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Trusight rna pan cancer panel

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The TruSight RNA Pan-Cancer Panel is a targeted RNA sequencing panel designed to analyze the expression of genes associated with various cancer types. The panel consists of a set of probes that target specific regions of the human transcriptome, enabling the detection and quantification of gene expression levels.

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Lab products found in correlation

Rneasy ffpe kit, by Qiagen (4 mentions) Sureselect rna capture kit, by Agilent Technologies (2 mentions) Nextseq platform, by Illumina (2 mentions) Nextseq reagent kit v3, by Illumina (2 mentions) Nextseq 550 system, by Illumina (2 mentions) Basespace, by Illumina (2 mentions) Pe nextseq flow cell, by Illumina (2 mentions) Tophat alignment, by Illumina (2 mentions) Trusight rna fusion panel, by Illumina (2 mentions) Dnase 1, by Thermo Fisher Scientific (2 mentions) Tapestation 2200, by Agilent Technologies (2 mentions) Nextseq 550, by Illumina (1 mentions) Hiseq 2500, by Illumina (1 mentions) Nextseq 500 sequencer, by Illumina (1 mentions) Rna seq alignment, by Illumina (1 mentions) Sureselectxt custom kit, by Agilent Technologies (1 mentions) Taqman copy number reference assay, by Thermo Fisher Scientific (1 mentions) Generead dna ffpe kit, by Qiagen (1 mentions) Nanodrop 1000, by Qiagen (1 mentions) Rna seq alignment workflow, by Illumina (1 mentions)

20 protocols using trusight rna pan cancer panel

1

Automated FFPE Transcriptome Profiling

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The Agencourt FormaPure Total 96-Prep Kit was used to extract DNA and RNA from the same FFPE tissue lysates using an automated KingFisher Flex following the protocols recommended by the manufacturers. Samples were selectively enriched for 1408 cancer-associated genes using reagents provided in the Illumina® TruSight® RNA Pan-Cancer Panel. cDNA was generated from the cleaved RNA fragments using random primers during the first and second-strand synthesis. Sequencing adapters were ligated to the resulting double-stranded cDNA fragments. The coding regions of the expressed genes were captured from this library using sequence-specific probes to create the final library. Sequencing was performed using an Illumina NextSeq 550 system platform. Ten million reads per sample in a single run were required, and the read length was 2 × 150 bp. The sequencing depth was 10×–1739× with a median of 41×. An expression profile was generated from the sequencing coverage profile of each individual sample using Cufflinks. Expression levels were measured as fragments per kilobase of transcript per million.
Albitar M., Zhang H., Goy A., Xu-Monette Z.Y., Bhagat G., Visco C., Tzankov A., Fang X., Zhu F., Dybkaer K., Chiu A., Tam W., Zu Y., Hsi E.D., Hagemeister F.B., Huh J., Ponzoni M., Ferreri A.J., Møller M.B., Parsons B.M., van Krieken J.H., Piris M.A., Winter J.N., Li Y., Xu B, & Young K.H. (2022). Determining clinical course of diffuse large B-cell lymphoma using targeted transcriptome and machine learning algorithms. Blood Cancer Journal, 12(2), 25.
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2

Comprehensive RNA Profiling from FFPE Samples

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Total RNA was extracted from FFPE samples using an RNeasy FFPE Kit (Qiagen) and was then treated with DNase I (Thermo Fisher Scientific). RNA quality was evaluated on a 2200 TapeStation using the HighSensitivity RNA Screen Tape system to calculate RNA integrities (RIN) and DV200. cDNA synthesis and library preparation for coding exon capture were conducted using the TruSight RNA Pan‐Cancer Panel (Illumina), according to the manufacturer's protocol. cDNA synthesis and library preparation for junction capture were conducted using a SureSelect RNA Capture kit (Agilent Technologies), according to the manufacturer's protocol. Custom‐made probes for the junction capture method were designed to hybridize and capture the junctional sequences of the target genes listed in Table S1. NGS sequencing was conducted from both ends of each cluster using a HiSeq2500 or Next‐seq platform (Illumina).
Kohsaka S., Tatsuno K., Ueno T., Nagano M., Shinozaki‐Ushiku A., Ushiku T., Takai D., Ikegami M., Kobayashi H., Kage H., Ando M., Hata K., Ueda H., Yamamoto S., Kojima S., Oseto K., Akaike K., Suehara Y., Hayashi T., Saito T., Takahashi F., Takahashi K., Takamochi K., Suzuki K., Nagayama S., Oda Y., Mimori K., Ishihara S., Yatomi Y., Nagase T., Nakajima J., Tanaka S., Fukayama M., Oda K., Nangaku M., Miyazono K., Miyagawa K., Aburatani H, & Mano H. (2019). Comprehensive assay for the molecular profiling of cancer by target enrichment from formalin‐fixed paraffin‐embedded specimens. Cancer Science, 110(4), 1464-1479.
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3

