Sequencing analysis viewer

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The Illumina Sequencing Analysis Viewer is a software tool designed to visualize and analyze data generated by Illumina sequencing platforms. It provides a comprehensive view of sequencing data, including sequence reads, quality scores, and alignment information. The core function of the Illumina Sequencing Analysis Viewer is to facilitate the interpretation and exploration of sequencing data.

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Lab products found in correlation

Qubit 2.0 fluorometer, by Thermo Fisher Scientific (12 mentions) Qubit dsdna hs assay, by Thermo Fisher Scientific (10 mentions) Nextseq 500, by Illumina (5 mentions) E220 focused ultrasonicator, by Thermo Fisher Scientific (3 mentions) Trusight oncology 500 kit, by Illumina (3 mentions) Qubit dsdna hs assay kit, by Thermo Fisher Scientific (3 mentions) 8 microtube 50 strip afa fiber v2, by Thermo Fisher Scientific (2 mentions) Basespace, by Illumina (2 mentions) Miseq reporter, by Illumina (2 mentions) Tso 500 kit, by Illumina (1 mentions) E220 focused ultrasonicator, by Covaris (1 mentions) Agilent tapestation, by Agilent Technologies (1 mentions) Bcl2fastq2 v2, by Illumina (1 mentions) Hiseq 3000 system, by Illumina (1 mentions) Rneasy ffpe kit, by Qiagen (1 mentions) Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions) Nextseq 550 system, by Illumina (1 mentions) Real time analysis software, by Illumina (1 mentions) Trusight oncology 500, by Illumina (1 mentions) Qubit dsdna high sensitivity assay kit, by Thermo Fisher Scientific (1 mentions)

16 protocols using sequencing analysis viewer

Optimized FFPE DNA Sequencing Protocol

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200 ng DNA were quantified with the Qubit dsDNA HS Assay (Thermo Fisher Scientific) on the Qubit 2.0 Fluorometer (Thermo Fisher Scientific) and sheared on the Covaris E220 Focused-ultrasonicator (Woburn, MA, USA) using the 8 microTUBE–50 Strip AFA Fiber V2 following manufacturer’s instructions. The treatment time was optimized for FFPE material. The treatment settings were the following: Peak Incident Power (W): 175; Duty Factor: 10%; Cycles per Burst: 200; Treatment Time (s): 200; Temperature (°C): 7; Water Level: 6. For DNA library preparation and enrichment the NEOplus v2 RUO kit (NEO New Oncology, Cologne, Germany) was used following manufacturer’s instructions with 100 ng DNA input. Post-enriched libraries were quantified, pooled and sequenced on a NextSeq 500 (Illumina Inc., San Diego, CA, USA).
Quality of the NextSeq 500 (Illumina) sequencing runs were assessed with the Illumina Sequencing Analysis Viewer (Illumina). Sequencing data were analyzed with the NEOonsite Data Analysis RUO (version 1.4.1) and the NEO software NEOdb 2.2 (NEO New Oncology).

Heydt C., Rehker J., Pappesch R., Buhl T., Ball M., Siebolts U., Haak A., Lohneis P., Büttner R., Hillmer A.M, & Merkelbach-Bruse S. (2020). Analysis of tumor mutational burden: correlation of five large gene panels with whole exome sequencing. Scientific Reports, 10, 11387.

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Comprehensive Tumor Profiling with TSO 500

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DNA (40 ng) was quantified using the Qubit dsDNA HS Assay (Thermo Fisher Scientific, Waltham, MA) on a Qubit 2.0 Fluorometer (Thermo Fisher Scientific), then sheared using a Covaris E220 Focused-ultrasonicator (Woburn, MA) and the 8 microTUBE–50 Strip AFA Fiber V2 following manufacturer’s instructions. The treatment time was optimized for FFPE material. The treatment settings were as follows: peak incident power (W), 75; duty factor, 15%; cycles per burst, 500; treatment time (seconds), 360; temperature (°C), 7; water level, 6. The DNA library was prepared and enriched using the TSO 500 Kit (Illumina); manufacturer’s instructions were followed. Post-enriched libraries were quantified, pooled, and sequenced using NextSeq 500 (Illumina). The quality of the NextSeq 500 sequencing runs was assessed using Illumina Sequencing Analysis Viewer (Illumina). Sequencing data were analyzed using the TSO 500 Local App ver. 1.3.0.39 (Illumina). The TSO 500 is a comprehensive tumor profiling assay designed to identify known and emerging tumor biomarkers, including small variants, splice variants, and fusions. Importantly, the TSO 500 measures tumor mutational burden (TMB) and microsatellite instability, which are potentially key biomarkers for immunotherapy. TMB was reported as mutations per megabase (Mb) sequenced, and a high TMB was defined as mutations of more than 10 per Mb (≥ 10 Mut/Mb).

