Seqscape2

We used the ReliaPrep gDNA Tissue Miniprep System (Promega Corporation, Madison, WI, USA) for DNA extraction and purification with standard protocol for animal tissue. Polymerase chain reaction (PCR) amplification of mitochondrial 16S rDNA and nuclear 28S rDNA, 18S rDNA and Histone 3 gene fragments were accomplished with the primers and the conditions described by Radashevsky et al. [26 (link),27 (link)]. In addition, we used the D1R/D2C primer pair to amplify 28S rDNA gene in some samples [28 (link)]. Purified PCR products were sequenced in both directions by the GnC Bio Company, Republic of Korea (www.gncbio.kr), and in the National Scientific Center of Marine Biology, Vladivostok, Russia, using the same primers as for PCR. Sequence editing and contig assembly were performed using SeqScape 2.5 (Applied Biosystems). GenBank accession numbers and brief information about sequences used in the present analysis are shown in Supplementary Materials Tables S3 and S5. To link sequences with complete corresponding data, unique numbers from the first author’s database (VIR) are given to samples in Supplementary Materials Tables S1 and S3. These numbers precede the collecting location names on the phylogenetic trees shown in Figure 2 and Figure 3A.

We screened for SNVs the entire coding sequence plus
exon–intron boundaries by direct sequencing of PCR products. Exons 1 to 9 of
the WWOX gene were PCR amplified (primer
sequences available upon request). Primers were modified by the addition of
either M13F (5’-tgtaaaacgacggccagt-3’) or M13R (5’-caggaaacagtcatgacc)
sequences at their 5’ end. The coding sequence was screened by direct DNA
sequencing with M13F and M13R primers as described
earlier.^{17 (link)} Sequences were automatically analyzed
with the Seqscape 2.5 software (Applied Biosystems).

The patients were genotyped for rs12979860 using direct sequencing (Applied Biosystems), as previously described [10 (link)]. Primers used are available on request. Free circulating DNA was extracted from 500μl serum sample (Qiagen). The PCR products were separated on an ABI3130 sequencer, and analysed with SEQSCAPE 2.6 (Applied Biosystems).

Matrilineal analysis was performed using PCR amplification of the complete mtDNA control region (16,024–16,576 bp) corresponding to the revised Human Mitochondria Cambridge Reference Sequence (rCRS) (Andrews et al. 1999 (link)). Sequencing was performed as described (Sandoval et al. 2013b (link)) using ABI 3130XL Genetic Analyzer (ABI) and Big Dye Terminator v.3.1. DNA sequences were aligned using SeqScape 2.6 (Applied Biosystems), and major haplogroups were assigned using MitoTool (Fan and Yao 2011 (link)) or haplogroup prediction tool from the Genographic Project (Behar et al. 2007 (link)). Indels and hotspot sites at nucleotide positions 303–315; 515–522; 16,182–16,193 and 16,519 were excluded from the statistical analyses.

DNAs of patients with ROH covering the 1p36.12 locus were sequenced for PINK1. All eight of the coding exons and intron–exon boundaries of PRKN and PINK1 were polymerase chain reaction (PCR) amplified and the PCR products were sequenced using Big Dye Terminator Cycle Ready Reaction 3.1 Kits and an ABI 3130xl automated sequencer for the patients and the 96 controls. The collected sequence data were analyzed using SeqScape2.1 software (Applied Biosystems, Foster City, CA, USA).

Since the G2019S mutation can be transmitted by either parent, and the uniparental markers can only be studied simultaneously in males, the 29 males carrying the G2019S mutation (18 Arab-speakers, 8 Berber-speakers and 3 mixed) were studied for their Y-chromosome and mtDNA markers. These markers are known to be inherited unchanged from one male or female generation to the next unless mutations occur.
The biallelic markers E-M81 and E-M78, reported as being the specific male lineage of autochthonous Berbers of North Africa [21 (link),22 (link)] and J1-M267, the most prominent genetic marker of males from the Levantine [23 (link),24 (link)], were analyzed in the 29 male carriers. All markers were amplified by PCR using primers as described previously [25 (link),26 (link)]. For the maternal lineages, we amplified the two mtDNA hypervariable regions, HV1 and HV2, using primers F15971/R16451 for HV1 and F15/R484 for HV2 as described by Levin BC et al. (1999) [27 (link)]. All PCR fragments were sequenced using Big Dye Terminator Cycle Ready Reaction 3.1 Kits and sequence analysis was done through an ABI 3130xl sequencer. Data were analyzed by SeqScape2.1 software (Applied Biosystems, Foster City, CA). The mtDNA haplogroup assignment was done using the HaploGrep2 software based on the underlying classification tree PhyloTree Build 17 [28 (link)].