Comprehensive Cancer Gene Profiling via Illumina RNA Sequencing

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RNA sequencing was performed using the TruSight RNA Pan-Cancer panel (Illumina, San Diego, CA) which contains 1385 cancer genes and enables fusion calling and variant detection within the panel. Twenty nanograms of RNA was processed according to the manufacturer’s protocol and was sequenced on a Next Seq 550 system (Illumina, San Diego, CA) using the NextSeq® Reagent Kit v3 (150 cycles) with a PE NextSeq® Flow Cell. Data analyses were performed using the Illumina BaseSpace apps TopHat Alignment (version 1.0.0, read mapping on hg19 reference genome by TopHat21, fusion calling by TopHat-Fusion2) and RNA-seq Alignment (version 1.1.0, read mapping on hg19 reference genome by STAR3, fusion calling by Manta4 using standard settings) (BaseSpace.illumina.com/apps">https://BaseSpace.illumina.com/apps). Fusion transcripts with a low number of split-reads were excluded as likely false positives. The raw data of the sequence variants were converted to variant call format (vcf) files and analysed in Variant Studio software v.4.0.
Bobeff K., Pastorczak A., Urbanska Z., Balwierz W., Juraszewska E., Wachowiak J., Derwich K., Samborska M., Kalwak K., Dachowska-Kalwak I., Laguna P., Malinowska I., Smalisz K., Gozdzik J., Oszer A., Urbanski B., Zdunek M., Szczepanski T., Mlynarski W, & Janczar S. (2023). Venetoclax Use in Paediatric Haemato-Oncology Centres in Poland: A 2022 Survey. Children, 10(4), 745.
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4

Fusion Transcript Detection in ALCL

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To detect fusion transcripts in ALCLs, the TruSight RNA Pan-Cancer panel (Illumina, San Diego, CA, USA) targeting 1385 cancer genes, including 507 known genes involved in fusions and 878 genes either mutated or deregulated in cancers, was used according to the provided protocol. The panel design covers all exons and 160 bp at the 5′ and 3′ UTR of every gene included in the panel. Briefly, double-stranded cDNA fragments generated from 50 ng of total RNA were ligated to sequencing adapters. The coding regions of expressed cancer-associated genes were captured using sequence-specific probes to create the final sequencing library. Paired-end RNA-sequencing was performed on a NextSeq500 sequencer using the NextSeq500 High Output Kit v2 chemistry (Illumina). Raw sequencing data converted to fastq file formats were analyzed.
Sequencing data are available at the Sequence Read Archive (https://www.ncbi.nlm.nih.gov/sra (accessed on 1 December 2021)).
Di Napoli A., Vacca D., Bertolazzi G., Lopez G., Piane M., Germani A., Rogges E., Pepe G., Santanelli Di Pompeo F., Salgarello M., Jobanputra V., Hsiao S., Wrzeszczynski K.O., Berti E, & Bhagat G. (2021). RNA Sequencing of Primary Cutaneous and Breast-Implant Associated Anaplastic Large Cell Lymphomas Reveals Infrequent Fusion Transcripts and Upregulation of PI3K/AKT Signaling via Neurotrophin Pathway Genes. Cancers, 13(24), 6174.
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5

