High-throughput targeted amplicon sequencing (Ion Torrent PGM platform) was used to detect mutation hotspots or full-length coding regions of 58 commonly mutated genes associated with hematological tumors [6 (link)]. For the mutation analysis of 58 genes implemented in this study, only hotspot mutations and mutations with clear pathological significance in hematological tumors were reported and analyzed. Average sequencing depth was greater than 1000×, single nucleotide variation (SNVs) and short fragment insertion/deletion mutations were identified by TVC 5.0-13 software. The SNVs recognition threshold was set to the minimum coverage depth of 100× and the variant allele frequency (VAF) was >5%. The dbSNP, 1000 Genomes, ExAC, ClinVar, and COSMIC databases and the bioinformatics software programs PolyPhen-2 and SIFT were used for mutation analysis. Mutations of FLT3-ITD, NPM1 and CALR genes were detected by fragment analysis (AB 3500XL sequencer), the results of which were analyzed using GeneMapper ID V3.2 software to calculate the length and VAF of the inserted/deleted fragments.
Pgm platform
Manufactured by Thermo Fisher Scientific
The PGM platform is a compact and flexible next-generation sequencing (NGS) system designed for targeted and small-scale genomic sequencing applications. It utilizes Ion Semiconductor Sequencing technology to generate high-quality sequencing data. The core function of the PGM platform is to provide researchers with a streamlined and efficient solution for targeted DNA or RNA sequencing.
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3 protocols using pgm platform
1
Targeted Amplicon Sequencing for Hematological Tumor Mutation Analysis
2
ITS2 Sequencing Protocol for Fungal Identification
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The ITS2 gene was sequenced using the fITS7b (5′-GTGARTCATCGAATCTTTG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) primers. Before sequencing, PCR was conducted utilizing the Phusion Flash High-Fidelity PCR master mix from Thermo Fisher Scientific. The following PCR protocol was followed: 2 min of initialisation, 35 repetitions of denaturation at 98 °C for 10 s, annealing at 54 °C for 20 s, and elongation at 72 °C for 30 s, with the final elongation lasting for 7 min. Amplicon sequencing was executed using an IonTorrent PGM platform. Sequencing was performed in four separate runs. Details about DNA extraction, PCR, and sequencing have been previously published [31 (link)].
3
Bovine Parainfluenza Virus 3 Isolation
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Six viral strains isolated from clinical samples from respiratory disease outbreaks submitted to the Texas A&M Veterinary Diagnostic Laboratory between 2007 and 2012 were used in the study. These viruses were isolated by serial passage in bovine turbinate cells (ATCC CRL-1390) using standard procedures of virus isolation in cell culture. The viruses were confirmed as BPI3V by fluorescent antibody staining [21 ]. After growth of the virus on the appropriate cell type, the medium was frozen/thawed thrice, cell debris removed by centrifugation, and aliquots frozen at −80 °C until further use. The virus stocks were treated with a nuclease cocktail, viral RNA isolated and sequencing libraries composed of 20 viral genomes were prepared and sequenced using the Ion Torrent PGM platform as previously described [16 (link)].
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