FISH was performed at diagnosis on immunomagnetic-enriched PCs from 169 out of the 225 cases with available phenotypic data. DNA from two PC clones FACS-purified according to their differentiation status from six newly diagnosed MM patients was analysed including the corresponding germline samples. DNA was extracted from cells using AllPrep DNA/RNA Micro Kit, Qiagen (Hilden, Germany). Targeted gene sequencing was performed using 20 ng of input DNA and applying the MM Mutation Panel Version 2.0 (M3P 2.0). Targeted panel consists of 1271 amplicons from 77 genes commonly mutated in MM. Enriched templates were sequenced using semiconductor technology (Ion Proton, Life Technologies, Waltham, MA, USA) and analysed with Ion Reporter Software v4.4 (Life Technologies). A median of 1700x depth coverage was obtained. Mutation calls were considered positive when called by ≥ 5% variant reads, with a minimum depth coverage of 10 reads.
Ion reporter software v4
Manufactured by Thermo Fisher Scientific
Sourced in United States
Ion Reporter Software v4.4 is a bioinformatics software tool designed to analyze and interpret data generated from next-generation sequencing (NGS) workflows. The software provides a comprehensive suite of tools for data processing, variant calling, annotation, and reporting.
Automatically generated - may contain errors
Lab products found in correlation
Torrent suite software v 4, by Thermo Fisher Scientific (2 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (2 mentions)
Ion torrent personal genome machine, by Thermo Fisher Scientific (2 mentions)
Onetouch 2, by Thermo Fisher Scientific (2 mentions)
Onetouch dl, by Thermo Fisher Scientific (2 mentions)
Torrent suite software v3, by Thermo Fisher Scientific (2 mentions)
Ion ampliseq library kit v2, by Thermo Fisher Scientific (2 mentions)
Ion proton, by Thermo Fisher Scientific (1 mentions)
Allprep dna rna micro kit, by Qiagen (1 mentions)
Ion pgm platform, by Thermo Fisher Scientific (1 mentions)
Recoverall total nucleic acid isolation kit, by Thermo Fisher Scientific (1 mentions)
Qubit 2, by Thermo Fisher Scientific (1 mentions)
Ion proton platform, by Thermo Fisher Scientific (1 mentions)
Ion ampliseq cancer hotspot panel v2, by Thermo Fisher Scientific (1 mentions)
Ion pi chip, by Thermo Fisher Scientific (1 mentions)
Agilent high sensitivity dna kit, by Agilent Technologies (1 mentions)
Ion ampliseq library kit 2, by Thermo Fisher Scientific (1 mentions)
Qiaamp dna ffpe tissue kit, by Qiagen (1 mentions)
Agilent 2100 bioanalyzer on, by Agilent Technologies (1 mentions)
6 protocols using ion reporter software v4
1
Targeted Sequencing of Mutated Genes in MM
2
FFPE Tumor DNA Extraction and Sequencing
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DNA was extracted from FFPE tumor samples using the RecoverAll™ Total Nucleic Acid Isolation Kit (Life Technologies) according to the manufacturer’s instructions. Quantity and quality were assessed using Qubit 2.0 (Life Technologies). Ten nanograms of DNA for each sample was used for library construction and template preparation with same procedures described above in the “Whole-exome sequencing” section. Target capture sequencing was carried out with customized panel using the Ion PGM™ platform (Life Technologies) according to the manufacturer’s instructions. The panel consisted of two separate PCR primer pools covering recurrent mutations in 96 genes with 1500X sequence coverage on Ion™ 318 chip. Sequencing data were analyzed with Ion Reporter™ software v4.4 (Life Technologies) using default parameters setting.
3
Comprehensive Cancer Hotspot Mutation Analysis
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DNA was extracted from 20 μm sections using QIAamp DNA FFPE Tissue Kit (Qiagen, Hilden, Germany). Library preparation was performed using Ion AmpliSeq Library Kit 2.0 and Ion AmpliSeq Cancer Hotspot Panel v2 (Thermo Fisher Scientific, Waltham, MA, USA). The panel target’s hotspot regions included more than 2800 COSMIC mutations of 50 cancer-related genes. After the library preparation, each amplicon library was quantified using the Agilent 2100 Bioanalyzer and Agilent High Sensitivity DNA Kit (Agilent Technologies, Santa Clara, CA, USA) and sequenced using the Ion Proton platform and Ion PI Chip (Thermo Fisher Scientific). The average read depths were approximately 1100.
Data were analyzed using Torrent Suite Software v4.2.191 (Thermo Fisher Scientific) and Ion Reporter Software v4.6 (Thermo Fisher Scientific). The read alignments were performed using the human reference genome hg19. Candidate pathogenic variants were filtered based on the number of reads in a target sequence and variant frequency in the total number of reads. Intronic, homogeneous, or synonymous variants were excluded. Mutations were analyzed using SIFT, PolyPhen, and Mutation Taster, and were considered relevant when scored as deleterious by at least two of these algorithms.