RNA Sequencing Pipeline for Pan-Cancer Profiling

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RNA sequencing was performed using the TruSight RNA Pan-Cancer panel (Illumina, San Diego, CA) on a Next Seq 550 system (Illumina, San Diego, CA) using NextSeq® Reagent Kit v3 (150 cycles) with a PE NextSeq® Flow Cell. Data analysis was performed using the Illumina BaseSpace apps TopHat Alignment (version 1.0.0, read mapping on hg19 reference genome by TopHat21, fusion calling by TopHat-Fusion2, and RNA-seq Alignment (version 1.1.0, read mapping on hg19 reference genome by STAR3, fusion calling by Manta4 using standard settings (https://basespace.illumina.com/apps). Raw data of sequence variants were analyzed in Variant Studio software v.4.0.
A detailed description of all methods can be found in the online Supplementary materials.
Urbańska Z., Lejman M., Taha J., Madzio J., Ostrowska K., Miarka-Walczyk K., Wypyszczak K., Styka B., Jakubowska J., Sędek Ł., Szczepański T., Stańczak M., Fendler W., Młynarski W, & Pastorczak A. (2022). The kinetics of blast clearance are associated with copy number alterations in childhood B-cell acute lymphoblastic leukemia. Neoplasia (New York, N.Y.), 35, 100840.
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6

Targeted DNA and RNA Sequencing from FFPE Samples

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Genomic DNA target sequencing and RNA sequencing using cDNA capture were previously described.7 Briefly, genomic DNA was isolated from FFPE samples using GeneRead DNA FFPE Kits (Qiagen). DNA quality was determined by the FFPE DNA QC Assay version 2, TaqMan Copy Number Reference Assay (Thermo Fisher Scientific). Total RNA was extracted from FFPE samples using RNeasy FFPE Kit (Qiagen). RNA quality was evaluated on a 2200 TapeStation (Agilent Technologies) to calculate DV200. DNA target sequencing library was prepared using the SureSelectXT custom kit (Agilent Technologies). Custom‐made probes were designed to hybridize and capture the genomic DNA of the target genes listed in Table S1 and intronic DNA of 4327 single nucleotide polymorphisms within the targeted gene regions. The cDNA library preparation was carried out using the TruSight RNA Pan‐Cancer Panel (Illumina). cDNA junction capture was undertaken using the SureSelect RNA Capture kit (Agilent Technologies) with custom‐made probes designed to hybridize and capture the junctional sequences of the target genes listed in Table S1. Next‐generation sequencing was carried out using the Next‐seq platform (Illumina) as previously described.7
Kage H., Shinozaki‐Ushiku A., Ishigaki K., Sato Y., Tanabe M., Tanaka S., Tanikawa M., Watanabe K., Kato S., Akagi K., Uchino K., Mitani K., Takahashi S., Miura Y., Ikeda S., Kojima Y., Watanabe K., Mochizuki H., Yamaguchi H., Kawazoe Y., Kashiwabara K., Kohsaka S., Tatsuno K., Ushiku T., Ohe K., Yatomi Y., Seto Y., Aburatani H., Mano H., Miyagawa K, & Oda K. (2023). Clinical utility of Todai OncoPanel in the setting of approved comprehensive cancer genomic profiling tests in Japan. Cancer Science, 114(4), 1710-1717.
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7

RNA Sequencing of FFPE Tissue Samples

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For Cases 1 and 2, (Table 1), RNA was isolated from formalin-fixed paraffin embedded (FFPE) tissue sections using RNeasy FFPE Kit of Qiagen (Hilden, Germany) and quantified spectrophotometrically using NanoDrop-1000 (Waltham, United States). Molecular analysis was performed using the TruSight RNA Fusion panel (Illumina, Inc., San Diego, CA, USA) with 500 ng RNA as input according to the manufacturer`s protocol. Libraries were sequenced on a MiSeq (Illumina, Inc., San Diego, CA, USA) with > 3 million reads per case, and sequences were analyzed using the RNA-Seq Alignment workflow, version 2.0.1 (Illumina, Inc., San Diego, CA, USA). The Integrative Genomics Viewer (IGV), version 2.2.13 (Broad Institute, REF) was used for data visualization [16 ]. Cases 3, 4, and 6 were subjected to targeted RNA sequencing using Illumina RNA Fusion assay as described previously [17 (link)]. The fusion in Case 4 was additionally independently confirmed by the Archer panel [18 (link)]. Case 5 was identified using the Illumina TruSight RNA PanCancer panel.
Tulving E., Kapur S., Markowitsch H.J., Craik F.I., Habib R, & Houle S. (1994). Neuroanatomical correlates of retrieval in episodic memory: auditory sentence recognition.. Proceedings of the National Academy of Sciences of the United States of America, 91(6), 2012-2015.
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8

