A bioinformatic filter for improved base-call accuracy and polymorphism detection using the Affymetrix GeneChip® whole-genome resequencing platform

Pandya, Gagan A.; Holmes, Michael H.; Sunkara, Sirisha; Sparks, Andrew; Bai, Yun; Verratti, Kathleen; Saeed, Kelly; Venepally, Pratap; Jarrahi, Behnam; Fleischmann, Robert D.; Peterson, Scott N.

Francisella tularensis genomic DNA was subjected to whole-genome amplification (WGA) by multiple displacement amplification (MDA). MDA was performed using f29 DNA polymerase using the Repli-g kit (Qiagen Inc, Valencia, CA) in 50 ml reaction volumes as follows. Genomic DNA (10 ng) in 2.5 ml of 1Â TE was denatured at room temperature for 3 min by the addition of an equal volume of 50 mM KOH, 1.25 mM EDTA (pH 8.0). The solution was neutralized by the addition of 5 ml of 1 M Tris-HCl, pH 4.0. A master mix (40 ml) containing 50 mM exonuclease-resistant random hexamers, 1 mM of each dNTPs, f29 DNA polymerase (800 U/ml final concentration) and yeast pyrophosphatase (1 U/ml final concentration) was added. The reaction was incubated at 308C for 16 h in a thermocycler PTC-225 (MJ Research, Waltham, MA). The reactions were terminated by heating at 658C for 3 min. The amplified DNAs were purified using 96-well purification microplates (Millipore, Billerica, MA). The DNAs were eluted for 1 h at room temperature on a shaker in 100 ml of 1Â TE. The amplified DNAs were examined on agarose gels and DNA concentrations were determined using Pico Green dsDNA quantitation kit (Invitrogen-Molecular Probes, Carlsbad, CA) using calf thymus DNA (Sigma-Aldrich, St Louis, MO) as a standard. The sample fluorescence was measured using a fluorescence microplate reader (TECAN, San Jose, CA) at excitation of 480 nm and measuring emission at 520 nm. Amplified DNA yields were typically 30-40 mg. The 7.5 kb plasmid DNA used as a positive control for resequencing (Tag IQ-EX template) was PCR amplified using the primers and conditions suggested in the CustomSeq Õ kit (Affymetrix, Inc., Santa Clara, CA).