We tested our hypothesis that APP variants with intra-exonic junctions (IEJs) reported in Lee at el. (Nature, 2018) could have arisen as PCR mis-pairing artifacts between partially homologous sequences during the PCR amplification of APP vector contaminants. We repeated the PCR assays following the Lee study protocols using 250 pg of recombinant vectors with two different isoforms of APP inserts (pCAX APP 751 [Addgene plasmid # 30138 ; http://n2t.net/addgene:30138 ; RRID:Addgene_30138] and pCAX APP 695 [Addgene plasmid # 30137 ; http://n2t.net/addgene:30137 ; RRID:Addgene_30137]) as templates, respectively. All combinations of three PCR enzymes (OneStep Ahead RT-PCR (Qiagen), FastStart PCR master mix (Sigma), Platinum SuperFi DNA polymerase (ThermoFisher)) and three reported PCR primer sets (APP 1-18, APP 1-18N, APP 2-17; provided by Supplementary Table 1 in Lee at el.) were tested with two different final primer concentration settings (0.5 μM and 1.0 μM).
For OneStep Ahead RT-PCR, the cycling program of low annealing stringency PCR was 45 °C for 15 min; 95 °C for 5 min; 40 cycles of 95 °C for 15 sec, 55 °C for 15 sec, 68 °C for 2.5 min; 68 °C for 5 min. High annealing stringency PCR was performed for 40 cycles using the FastStart PCR master mix with the following cycle settings: 95 °C for 30 sec, 65 °C for 30 sec, and 72 °C for 2.5 min. We used the Platinum SuperFi DNA polymerase with the following cycle settings: 98 °C for 10 sec, 65 °C for 10 sec, and 72 °C for 1.5 min. All PCR products were run on 2% agarose gels. For all PCR combinations, we observed multiple chimeric amplification bands that were clearly distinct from the correct band of APP inserts.
We then sequenced these non-specific APP vector PCR amplicons to confirm the existence of IEJs. All of the chimeric amplification bands were cut and recovered from 2% agarose gels using a QIAquick Gel Extraction Kit (28706; QIAGEN). Extracted DNA was sonicated into fragments with an average size of 200 bp using Covaris S2 with the following settings: sample volume, 50 μL; water level, 12; temperature, 7°C; intensity: 5; duty cycle, 10%; cycles per burst, 200; and treatment time, 120 s. Amplicon fragments were end-repaired, dA-tailed, and adaptor-ligated using the KAPA Hyper Prep Kit (KK8503; KAPA Biosystems). Amplicon libraries for each combination were labeled with unique dual index and paired-end sequenced (2×151 bp) on the Illumina HiSeq platform at Microgen, Inc. Less...