Lee S.E., Lee M.S., Jeon Y.K., Shim H.S., Kang J., Kim J, & Choi Y.L. (2022). Interlaboratory Comparison Study (Ring Test) of Next-Generation Sequencing–Based NTRK Fusion Detection in South Korea. Cancer Research and Treatment : Official Journal of Korean Cancer Association, 55(1), 28-40.

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FFPE RNA Extraction and Sequencing

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To isolate RNA, 10 µM-thick paraffin slices were trimmed from each FFPE tissue block using a microtome. Eight paraffin slices were used for RNA extraction. RNA was extracted from FFPE slices using a QIAGEN RNeasy FFPE Kit following the manufacturer’s protocol. RNA 6000 Nano or Qubit RNA Assay kits were used to measure RNA concentration. RNA Integrity Number (RIN) was measured using Agilent 2100 bio-analyzer. For depletion of ribosomal RNA and library construction, KAPA RNA Hyper with an rRNA erase kit (HMR only) was used. Different adaptors were used for multiplexing samples in one sequencing run. Library concentrations and quality were measured using the Qubit dsDNA HS Assay kit (Life Technologies) and Agilent Tapestation (Agilent). Single-end RNA sequencing, 50 bp read length, for ~30 million raw reads per sample, was performed at Omicslab LLC, Moscow and at the Department of Pathology and Laboratory Medicine, University of California Los Angeles, using the Illumina HiSeq 3000 System. A data quality check was performed using the Illumina Sequencing Analysis Viewer and de-multiplexing was performed with Illumina Bcl2fastq2 v 2.17 software. Sequencing data were deposited to NCBI Sequencing Read Archive (SRA) under accession ID PRJNA565016 and PRJNA578290.

Sorokin M., Ignatev K., Poddubskaya E., Vladimirova U., Gaifullin N., Lantsov D., Garazha A., Allina D., Suntsova M., Barbara V, & Buzdin A. (2020). RNA Sequencing in Comparison to Immunohistochemistry for Measuring Cancer Biomarkers in Breast Cancer and Lung Cancer Specimens. Biomedicines, 8(5), 114.

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Small RNA Sequencing Protocol

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Equimolar amounts (1 nM) of pooled libraries normalized to 10 nM were generated to sequence in multiplexing. PhiX DNA 1.5 pM was added to pooled libraries prior to sequencing at a final concentration of 10% in order to increase the sequence diversity of the libraries. Pooled small RNA libraries (1.7 pM) were sequenced using NextSeq™ 550 System (Illumina, San Diego, California, USA) following manufacturer’s instructions. NextSeq 500/550 High Output Kit v2.5 (75 Cycles) was used for sequencing in single reads of 75 pb fragments for small RNA library. This flow cell allows generating around 400 million reads per run, therefore 45 libraries per run were loaded to guarantee around 9 million reads per sample. Calculation of qualitative scores of the NGS runs (cluster density, Passing Filter clusters, % PF, and Q-score) was done with the Real-Time Analysis software (Illumina) and checked by using the Illumina Sequencing Analysis Viewer (Illumina). In our experiments, we obtained 10,313.55 ± 142.6 Kreads/sample, with an optimal cluster density (242.67± 5.03 K/mm2), high % PF (80.38 ± 1.12) and Q30 (Q-Score) with an average value of 91.93% ± 0.58. Finally, the data were collected as FastQ files.

Giannella A., Riccetti S., Sinigaglia A., Piubelli C., Razzaboni E., Di Battista P., Agostini M., Dal Molin E., Manganelli R., Gobbi F., Ceolotto G, & Barzon L. (2022). Circulating microRNA signatures associated with disease severity and outcome in COVID-19 patients. Frontiers in Immunology, 13, 968991.

