Genomic alterations in cfDNA were analyzed by the Oncomine™ Pan-Cancer Cell-Free assay that is designed to detect single-nucleotide variants (SNV), insertions/deletions (indel), copy number variants (CNV), and gene fusions across 52 genes. Target regions from cfDNA were amplified using the Oncomine™ cfDNA Assay (Thermo Fisher Scientific). Library construction of the amplicons was performed according to the Oncomine cfDNA Assays Part I: Library Preparation or Oncomine Cell-Free Research Assay (Thermo Fisher Scientific) User Guide. Template preparation and chip loading were conducted with the Ion 530 Kit-Chef (Thermo Fisher Scientific). The Ion 530 Kit-Chef was used with the Ion S5 XL sequencer (Thermo Fisher Scientific), as described in the Ion 530 Kit-Chef User Guide.
Ion s5 xl sequencer
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Ion S5 XL Sequencer is a high-performance DNA sequencing instrument designed for a wide range of applications. It utilizes semiconductor-based sequencing technology to generate sequencing data. The Ion S5 XL Sequencer is capable of producing sequencing reads and supports multiple sample processing.
Automatically generated - may contain errors
Lab products found in correlation
Ion chef system, by Thermo Fisher Scientific (10 mentions)
Ion chef instrument, by Thermo Fisher Scientific (7 mentions)
Ion library taqman quantitation kit, by Thermo Fisher Scientific (6 mentions)
Ion 540 chip, by Thermo Fisher Scientific (4 mentions)
Ion ampliseq library kit 2, by Thermo Fisher Scientific (3 mentions)
Ion 540 kit chef, by Thermo Fisher Scientific (3 mentions)
Torrent suite software, by Thermo Fisher Scientific (2 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (2 mentions)
Ion s5 sequencing kit, by Thermo Fisher Scientific (2 mentions)
Ion ampliseq technology, by Thermo Fisher Scientific (2 mentions)
Ion 520 chip, by Thermo Fisher Scientific (2 mentions)
Ion 520 kit chef kit, by Thermo Fisher Scientific (2 mentions)
Qubit 3.0 fluorometer, by Thermo Fisher Scientific (2 mentions)
Ion 530 chip, by Thermo Fisher Scientific (2 mentions)
Ion ampliseq library kit plus, by Thermo Fisher Scientific (2 mentions)
2100 bioanalyzer, by Agilent Technologies (2 mentions)
Oncomine immune response research assay, by Thermo Fisher Scientific (2 mentions)
Superscript vilo cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Ion 530 kit chef, by Thermo Fisher Scientific (1 mentions)
Ampliseq rna plugin, by Thermo Fisher Scientific (1 mentions)
39 protocols using ion s5 xl sequencer
1
Oncomine Pan-Cancer Cell-Free Sequencing
2
RNA-Seq Data Analysis Pipeline
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All sequencing data was processed on Ion S5xl Sequencer (Thermo Fisher Scientific, Waltham, MA, USA) and transferred to the Ion Proton™ Torrent Server for primary data analysis with gene-level transcript quantification from sequence read data performed using AmpliSeq RNA Plug in (ver 5.6.0.3) by Torrent Suite Software (Thermo Fisher Scientific, Waltham, MA, USA). Identification of up or down-regulated genes was performed using the Excel-based Differentially Expressed Gene Analysis software (ExDEGA version 1.6.3, e-Biogen, Seoul, Korea). The DEG list was filtered using a 5% false discovery rate (FDR) cutoff and |Fold change (FC)| > 2.0 for upregulated and downregulated genes. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG, Kyoto, Japan) Pathway Enrichment analysis for OTA was executed using the database for annotation, visualization, and integrated discovery (DAVID, Frederick, MA, USA) version 6.8 (https://david.ncifcrf.gov , accessed on 10 January 2021) functional annotation system [72 (link)]. The gene set enrichment analysis (GSEA; Broad Institute) software platform (MSigDB version 6.1, Massachusetts, CA, USA) was used to identify different expression pathways from control cells in OTA-treated cells by comparing them with hallmark gene sets representing specific well-defined biological states [73 (link)].
