Torrent suite software v3

Manufactured by Thermo Fisher Scientific

Sourced in United States

Torrent suite software v3.6.2 is a software tool designed for data analysis and management in the field of life sciences. The software provides a comprehensive platform for processing and analyzing data generated from various sequencing platforms. It offers a range of features and functionalities to streamline the data analysis workflow.

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7 protocols using torrent suite software v3

Targeted NGS Variant Identification

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Alignment of sequencing reads with the reference genome and base calling was performed using Torrent Suite software v3.6.2 (Life Technologies). Human genome build 19 was used as the reference for alignment. Identification of sequence variants was facilitated via IT Variant Caller Plugin software v3.6.59049 (Life Technologies) and coverage of each amplicon was obtained by the Coverage Analysis Plugin software v3.6.58977 (Life Technologies). The Integrative Genomics Viewer (IGV) (Thorvaldsdottir et al, 2012 (link)) was used to visualise the read alignment and the presence of variants against the reference genome as well as to confirm variant calls by checking for strand biases and sequencing errors. A custom in-house developed software (OncoSeek) described earlier (Singh et al, 2013 (link)) was used to interface the data generated by the Ion Torrent Variant Caller (Life Technologies) with the IGV, filter repeat errors due to nucleotide homopolymer regions, compare replicate samples, and annotate the sequencing information to generate a clinical report. Torrent Suite v4.0.2 (Variant Caller plugin v4.0-r76860 and Coverage Analysis plugin v4.0-r77897) (Life Technologies) was used to reanalyse the sequencing data for a subset of samples.

Singh R.R., Patel K.P., Routbort M.J., Aldape K., Lu X., Manekia J., Abraham R., Reddy N.G., Barkoh B.A., Veliyathu J., Medeiros L.J, & Luthra R. (2014). Clinical massively parallel next-generation sequencing analysis of 409 cancer-related genes for mutations and copy number variations in solid tumours. British Journal of Cancer, 111(10), 2014-2023.

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Comprehensive Somatic Mutation Analysis

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The raw data were analyzed using the torrent suite software v3.6.2 (Life technologies). The coverage analysis was performed using the coverage analysis plug-in v3.6. Cases for which the number of mapped reads was <100000 and/or the average base coverage was <500x were considered as non informative. Mutations were detected using the Variant Caller plug-in v3.6 with low stringency settings (Life Technologies). In the variant list obtained, each mutation was verified in the Integrative genome viewer (IGV) from the Broad Institute (http://www.broadinstitute.org/igv/) [19 (link)]. Only mutations reported in the COSMIC (Sanger Institute Catalogue of Somatic Mutations in Cancer) database (http://www.sanger.ac.uk/cosmic) were taken into account and silent or intronic mutations were not reported.

D’Haene N., Le Mercier M., De Nève N., Blanchard O., Delaunoy M., El Housni H., Dessars B., Heimann P., Remmelink M., Demetter P., Tejpar S, & Salmon I. (2015). Clinical Validation of Targeted Next Generation Sequencing for Colon and Lung Cancers. PLoS ONE, 10(9), e0138245.

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Comprehensive Genomic Variant Analysis Protocol

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The raw data were analyzed using the torrent suite software v3.6.2 (Life technologies). The coverage analysis was performed using the coverage analysis plug-in v3.6. Cases for which the number of mapped reads was <100000 and/or the average base coverage was <500x were considered as non-informative. Mutations were detected using the Variant Caller plug-in v3.6 with low stringency settings (Life Technologies). In the variant list obtained, each mutation was verified in the Integrative genome viewer (IGV) from the Broad Institute (http://www.broadinstitute.org/igv/). Only mutations reported in the COSMIC (Sanger Institute Catalogue of Somatic Mutations in Cancer) database (http://www.sanger.ac.uk/cosmic) were taken into account and silent or intronic mutations were not reported. Locis were further analyzed for functional prediction of amino acid changes using two different prediction algorithms (Provean and SIFT) [17 (link), 18 (link)].

Lim S.M., Kim H.R., Cho E.K., Min Y.J., Ahn J.S., Ahn M.J., Park K., Cho B.C., Lee J.H., Jeong H.C., Kim E.K, & Kim J.H. (2016). Targeted sequencing identifies genetic alterations that confer primary resistance to EGFR tyrosine kinase inhibitor (Korean Lung Cancer Consortium). Oncotarget, 7(24), 36311-36320.