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Tumor Mutational Burden Measurement

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Pre-treatment biopsy specimens were obtained by incisional biopsy (62 cases) or surgical resection (29 cases). TMB was measured by TruSight Oncology 500 (Illumina Inc., San Diego, CA, USA) as described previously [22 (link)]. Briefly, 40 ng of DNA was quantified with the Qubit dsDNA HS Assay (Thermo Fisher Scientific, Inc., Waltham, MA, USA) on the Qubit 2.0 Fluorometer (Thermo Fisher Scientific) and then sheared using a Coraris E220 focused-ultrasonicator (Woburn, MA, USA) and an 8 microTUBE–50 Strip AFA Fiber V2 following the manufacturer’s instructions. For DNA library preparation and enrichment, the TruSight Oncology 500 Kit (Illumina) was used following the manufacturer’s instructions. Post-enriched libraries were quantified, pooled, and sequenced on a NextSeq 500 (Illumina). The quality of the NextSeq 500 (Illumina) sequencing runs was assessed with the Illumina Sequencing Analysis Viewer (Illumina). Sequencing data were analyzed with the TruSight Oncology 500 Local App Version 1.3.0.39 (Illumina). TMB was reported as mutations per megabase (Mb) sequenced, and high TMB was defined as more than 10 mutations per Mb (≥10 Mut/Mb).

Lee H., Moon S.H., Hong J.Y., Lee J, & Hyun S.H. (2023). A Machine Learning Approach Using FDG PET-Based Radiomics for Prediction of Tumor Mutational Burden and Prognosis in Stage IV Colorectal Cancer. Cancers, 15(15), 3841.

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FFPE DNA Extraction and NGS Analysis for Oncology

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DNA was extracted from formalin-fixed and paraffin-embedded (FFPE) tumor tissue and non-lesional adjacent liver tissue using Maxwell CSC instrument (Promega, Madison, WI, USA) with the Maxwell RSC DNA FFPE kit (Promega, Madison, WI, USA) according to the manufacturer’s protocols; DNA concentrations were measured on a Qubit 2.0 fluorometer (Thermofisher Scientific, Waltham, MA, USA) using the Qubit dsDNA High Sensitivity Assay Kit.
For DNA library preparation and enrichment, the TruSight™ Oncology 500 Kit (Illumina) was used following the manufacturer’s instructions. Post-enriched libraries were quantified, pooled, and sequenced on a NextSeq 550 (Illumina Inc., San Diego, CA, USA). The quality of the NextSeq 550 (Illumina) sequencing runs was assessed with the Illumina Sequencing Analysis Viewer (Illumina). NGS data were analyzed with Illumina TruSight Oncology 500 Local App v2.1 [11 ], and variant report files were uploaded into the Pierian Clinical Genomics Workspace cloud (Pierian DX software CGW_V6.21.1).

Francalanci P., Giovannoni I., Tancredi C., Gagliardi M.G., Palmieri R., Brancaccio G., Spada M., Maggiore G., Pietrobattista A., Monti L., Castellano A., Giustiniani M.C., Onetti Muda A, & Alaggio R. (2024). Histopathological Spectrum and Molecular Characterization of Liver Tumors in the Setting of Fontan-Associated Liver Disease. Cancers, 16(2), 307.

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FFPE DNA Shearing, Library Prep, and NGS Sequencing

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40 ng DNA were quantified with the Qubit dsDNA HS Assay (Thermo Fisher Scientific) on the Qubit 2.0 Fluorometer (Thermo Fisher Scientific) and sheared on the Covaris E220 Focused-ultrasonicator (Woburn, MA, USA) using the 8 microTUBE–50 Strip AFA Fiber V2 following manufacturer’s instructions. The treatment time was optimized for FFPE material. The treatment settings were the following: Peak Incident Power (W): 75; Duty Factor: 15%; Cycles per Burst: 500; Treatment Time (s): 360; Temperature (°C): 7; Water Level: 6. For DNA library preparation and enrichment the TruSight Oncology 500 Kit (Illumina) was used following manufacturer’s instructions. Post-enriched libraries were quantified, pooled and sequenced on a NextSeq 500 (Illumina Inc., San Diego, CA, USA).
Quality of the NextSeq 500 (Illumina) sequencing runs were assessed with the Illumina Sequencing Analysis Viewer (Illumina). Sequencing data was analyzed with the TruSight Oncology 500 Local App Version 1.3.0.39 (Illumina).

