Sequencing analysis software

PCR amplifications were performed in a final volume of 25 μL, including 20 ng of purified gDNA, 2.5 μL of 10× PCR buffer, 2 mM of MgCl2 and 200 μM of dNTP, 1.5 U of Platinum Taq DNA polymerase (Life Technologies) and 400 nM each of the forward and reverse primers. The primer sequences were as follows: forward 5’-TGTTTTCCTTTACTTACTACACCTCA-3’ and reverse 5’-CCACAAAATGGATCCAGACA-3’. The cycling conditions used were as follows: 95°C for 10 min, 35 cycles of 94°C for 30 s, 60°C for 30 s and 72°C for 30 s and a final extension at 72°C for 10 min. The products obtained were then treated with ExoSAP-IT (Affymetrix, Santa Clara, CA, USA) according to the manufacturer’s recommendations. The sequencing reactions were performed in a final volume of 10 μl as follows: 2 μl of diluted PCR product, BigDye Terminator v1.1 Sequencing reagents (Life Technologies), and 150 nM each of the forward and reverse primers. The cycling conditions were as follows: 96°C for 2 min, followed by 25 cycles of 96°C for 10 s, 50°C for 5 s, and 60°C for 2 min. Sequencing products were purified using the BigDye Xterminator Purification Kit (Life Technologies) as recommended. Products were then sequenced on a 3130 Genetic Analyzer (Applied Biosystems) and analyzed with the sequencing analysis software (Applied Biosystems).

PCR products were purified of unused primers and nucleotides by treatment with ExoSAP-IT® (Amersham-Pharmacia Biotech), followed by direct sequencing using the ABI PRISM® BigDye^TM Terminator Cycle Sequencing kit (Applied Biosystems) and analysis on an ABI 3130 Genetic Analyzer with Sequencing Analysis software (Applied Biosystems).

APOE genotyping was performed by direct sequencing. PCR amplification of the region containing the rs429358 and rs7412 sites that determine the 𝜀2, 𝜀3, or 𝜀4 variants of the gene was carried out using the primers pair Forward: 5′-TAAGCTTGGCACGGCTGTCCAAGGA-3′ and Reverse: 5′-ACAGAATTCGCCCCGGCCTGGTACAC-3′, resulting in a 244 bp fragment (Hixson and Vernier, 1990 (link)). Purified PCR products were sequenced by the BigDye Terminator v3.1 Cycle Sequencing Kit (Life Technologies, Carlsbad, CA, United States) according to the manufacturer protocol. Sequence products were then separated on an ABI 3130xl automatic sequencer (Applied Biosystems, Paisley, United Kingdom) and analyzed using Sequencing Analysis Software (Applied Biosystems, Paisley, United Kingdom).

Exons where the variants of interest located were amplified using primers listed in Supplementary Data 10 on Dual 96-well GeneAmp PCR System 9,700 (Applied Biosystems, Courtaboeuf, France), with 20 ng of template DNA from each sample used per reaction. The products were sequenced by 3730 × l DNA Analyzer (Applied Biosystems, Courtaboeuf, France). All sequences were analysed by the Sequencing Analysis Software (version 5.2, Applied Biosystems, Courtaboeuf, France). All variants of interest were manually confirmed in the resulting trace files.

The purified plasmids were sequenced using M13 forward primer as well as insert specific forward and reverse primers using Sanger’s dideoxy chain terminator cycle sequencing method. In case of colony PCR, PCR products were purified by treating with Exonuclease I and Shrimp Alkaline Phosphatase (ExoSAP-IT®; USB Corporation, Cleveland, Ohio, USA) and incubated at 37°C for 15 min. then at 80°C for 15 min. Purified PCR products (1.0 μl) were subjected to sequencing reaction, by adding 0.65–2.0 pmoles of the primer and 3.2 μl of BigDye^TM (Applied Biosystems, Foster City, CA, USA; containing fluorescently labeled ddNTPs and unlabeled dNTP). The sequencing PCR conditions include: 30 cycles of 94°C for 10 sec, 55°C for 5 sec and 60°C for 4 min. The PCR product was precipitated using 3 M sodium acetate (pH 5.2) and ethanol, and centrifuged at 4,000 rpm at 4°C for 15 min. The pellet was washed in 70% alcohol, air dried and suspended in 10 μl of HiDi^TM formamide (Applied Biosystems, Foster city, USA) and loaded on to ABI 3730 Automated DNA analyzer, after denaturation at 94°C for 10 min. The samples were run using POP-7^TM polymer and analyzed using ‘Sequencing Analysis’ software (Applied Biosystems, Foster city, USA) and MEGA 6.