3
NGS Acute Leukemia Panel Analysis
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NGS was performed using a St. Mary’s customized NGS panel for acute leukemia, i.e., the “SM Acute leukemia panel”. Ion AmpliSeq Technology was used to amplify 67 genes (Supplementary Table S1 ) using an Ion Chef™ system and an Ion S5 XL Sequencer (all from Thermo Fisher Scientific, Waltham, MA, USA) [20 (link)]. Sequenced reads were mapped to the human reference genome (hg19, Genome Reference Consortium, February 2009). Annotated variants were classified into four tiers according to the Standards and Guidelines of the Association for Molecular Pathology [21 (link)]. Bioinformatics analysis was carried out using both customized and manufacturer-provided pipelines. Variants were selected and annotated using analytics algorithms and public databases [22 (link)]. All mutations were manually verified using the Integrative Genomic Viewer [23 (link)]. Among the 139 patients, 106 patients and 19 patients were analyzed with samples at diagnosis and at relapse, respectively, while 14 patients were analyzed with both samples.
4
Automated Multiomics Workflow for Biomarker Discovery
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A 150 m2 fully equipped laboratory supports the pivotal work on the samples collected for our project. All procedures of platelet separation and plasma processing, cfDNA and RNA extraction, quality control, amplification, library preparation and sequencing happen in separate, clean environments optimised for such tasks. Every operation, from sample processing, to nucleic acid extraction and sequencing, is semi-automated in order to minimise cross-sample contamination and ensure optimal reproducibility and consistency of operations. A dedicated freezer is available in the laboratories for sample storage. The laboratory is equipped with a ThermoFisher Scientific Ion S5 XL sequencer. Data collection and the dry-lab part of the analyses are performed on high performance workstations maintained in high-security, dedicated environments to avoid the risk of data loss and sensible data cyber-theft.
5
Ion RNA-seq Library Preparation and Sequencing
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An Ion Total RNA-Seq Kit v2 was used to prepare a reconstructive cDNA library for preparation of small RNA sequencing. The size and concentration of base pairs of the cDNA library were measured with an Agilent 2100 Bioanalyzer (Agilent Technologies). Preparation for deep sequencing such as emulsion PCR, bead enrichment, and chip loading were automatically performed on an Ion Chef− instrument (Thermo Fisher Scientific). In the final step of sample preparation for sequencing, the chip was loaded with the Ion Sphere Particle (ISP) sequencing reaction mixture. Synthesized templates were sequenced on an Ion S5−XL sequencer (Thermo Fisher Scientific) using an Ion 540− chip.
6
Oncomine Immune Response Assay
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Only tissue samples derived from primary tumors were sequenced, not considering lymph node-derived samples. Briefly, 10 ng of input RNA was reverse transcribed into cDNA using the SuperScript IV VILO Master Mix (Cat. No. 11756050), and libraries were prepared using the Ion AmpliSeq Library Kit 2.0 (Life Technologies Cat. No. 4475345) and the Oncomine Immune Response Research Assay (Cat. No. A31930) panel that targets 395 genes related to immunological processes (online supplemental table S2 ). Equal volumes from eight samples at 50 pM were pooled together for sequencing on an Ion 530 Chip. Template preparation was performed using an Ion Chef System (Thermo Fisher Scientific) and Ion 520 and Ion 530 Kit (Thermo Fisher Scientific). Sequencing was performed on an Ion S5 XL Sequencer (Thermo Fisher Scientific). The ImmuneResponse RNA plugin was used to align the sequences to the reference genome (ImmuneResponse_v3.1) and to count the sequencing reads with the Torrent Suite software.
7
RNA-Seq Analysis of Porcine Myocardium
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A total of 400 ng of extracted RNA from the remote LV myocardium was used for RNA sequencing after ribosomal and mitochondrial RNA depletion. Ion Torrent sequencing libraries were prepared, and sequencing was performed in an Ion S5XL™ sequencer (Thermo-Fisher). On average, about 15 million reads were obtained for each sample. Sample read quality was determined using FASTQC, low quality sequences were dropped (Phred score <30) and Trimmomatic was used to remove adapters (21 (link)). Resulting reads were aligned to the reference pig genome (Sus_scrofa.Sscrofa11.1.92) with HISAT2 (22 (link)). FeatureCounts was used to obtain gene counts in Galaxy online software (23 (link)). Differentially expressed gene (DEG) analysis was performed using edgeR in Galaxy (ver.3.36.0), using DEG selection criteria of [log2(Fold Change)] >1 or <−1 and false-discovery rate (FDR) <0.05. Gene ontology analysis was performed using Enrichr (GO Biological Process 2021) (24 , 25 (link)). Principal coordinate analysis (PCA) coordinates were obtained in Galaxy using ggplot2 (ver. 3.3.5). Expression heatmaps were assembled using Heatmapper online software (26 (link)), using complete clustering and Pearson distance measuring method.
