Abi prism 3500xl genetic analyzer

PCR products were excised from the gel and purified using the GFX™ PCR DNA and Gel Band Purification Kit (GE Healthcare). Amplicons were sequenced in both directions using the PCR primers described above. Sequencing reactions were performed on an ABI Prism 3500xL Genetic Analyzer (Applied Biosystems). Quality assessment, edition, assembly and multiple alignments of data derived from sequencing were performed with CodonCode Aligner (v.3.7.1) and BioEdit/ClustalW⁴⁴ . The BLAST tool (www.ncbi.nlm.nih.gov/BLAST) was used to confirm the sequence identities by comparing our sequences to those deposited in GenBank database.

The analysis of hotspot mutations of IDH1 (exon 4) was performed by PCR followed by direct sequencing. Briefly, the IDH1 region of interest was amplified by PCR using the primers: 5′-CGGTCTTCAGAGAAGCCATT-3′ (forward) and 5′-CACATTATTGCCAACATGAC-3′ (reverse). An amplification PCR reaction was performed in a total volume of 15 μl, comprising: 1 μl of DNA, 1 × buffer solution, 2 mM MgCl₂, 200 μM of each dNTP, 0.3 μM of each set primer, and 0.5 U Taq DNA polymerase (Invitrogen), and was performed in a Veriti 96-well Thermal Cycler with an initial denaturation at 95° for 10 min, amplified for 40 cycles of denaturation at 95° for 45 sec, annealing at 58° for 45 sec, and extension at 72° for 45 sec, and a final extension at 72° for 10 min. Amplification of PCR products was confirmed by gel electrophoresis. Sequencing PCR was performed using a Big Dye terminator v3.1 cycle sequencing ready reaction kit (Applied Biosystems) and an ABI PRISM 3500 xL Genetic Analyzer (Applied Biosystems).

MSI NGS accuracy was evaluated by comparison with a PCR based NYS CLEP-approved assay for all samples. For gold standard PCR analysis, 4 sets of fluorescently-labeled primers were used for amplification of five markers (BAT-25, BAT-26, D2S123, D5S346, and D17S250). Internal lane size standards added to PCR products assured accurate sizing of alleles and to adjust for run-to-run variation. PCR products were separated by capillary electrophoresis using ABI PRISM 3500xl Genetic Analyzer and output data was analyzed with GeneMapper Software 5 (both Applied Biosystems, Foster City, CA, USA).

Genomic DNA was extracted from whole blood with EDTA using the GeneJET Genomic DNA Purification Kit (Thermo Fisher Scientific, Waltham, MA, USA). Direct sequencing was performed on an ABI PRISM 3500xL Genetic Analyzer (Applied Biosystems, Waltham, MA, USA). Massive parallel sequencing was performed with Ampliseq technology on Ion S5 (Thermo Fisher Scientific, Waltham, MA, USA). We used the panel targeting coding exons of 52 genes that are associated with inherited diseases with cholestasis, including the JAG1 and NOTCH2 genes. Nucleotide variants were initially prioritized by minor allele frequency <2% in the general population and selected based on clinical diagnosis using human phenotype ontology (HPO) terms. Sanger sequencing was used to verify the mutation in the affected individuals and family members.
All founded genetic variants were interpreted using the ACMG/AMP Interpreting Sequence Variant Guidelines in accordance with the SVI Recommendations (https://www.clinicalgenome.org/working-groups/sequence-variant-interpretation/ (accessed on 10 July 2023).

Genomic DNA was extracted from whole blood with EDTA using GeneJET Genomic DNA Purification Kit (Thermo Fisher Scientific, USA). Direct sequencing was performed on ABI PRISM 3500xL Genetic Analyzer (Applied Biosystems, USA). Massive parallel sequencing was performed with Ampliseq technology on Ion S5 (Thermo Fisher Scientific, USA).
For the patient P –IV/2 we used the panel targeting coding exons of 56 genes which associated with inherited diseases with cholestasis and including the 6 PEX genes (PEX1, PEX10, PEX12, PEX26, PEX6, PEX7). For the other patient (P-IV/4) we performed targeted sequencing of 587 genes associated with metabolic disorders including 14 PEX genes (PEX1, PEX10, PEX11B, PEX12, PEX13, PEX14, PEX16, PEX19, PEX2, PEX26, PEX3, PEX5, PEX6, PEX7). Nucleotide variants were initially prioritized by minor allele frequency <2% in the general population and selected based on clinical diagnosis using human phenotype ontology (HPO) terms. Sanger sequencing was used to verify the mutation in affected individuals and family members.

Genomic DNA was extracted from whole peripheral blood samples using the Qiamp Blood extraction mini kit (Qiagen GmbH, Hilden, Germany).
CES analysis was performed using the Clinical Exome solution by the SOPHiA™ Genetics kit (Sophia Genetics SA, Saint-Sulpice, Switzerland), on a Miseq platform (Illumina), following the manufacturer’s instruction. Raw data processing, and variant calling and annotation were conducted using SOPHiA™ DDM v5.7.5 (Sophia Genetics SA, Saint-Sulpice, Switzerland) bioinformatics pipelines. The CES panel includes 4493 clinically relevant genes. The identified mutations were confirmed in the parents whenever possible and were validated by specific long-range PCR amplification, using primers designed to discriminate between GBA and GBAP1 sequences (GenBank AH006907.2) [28 (link)], followed by automated Sanger sequencing (ABI Prism 3500xl genetic analyzer, Applied Biosystems, Foster City, CA, USA).