μl 2 × PCR mix (DreamTaq^tm Green PCR Master Mix, Fermentas). Amplification reactions were performed in a Tprofessional Standard Thermocycler (Biometra, Göttingen, Germany) under the following conditions: 95°C for 4 min; then 35 cycles of denaturation at 94°C for 60s, annealing at 53°C for 60s, and extension at 72°C for 60s; with a final extension at 72°C for 8 min. The PCR products were electrophoresed on 1% agarose gels and sequencing was performed on an ABI Prism® 3730 Genetic Analyser (PE Applied Biosystems, Foster, CA, USA) at the Beijing Genomic Institute (BGI) using the same PCR primers. The raw sequences were assembled and checked with SeqMan implemented in Lasergene v7.1 (DNASTAR Inc., USA). The newly generated sequences in this study were submitted to GenBank.

The success of PCR was checked on 2% agarose gels. The successful amplicons were sequenced using a Big Dye Terminator cycle sequencing kit on an ABI PRISM 3730 Genetic Analyser (Applied Biosystems, Foster City, CA, USA). The obtained sequences were identified using BLASTN against the NCBI GenBank database.

Ancient and modern samples' Y-chromosomal haplotypes were obtained using the PowerPlex Y23 System (Promega) and analysed by capillary electrophoresis on an ABI Prism 3730 Genetic Analyser (Applied Biosystems) at the genomic technical platform PlaGe (Genopole) and on an ABI Prism 3130xl Genetic Analyser (Applied Biosystems) at the University of Leicester. For Skeleton 1, this was carried out on three separate extracts (RM2, LM1 and LM3) in two different ancient DNA laboratories (York and Toulouse). For the modern relatives, this was carried out on two different extracts in two different modern laboratories (Leicester and Toulouse).

Genotyping of the serotonin transporter-linked promoter region (5-HTTLPR) was performed using polymerase chain reaction according to standard protocols at the Department of Human Genetics of the Radboud University Medical Center in Nijmegen, Netherlands. After the PCR, fragment length analysis was performed on the ABI Prism 3730 Genetic Analyser (Applied Biosystems, Nieuwerkerk aan den IJssel, Netherlands), and results were analyzed with GeneMapper Software, version 4.0 (Applied Biosystems).

After obtaining the informed consent, 10 ml peripheral vein blood was extracted from patients with MMD and normal control participants, placed in EDTA anticoagulant tubes and stored in a freezer at −80°C until analysis. DNA was isolated using the salting-out method [12 (link)]. The extracted DNA was spectro-photometrically quantified and checked for purity at an absorbance of 260nm and 280nm. Resequencing of the 17-q.25-ter’ region spanning the RNF 213 region covering 21 SNPs was carried out. The selection of SNPs was based on functional significance, minor allele frequency, and their tagging status. The details of the SNPs selected, PCR primers and in silico functional prediction are shown in “Table 1”. The PCR primers were designed using Primer-BLAST and verified by UCSC in silico PCR and synthesised by Sigma-Genosys. Genotyping was done by Gradient PCR amplification at a temperature of 55–65°C. This was followed up by Sanger sequencing using ABI PRISM Big Dye Terminator v3.1 sequencing kit, (Applied Bio-systems, Foster City, CA, USA) according to the manufacturer’s instructions and was run on ABI PRISM3730 Genetic Analyser (Applied Bio-systems, Foster City, CA, USA). Sequence analysis was done using Applied Bio-systems sequence scanner V.1.1.

The capture approach yielded insufficient coverage for all HIrisPlex and Y-chromosome SNPs and therefore primers were designed to generate amplicons containing these SNPs as well as two SNPs, which further define Y-chromosome haplogroup G: M285 (G1) and P287 (G2) (ref. 14 (link)). These were amplified as part of multiplex reactions following Römpler et al.44 (link) or singleplex reactions (using 40 cycles and with no secondary amplification) and sequenced on the Ion Torrent following library preparation using Ion PGM 200 Xpress Template Kit and PGM 200 Sequencing Kit. To increase coverage, singleplex PCR and sequencing of one marker (rs28777) was carried out according to Binladen et al.45 (link)
Typing of the haplogroup G defining SNPs (M201, M285 and P287) was repeated in Toulouse using singleplex PCRs. Sequencing of these PCR products was carried out using Big-Dye Terminator V3.1 cycle sequencing kit (Applied Biosystems) analysed by capillary electrophoresis on an ABI Prism 3730 Genetic Analyser (Applied Biosystems) at the genomic technical platform PlaGe (Genopole).