Sequencer v 4

From the samples confirmed, by microscopy and PCR amplification, to be positive for intracellular H. pylori, one sample was randomly selected, and total DNA was extracted from yeasts using the UltraClean Microbial DNA Isolation kit (M.O. BIO, Carlsbad, CA, USA) following the instructions of the manufacturer. The 16S rDNA was amplified by PCR using universal bacterial primers 8F-(5′-AGTTTGATCCTGGCTCAG-3′), 1492R (5′-ACCTTGTTACGACTT-3′). The amplified fragments were purified and sequenced by Macrogen Inc. (Seoul, Korea). To determine the taxonomic allocation of the fragments sequenced, their phylogenetic affiliation was analysed comparing the 16S rRNA gene sequence. Sequences were revised and corrected using Sequencer v4.7 software (Gene Codes Corp, Ann Arbo, MI, USA). The sequences were added to the updated and prealigned 16S rRNA gene database Silva (http://www.arb-silva.de/projects/living-tree/), compiling all sequences of all type strains for which an entry of high quality was found [34 (link)]. The sequences were aligned using the ARB software package (http://www.arb-home.de) [35 (link)] and manually improved. The tree reconstruction was performed using the neighbor-joining algorithm implemented in the ARB software package. The sequences were submitted to GenBank with the following access number MT477178.

Prior to further investigation, all potential disease-causing variants were validated using Sanger sequencing. Primers for exon 6 of the CIB2 gene (RefSeq: NM_006383.2 and transcript ID ENST00000258930) were designed using primer3 v0.4.0 (bioinfo.ut.ee/primer3-0.4.0/) and any SNP’s in the primer-binding site were ruled out using the NGRL SNPCheck database (https://ngrl.manchester.ac.uk/SNPCheckV3/snpcheck). PCR amplification was performed using the FASTstart High Fidelity PCR system (Roche, Madison, WI) at 59°C annealing temperature. Amplified PCR products were purified using the Agencourt AMPure XP Purification System (Beckman Coulter, Indianapolis, IN) and sequenced on the Applied Biosystems 3730 sequencer (Genomics Core at Einstein, NY). The Sequencer v4.0.1 software (Gene Codes, Ann Arbor, MI) was used to compile and compare the data to the CIB2 sequence.

A TP53 variant, previously reported as pathogenic in ClinVar in relation to Li-Fraumeni syndrome and/or hereditary cancer pre-disposing syndrome, was found in P65 and validated using Sanger sequencing. Primers were designed using primer3 v0.4.0 (bioinfo.ut.ee/primer3-0.4.0/). SNPs in the primer-binding site were ruled out using the NGRL SNPCheck database (https://ngrl.manchester.ac.uk/SNPCheckV3/snpcheck) prior to ordering. PCR amplification was performed using the FASTstart High Fidelity PCR system (Roche, Madison, WI) at 59°C annealing temperature. Amplified PCR products were then purified using the AMPure Purification System (Beckman Coulter, Indianapolis, IN). The purified products were sequenced on the Applied Biosystems 3730 sequencer (Genomics Core at Einstein, NY). The Sequencer v4.0.1 software (Gene Codes, Ann Arbor, MI) was used to analyze sequencing files.