8
Oncomine Tumor Mutation Load Assay
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The Oncomine™ Tumor Mutation Load Assay is a PCR-based target enrichment next-generation sequencing assay performed on the Ion Torrent platform. The panel covers 1.65 Mb with 1.2 Mb of exonic bases across 409 oncogenes relevant across major cancer types. DNA was extracted from formalin-fixed paraffin-embedded tissue using the RecoverAll Multi-Sample RNA/DNA Workflow kit (Invitrogen, Carlsbad, CA, USA), following the manufacturer’s protocol. Genomic DNA was quantified by quantitative real-time reverse transcription (RT)-PCR using a TaqMan® RNase P Detection Reagents kit (Applied Biosystems, Foster City, CA, USA). The Libraries were prepared by automation using The Ion AmpliSeq™ Kit for Chef DL8 (Ion Torrent, Thermo Fisher Scientific). Library preparation and templating were performed on the Ion Chef using an automated Ion AmpliseqTM Kit for Chef DL8 (Ion Torrent) and Ion 540 chip - Chef kit (Ion Torrent) respectively. Sequencing was performed on the Ion S5™XL Sequencer using an Ion S5™sequencing kit (Thermo Fisher Scientific) with Torrent Suite software (Version 5.10). Variants were identified using the Ion Torrent Variant Caller plug-in (Version 5.10) and annotated using Ion Reporter software (Version 5.10).
9
Ion Torrent Sequencing Workflow
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Emulsion PCR, bead enrichment, and chip loading procedures were automatically performed on an Ion Chef instrument (Thermo Fisher Scientific) using Ion 510, 520, and 530 Kits (Thermo Fisher Scientific). A planned run was created for each chip within Ion Torrent Suite Software v5.12.1 (Thermo Fisher Scientific) with the template size set at 200 bp. The NGS libraries were then sequenced on an Ion S5 XL sequencer (Thermo Fisher Scientific) [18 (link)].
10
NGS-based Acute Leukemia Variant Analysis
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NGS analysis was performed using St. Mary’s customized NGS panel for acute leukemia, i.e., the “SM Acute leukemia panel.” Ion AmpliSeq Technology (Thermo Fisher Scientific) was used to amplify 67 genes (Supplementary Table S2 ) using an Ion Chef™ system (Thermo Fisher Scientific) and an Ion S5 XL Sequencer (Thermo Fisher Scientific)16 (link).
Annotated variants were classified into four tiers according to the Standards and Guidelines of the Association for Molecular Pathology17 (link). Bioinformatics analysis was carried out using both customized and manufacturer-provided pipelines. Variants were selected and annotated using analytics algorithms and public databases18 (link). Subsequently, trackable somatic mutations specific to each patient were selected. For NGS-MRD, we carefully inspected the mutations and determined the residual variant allele fraction (% VAF), which was calculated by dividing the number of mutant sequencing reads with the number of total sequencing reads. All mutations were manually verified using the Integrative Genomic Viewer19 (link). Across all time points, the mean of on-target reads, depth of on-target regions, and uniformity were 99.4%, 2406×, and 96.9%, respectively. Details of the quality control matrices are summarized in Supplementary TableS3 .
Annotated variants were classified into four tiers according to the Standards and Guidelines of the Association for Molecular Pathology17 (link). Bioinformatics analysis was carried out using both customized and manufacturer-provided pipelines. Variants were selected and annotated using analytics algorithms and public databases18 (link). Subsequently, trackable somatic mutations specific to each patient were selected. For NGS-MRD, we carefully inspected the mutations and determined the residual variant allele fraction (% VAF), which was calculated by dividing the number of mutant sequencing reads with the number of total sequencing reads. All mutations were manually verified using the Integrative Genomic Viewer19 (link). Across all time points, the mean of on-target reads, depth of on-target regions, and uniformity were 99.4%, 2406×, and 96.9%, respectively. Details of the quality control matrices are summarized in Supplementary Table
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