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Differential Gene Expression in PD Blood

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Based on the data from whole transcriptome RNA sequencing, gene expression of 24 samples, including 12 blood samples of PD and 12 blood samples of controls were analyzed using Illumina Sequencing Analysis Viewer (Illumina, San Diego, USA). The DEGs were first screened with the restriction of |log2 (Fold-Change)| > 0.5 and p < 0.05 [14 (link)–16 (link)].

Wu Y., Bai Y., Lu Y., Zhang Z., Zhao Y., Huang S., Tang L., Liang Y., Hu Y, & Xu C. (2023). Transcriptome sequencing and network pharmacology-based approach to reveal the effect and mechanism of Ji Chuan Jian against Parkinson’s disease. BMC Complementary Medicine and Therapies, 23, 182.

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Tumor Mutational Burden Profiling

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40 ng DNA quantified with the Qubit dsDNA HS Assay (Thermo Fisher Scientific) on the Qubit 2.0 Fluorometer (Thermo Fisher Scientific) was used for library preparation with the QIAseq Human Tumor Mutational Burden Panel (Qiagen, Hilden, Germany) according to manufacturer’s instructions. Final libraries were quantified Qubit dsDNA HS Assay (Thermo Fisher Scientific), pooled and sequenced on a NextSeq 500 (Illumina).
Quality of the NextSeq 500 (Illumina) sequencing runs were assessed with the Illumina Sequencing Analysis Viewer (Illumina). Sequencing data was analyzed with ‘Identify QIAseq DNA Somatic Variants with TMB Score (Illumina)’ v1.47 in the plugin ‘Biomedical Genomics Analysis v 1.2′ on the CLC Genomics Workbench v12.0.2 (Qiagen).
In addition to the Qiagen software, we also analyzed the data with our in-house pipeline (see description above) with minor modifications regarding the extraction of the umi (unique molecular index). Due to the different chemistry for library preparation, we also sequenced 15 normal samples independent from tumors that served as a panel of normal.
Variant annotation for filtering was done with Mutect2 FilterMutectCalls. Read_position and strand_artifact filter flags were removed for subsequent analysis. Further we employed the LearnReadOrientationModel of GATK to filter strand biases.

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Illumina Shotgun Metagenomics Sequencing Protocol

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Illumina shotgun metagenomics sequencing was performed at the Next Generation Sequencing Platform, University of Bern. The extracted DNA was assessed for quantity, purity, and length using a Thermo Fisher Scientific Qubit 4.0 fluorometer with the Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific, Q32854), a DeNovix DS-11 FX spectrophotometer, and an Agilent FEMTO Pulse System with a Genomic DNA 165 kb Kit (Agilent, FP-1002-0275), respectively. Sequencing libraries were made using an Illumina DNA Prep Library Kit (Illumina, 20,018,705) in combination with IDT for Illumina DNA/RNA UD Indexes Set B, Tagmentation (Illumina, 20,027,214) according to the Illumina DNA Prep Reference Guide (Illumina, 10,000,000,254 16v09). Six PCR cycles were employed to amplify 30 ng of tagemented DNA. Pooled DNA libraries were sequenced paired-end on a NovaSeq 6000 SP Reagent Kit v1.5 (300 cycles; Illumina, 20,028,400) on an Illumina NovaSeq 6000 instrument. The run produced, on average, 159 million reads/sample. The quality of the sequencing run was assessed using Illumina Sequencing Analysis Viewer (Illumina version 2.4.7) and all base call files were demultiplexed and converted into FASTQ files using Illumina bcl2fastq conversion software v2.20.
Raw data from Illumina shotgun metagenomics sequencing were uploaded to the SRA NBI databank.

Rieder J., Kapopoulou A., Bank C, & Adrian-Kalchhauser I. (2023). Metagenomics and metabarcoding experimental choices and their impact on microbial community characterization in freshwater recirculating aquaculture systems. Environmental Microbiome, 18, 8.

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