3730xl dna sequencer

Exonic indels (n = 2 of 6 tested) and a subset of candidate SNVs (n = 50 of 60 tested) identified by WGS were validated by PCR and Sanger sequencing. Fifty nanograms of genomic DNA was whole genome amplified using the REPLI-g Midi Kit (Qiagen). PCR primers were designed using Primer3Plus [Untergasser et al., 2007 (link)]. ALL and remission samples were amplified by PCR and the products were sequenced with BigDye Terminator 3.1 chemistry using an Applied Biosystems 3730XL DNA sequencer. The sequence traces were analyzed with the Sequencher software (Applied Biosystems, Foster City, CA, USA). For validation of remaining SNVs, the allele fraction (AF) from the target capture experiment was calculated with a custom Python script (available at https://github.com/Molmed/Berglund-Lindqvist-2013). A candidate SNV was considered validated if the AF was ≥ 0.1 in the ALL sample, the AF was < 0.01 in the matched remission sample, and the sequence depth was ≥10 in both samples. Bases with a Phred quality score < 20 and bases in a read with mapping quality = 0 were disregarded. Positions where an SNV was called in more than one patient were manually inspected in the Integrative Genomics Viewer [Thorvaldsdottir et al., 2013 (link)].

All PCR products were separated by electrophoresis on a 2% agarose gel. The different fragments of expected size (240, 367 and 373 bp) were extracted and purified using a gel extraction kit (Qiagen, Inc.) and then sequenced directly using a 3730xl DNA sequencer (Applied BioSystems; Thermo Fisher Scientific, Inc.).

The genotypes of study participants were validated at the BGI Laboratory using Sanger sequencing. PCR primers (*2 forward: 5′-CAGAGCTTGGCATATTGTATC-3′ and *2 reverse: 5′-GTAAACACAAAACTAGTCAATG-3′, *3 forward: 5′-TGTGCTCCCTGCAATGTGAT-3′ and *3 reverse: 5′-TTTGGGGCTGTCACCAAAGT-3′,*17 forward: 5′-GCCCTTAGCACCAAATTCTC-3′ and *17 reverse: 5′-ATTTAACCCCCTAAAAAAACACG-3′) were designed to amplify CYP2C19*2, *3, and *17 genomic fragments. The PCR conditions were 96°C for 5 min, followed by 10 cycles of 96°C for 20 s, 62°C–52°C touchdown for 20 s and 72°C for 30 s and 35 cycles of 96°C for 20 s, 55°C for 20 s and 72°C for 30s. The final extension was performed at 72°C for 5 min. Peripheral blood samples were processed and subsequently analysed using a 3730XL DNA sequencer (Applied Biosystems, Massachusetts, USA).