Seqscape software v3

Samples for genotyping were selected to represent all the different geographical regions of the country affected in a certain year. No other selection criteria such as age of the patients or sex were considered. DNA was extracted according to the manufacturer’s protocol from 200 µl of serum with the QIAamp DNA Blood Mini Kit (Qiagen). Viral DNA for sequencing was prepared by nested-PCR using previously described primers e1855f, e1863f, B19-R1 and B19-R2 for amplification of a 1,100-bp region spanning the NS1/VP1-unique region junction (NS1/VP1u)^{28 (link),33 (link)}. The PCR products were analysed in a 1.5% agarose gel stained with ethidium bromide. Products for sequencing were purified with the QIAquick PCR Purification Kit (Qiagen).
PCR products were sequenced with the BigDye Terminator v3.1 Cycle Sequencing Kit (Life Technologies), using the nested PCR primers on a capillary sequencer (models 3130/3500 Avant, Applied Biosystems). Sequences were edited using SeqScape Software v3.0 (Applied Biosystems) and then aligned with references in ClustalW (integrated in MEGA version 5).

All the coding exons and 50 bp of the flanking intron–exon boundaries of SOD1 were PCR-amplified, sequenced using the BigDye Terminator v3.1 sequencing kit (Applied Biosystems Inc.), and run on an ABIPrism 3500 genetic analyzer. The exon 2 of HFE was amplified by PCR and analyzed by denaturing high-performance liquid chromatography (DHPLC) (Transgenomic, Inc., Omaha, NE, USA). PCR products with heteroduplex profiles were sequenced on an ABI 3500 sequencer (Life Technologies, Foster City, CA, USA) with BigDye termination v.1.1 (Life Technologies) technologies according to standard protocols. Samples with homozygous profiles were coupled with a wild-type reference, denaturated and re-analyzed by DHPLC in order to also detect homozygous sequence alterations. If a mixed profile was positive, the original sample was sequenced. All sequencing products were analyzed with SeqScape Software v.3.0 (Applied Biosystems—Life Technologies). Other ALS-related genes were screened according to previously described procedures [10 (link)].

To confirm the mutations in the patient and both parents, the extracted genomic DNA was used for Sanger sequencing (Estrada‐Rivadeneyra, 2017). For each gene, specific primers were designed. The complete list of primers is presented in Table S1. Applied Biosystems Sequencing Analysis Software v6.0 (Sequencing Analysis Software v6.0, Applied Biosystems, Cat No.: 4474950), and Applied Biosystems SeqScape Software v3.0 were used for Sanger sequencing analysis (SeqScape^TM Software v3.0, Applied Biosystems, Cat No.: 4474978).