Data were analyzed using Torrent Suite Software v4.2.191 (Thermo Fisher Scientific) and Ion Reporter Software v4.6 (Thermo Fisher Scientific). The read alignments were performed using the human reference genome hg19. Candidate pathogenic variants were filtered based on the number of reads in a target sequence and variant frequency in the total number of reads. Intronic, homogeneous, or synonymous variants were excluded. Mutations were analyzed using SIFT, PolyPhen, and Mutation Taster, and were considered relevant when scored as deleterious by at least two of these algorithms.
4
Targeted NGS Variant and Copy Number Analysis
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Data were analyzed using Torrent Suite Software v4.2.191 (Thermo Fisher Scientific) and Ion Reporter Software v4.6 (Thermo Fisher Scientific). The read alignments were performed using the human reference genome hg19. Detected variants with quality scores of <20 and allele frequencies of <4.0%9 were eliminated. Further, we filtered out possible germline mutations using the databases of the 1000 genomes project (http://www.internationalgenome.org/ ) and 5000 exomes project (http://evs.gs.washington.edu/EVS/ ), as matched normal tissue samples were not analyzed in this study. Copy number analyses were performed using Biomedical Genomics Workbench 2.5 (Qiagen). The algorithm implemented is based on a CNA detection tool called COpy Number Targeted Resequencing Analysis (CONTRA), which includes a module for efficiently creating a pseudo‐control from multiple samples.10 Non‐cancer samples derived from FFPE specimens of nine patients with oral leukoplakia were used to create pseudo‐control data. Each target region copy number was calculated using a log2 ratio. We used |2| ≤ adjusted fold‐change (log2) as the criterion for CNAs. More details about this method are given in the Supporting information (Doc. S1 , Fig. [Link] , [Link] a–b).
5
Multiplex PCR and Ion Torrent Sequencing
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Ten nanograms of DNA was used for multiplex PCR amplification. Libraries were constructed using the Ion AmpliSeq Library Kit v2.0 (Thermo Fisher Scientific) according to the manufacturer’s instructions. The quality of the obtained libraries was evaluated using Agilent 2100 Bioanalyzer on-chip electrophoresis (Agilent Technologies, Palo Alto, CA, USA). Emulsion PCR was performed with the OneTouch DL or OneTouch 2 system (Thermo Fisher Scientific). Sequencing was run on the Ion Torrent Personal Genome Machine (Thermo Fisher Scientific), loaded with a 316 or 318v2 chip as per the manufacturer’s protocol. Data analysis, including alignment to the hg19 human reference genome as well as variant calling and filtering, was completed using Torrent Suite Software v3.6 (Thermo Fisher Scientific). Filtered variants were annotated using Ion Reporter software v4.4 (Thermo Fisher Scientific).
6
Targeted Deep Sequencing of GDF15 and TP53
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Ten nanogram of DNA were used for multiplex PCR amplification. Libraries were constructed using the Ion AmpliSeq Library Kit v2.0 (Thermo Fisher Scientific, USA) according to the manufacturer's instructions. The quality of obtained library was evaluated by the Agilent 2100 Bioanalyzer on-chip electrophoresis (Agilent Technologies, USA).
Emulsion PCR was performed with the OneTouch DL or OneTouch 2 system (Thermo Fisher Scientific, USA). Sequencing was run on the Ion Torrent Personal Genome Machine (Thermo Fisher Scientific, USA), loading with 316™ or 318™v2 chip as per manufacturer's protocol. Data analysis, including alignment to the hg19 human reference genome as well as variant calling and filtering, was done using the Torrent Suite Software v.3.6 (Thermo Fisher Scientific, USA). Filtered variants were annotated using Ion Reporter software v4.4 (Thermo Fisher Scientific, USA). Alignments were visually verified with the Integrative Genomics Viewer; v.2.3. The mean coverage achieved was 1361-fold and 2338-fold in the tumor tissues for GDF15 and TP53 sequencing, respectively, and the same deep sequencing was done (two sample in 316™ chip or four sample in 318™v2 chip) on the control tissues. 90% and 95% of the targeted bases were represented by at least 10 reads for GDF15 and TP53, respectively.
Emulsion PCR was performed with the OneTouch DL or OneTouch 2 system (Thermo Fisher Scientific, USA). Sequencing was run on the Ion Torrent Personal Genome Machine (Thermo Fisher Scientific, USA), loading with 316™ or 318™v2 chip as per manufacturer's protocol. Data analysis, including alignment to the hg19 human reference genome as well as variant calling and filtering, was done using the Torrent Suite Software v.3.6 (Thermo Fisher Scientific, USA). Filtered variants were annotated using Ion Reporter software v4.4 (Thermo Fisher Scientific, USA). Alignments were visually verified with the Integrative Genomics Viewer; v.2.3. The mean coverage achieved was 1361-fold and 2338-fold in the tumor tissues for GDF15 and TP53 sequencing, respectively, and the same deep sequencing was done (two sample in 316™ chip or four sample in 318™v2 chip) on the control tissues. 90% and 95% of the targeted bases were represented by at least 10 reads for GDF15 and TP53, respectively.
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