RNA-seq Analysis of Formalin-Fixed Tissue

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Scrolls (3–4 at 10 microns) of formalin-fixed paraffin-embedded tissue blocks were cut into Eppendorf tubes. Cases 1–2: RNA was extracted using ExpressArt FFPE Clear RNA Ready kits (Amsbio, Cambridge, MA), and RNA-seq libraries prepared using 20–100 ng RNA with the TruSight RNA Fusion Panel (Illumina, San Diego, CA). Cases were sequenced with 76 base-pair paired-end reads on an Illumina MiSeq with 8 samples per flow cell (~3 million reads per sample). Analysis was performed using both the STAR aligner and Manta fusion caller, and the JAFFA fusion caller utilizing BOWTIE2 aligner;9 (link),10 (link) Case 3: RNA-seq libraries were prepared with the TruSight RNA Pan-Cancer Panel (Illumina, San Diego, CA). Sequencing with 76 base-pair paired-end reads on an Illumina NextSeq 500 System (Illumina, San Diego, CA). Analysis was performed using the STAR aligner and STAR fusion caller11 (link) Case 4 was confirmed by Caris Life Sciences, (Irving, TX).
Lacambra M.D., Antonescu C.R., Chit C., Chiu W.K., Demicco E.G., Ferguson P.C., Swanson D., To K.F., Zhang L, & Dickson B.C. (2022). EXPANDING THE SPECTRUM OF MESENCHYMAL NEOPLASMS WITH NR1D1-REARRANGEMENT. Genes, chromosomes & cancer, 61(7), 420-426.
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9

Tumor RNA Extraction and Sequencing

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Total RNA from tumor tissue was extracted using mirVana miRNA Isolation Kit (Thermo Fisher Scientific, MA, USA). Quantity and quality of extracted RNA were checked by Qubit® 2.0 Fluorometer system (Thermo Fisher Scientific, MA, USA) and NanoDrop 2000c Spectrophotometer (Thermo Fisher Scientific, MA, USA). For sequencing libraries preparation, TruSight RNA Pan-Cancer Panel (Illumina, CA, USA), which targets fusions in 1385 genes, was used. Sequencing libraries were subsequently loaded on NextSeq 500/550 Mid Output Kit v2 (150 cycles) and NextSeq 500 sequencing device (both Illumina, CA, USA). All processes were performed according to the manufacturer's instructions. Quantity and quality of sequencing libraries were checked by Qubit® 2.0 Fluorometer system (Thermo Fisher Scientific, MA, USA) and TapeStation 2200 (Agilent Technologies, CA, USA). For data analysis, BreakingPoint tool was used.
Gojo J., Pavelka Z., Zapletalova D., Schmook M.T., Mayr L., Madlener S., Kyr M., Vejmelkova K., Smrcka M., Czech T., Dorfer C., Skotakova J., Azizi A.A., Chocholous M., Reisinger D., Lastovicka D., Valik D., Haberler C., Peyrl A., Noskova H., Pál K., Jezova M., Veselska R., Kozakova S., Slaby O., Slavc I, & Sterba J. (2020). Personalized Treatment of H3K27M-Mutant Pediatric Diffuse Gliomas Provides Improved Therapeutic Opportunities. Frontiers in Oncology, 9, 1436.
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10

TruSight RNA Pan-Cancer Sequencing

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Library preparation was performed from 20 ng of RNA using TruSight RNA Pan-Cancer Panel (Illumina, SanDiego, California) targeting 1385 genes involved in cancer biology (panel available at https://www.illumina.com/content/dam/illumina-marketing/documents/products/gene_lists/gene_list_trusight_pan_cancer.xlsx). Libraries from 16 samples were multiplexed and sequenced on a Nextseq 500 device (Illumina) with a 2 × 81 paired-end run on a mid-output flowcell according to the manufacturer’s instructions (mean number of reads by sample: 32 × 106; range 20–59 × 106).
Hayette S., Grange B., Vallee M., Bardel C., Huet S., Mosnier I., Chabane K., Simonet T., Balsat M., Heiblig M., Tigaud I., Nicolini F.E., Mareschal S., Salles G, & Sujobert P. (2021). Performances of Targeted RNA Sequencing for the Analysis of Fusion Transcripts, Gene Mutation, and Expression in Hematological Malignancies. HemaSphere, 5(2), e522.
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