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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).

Ma J., Fu Y., Tu Y.Y., Liu Y., Tan Y.R., Ju W.T., Pickering C.R., Myers J.N., Zhang Z.Y, & Zhong L.P. (2018). Mutation allele frequency threshold does not affect prognostic analysis using next-generation sequencing in oral squamous cell carcinoma. BMC Cancer, 18, 758.

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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.

Ma J., Tang X., Sun W.W., Liu Y., Tan Y.R., Ma H.L., Zhu D.W., Wang M., Wang L.Z., Li J., Tu Y.Y., Zhang C.P., Zhang Z.Y, & Zhong L.P. (2015). Mutant GDF15 presents a poor prognostic outcome for patients with oral squamous cell carcinoma. Oncotarget, 7(2), 2113-2122.

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Ion Torrent Deep Sequencing of Cancer Genes

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Amplicons. Deep sequencing was performed using the Ion Torrent platform (Life Technologies). Briefly, 10 ng of purified genomic DNA were used for library construction with the Ion AmpliSeq Colon and Lung Cancer Panel v2 (Life Technologies) that targets 504 mutational hotspot regions of the following 22 cancer-associated genes, in alphabetical order: AKT1, ALK, BRAF, CTNNB1, DDR2, EGFR, ERBB2, ERBB4, FBXW7, FGFR1, FGFR2, FGFR3, KRAS, MAP2K1, MET, NOTCH1, NRAS, PIK3CA, PTEN, SMAD4, STK11, TP53. Emulsion PCR was performed either manually or with the OneTouch DL system (Life Technologies). The quality of the obtained library was evaluated by the Agilent® 2100 Bioanalyzer on-chip electrophoresis (Agilent Technologies; Santa Clara, CA). Sequencing was run on the Ion Torrent Personal Genome Machine™ (PGM, Life Technologies) loaded with a 316 chip as per manufacturer's protocol. Data analysis, including alignment to the hg19 human reference genome and variant calling, was done using the Torrent Suite Software v.3.2 (Life Technologies). Filtered variants were annotated using both the Ion Reporter software v1.2 (Life Technologies) and the SnpEff software v.3.0 (alignments visually verified with the Integrative Genomics Viewer; IGV v.2.1, Broad Institute) [46 (link)].

Bria E., Pilotto S., Amato E., Fassan M., Novello S., Peretti U., Vavalà T., Kinspergher S., Righi L., Santo A., Brunelli M., Corbo V., Giglioli E., Sperduti I., Milella M., Chilosi M., Scarpa A, & Tortora G. (2015). Molecular heterogeneity assessment by next-generation sequencing and response to gefitinib of EGFR mutant advanced lung adenocarcinoma. Oncotarget, 6(14), 12783-12795.

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Custom Multiplex PCR Sequencing Protocol

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Genomic sequencing was performed using a custom multiplex PCR designed to include the entire coding sequence of the majority of altered genes in the whole-genome sequencing discovery cohort as well as TP53 and in total encompassing 106.3 kB of target region (Table S11). Primers for the targeted sequencing were designed using the Ion Ampliseq designer (Life Technologies) and sequencing was performed on the IonTorrent PGM (Life Technologies). PGM sequencing data was analyzed using Torrent Suite software v3.2 (Life Technologies) and ANNOVAR. Genomic sequencing was performed to an average mean coverage of 311× and variants were called using high-confidence thresholds and filtered to include only those that are protein altering and unreported or rare (population allele frequency <0.005) in the dbSNP and 1000 genomes databases. Mutations of interest were verified by capillary sequencing with a <5% false positive rate. Sequence coverage data was calculated at a position, exon and gene levels to look for structural alterations of the recurrently mutated genes (Figure S8). Coverage data was visualized using the Integrated Genomics Viewer.

Brohl A.S., Solomon D.A., Chang W., Wang J., Song Y., Sindiri S., Patidar R., Hurd L., Chen L., Shern J.F., Liao H., Wen X., Gerard J., Kim J.S., Lopez Guerrero J.A., Machado I., Wai D.H., Picci P., Triche T., Horvai A.E., Miettinen M., Wei J.S., Catchpool D., Llombart-Bosch A., Waldman T, & Khan J. (2014). The Genomic Landscape of the Ewing Sarcoma Family of Tumors Reveals Recurrent STAG2 Mutation. PLoS Genetics, 10(7), e1004475.

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