Ion personal genome machine system

The Oncomine™ Solid Tumour DNA kit (OST, Thermo Fisher Scientific), a CE-IVD-marked version of the Ion Ampliseq Colon and Lung Cancer Research Panel v2 (referred to as the Colon and Lung panel), was used to prepare sequencing libraries from cfDNA (1.1-10 ng). Sample preparation was performed using the Ion Chef™ Instrument, and sequencing was conducted on the Ion Personal Genome Machine® (PGM™) System (both Thermo Fisher Scientific, Watham, MA; USA). Each Ion 316™ v2 BC chip was loaded with eight samples. If samples did not meet the criteria of mean depth ≥ 2000, they were disqualified. Variants were called if they were exonic, previously observed, reported to COSMIC, and if the allele frequency ≥ 1% [14 (link)]. Benign SNPs were not reported. All variants were visualized and manually inspected using the Integrative Genomics Viewer [31 (link)]. The data analysis is described in Supplementary File 1.
The performances of each of the 92 amplicons were evaluated by investigating the mean coverage over the 36 patient samples successfully sequenced. Five amplicons generally performed poorly and did not meet the defined mean coverage (CHP2_ERBB4_1, CHP2_PTEN_2, ON_DDR2_3, CHP2_AKT1_1, CHP2_NOTCH1_1). These were excluded from further analyses. The majority of the sequence data has been previously published [9 (link)].

RNA was extracted from IECs using a miRNeasy Mini Kit (Qiagen, Germantown, MD, USA). Library construction and sequencing of small RNAs (including miRNAs) were achieved by using an Ion Total RNA-Seq Kit v2 (Thermo Fisher Scientific, Waltham, MA, USA) and the Ion Personal Genome machine (PGM) system (Thermo Fisher Scientific) according to the manufacturer’s instructions at the Mie University Center for Molecular Biology and Genetics (Tsu, Japan) as previously described [30 (link)]. Data collection was performed with Torrent Suite v4.0.1 software. The assessment of miRNA profiling was conducted as previously described [30 (link)]. In brief, detectable miRNAs (>0, RPKM) across all samples were chosen for differential expression and downstream pathway analysis [32 (link)]. Individual fold changes (RPKM in CS-injected sepsis mouse/RPKM in sham mouse) were calculated by taking the ratio of the candidate miRNA expression values with one sham control. Those miRNAs and mRNAs with a fold change of 2 or greater (FC > 2) were classified as upregulated miRNAs and mRNAs in the sepsis group compared to sham, while those with (FC < −2) were classified as downregulated miRNAs and mRNAs in the sepsis group compared to sham.

Panel-based next-generation sequencing was performed following the manufacturer's instructions. Briefly, 10 ng of genomic DNA were used for preparation of barcoded PCR-libraries using the comprehensive cancer panel. Libraries (100 pM of each primer pool per cell line) were mixed, amplified and bead-coupled by emulsion PCR using the OneTouch 2 system. Sequencing of duplexed samples was performed on the Ion Personal Genome Machine (PGM) System and a 318v2 chip (all equipment from Thermo Fisher Scientific, Waltham, MA, USA). Variant call files were analyzed by our in-house pipeline and mutations were selected regarding prediction of potential impact on protein function by algorithms SIFT/PolyPhen^{54 (link), 55} and MutationTaster.^{56 (link)} Analysis of CNV was performed with R package CNVPanelizer using BAM/BAI sequencing output files. Determination of CNVs of resistant MeWo cells was referenced to sensitive (MeWo^Par) cells.

The genomic library was diluted to 100 pM. The concentration was measured on the Qubit™ 2.0 Fluorometer using Qubit™ dsDNA HS Assay Kit (Pub. No. MAN0002326 Revision: B.0) (Life Technologies). The P. uncinata template preparation was performed according to protocol: Ion PGM™ Hi-Q™ View OT2 Kit (Cat. No. A29900, Pub. No. MAN0014580 Rev. C.0). P. mugo and P. rotundata templates preparation were performed according to protocol: Ion 540™ Kit – OT2 (Cat. No A27753 Pub. No. MAN0010852 Rev. E.0). Evaluation of the templated Ion Sphere™ Particles (ISPs) was conducted using Ion Sphere™ Quality Control Kit (Cat.No. 4468656), according to protocol Ion Sphere™ Assay on the Qubit ™ 2.0 Fluorometer (Pub. No. MAN0016387 Revision A.0) (ThermoFisher Scientific, Waltham, MA, USA). P. uncinata genome sequencing was conducted on Ion 318™ Chip v2 BC by Ion Personal Genome Machine™ (PGM™) System (Thermo Fisher Scientific, Waltham, MA, USA) according to manufacturer’s recommendations using protocol: Ion PGM™ Hi-Q™ View Sequencing Kit user guide (Cat. No. A30044, Pub. No. MAN0014583). Then, P. mugo and P. rotundata genome sequencing was conducted on Ion 540™ Chip by GeneStudio™ S5 System (Thermo Fisher Scientific, Waltham, USA) according to manufacturer’s recommendations using protocol: Ion 540™ Kit – OT2 User Guide (Cat. No A27753, Pub. No MAN0010850, Rev. D).

HTS for the round 9 and 11 fentanyl pools, round 8 and 10 acetyl fentanyl pools, and round 7 and 10 furanyl fentanyl pools was performed at FIU’s DNA Core Facility with an Ion Personal Genome Machine System with an Ion 318 v2 chip (Thermo Fisher Scientific). To prepare samples for sequencing, the library pool (final concentration: 10 nM) was mixed with GoTaq Hot Start Colorless Master Mix, forward primer (final concentration: 1 μM) and reverse primer (final concentration: 1 μM) and diluted with PCR-quality water to a final volume of 50 μl. Nine cycles of PCR were performed using the following conditions: 2 min at 95°C; 9 cycles of 95°C for 15 s, 58°C for 30 s and 72°C for 45 s; and finally 5 min at 72°C. 40 μl of PCR product was added into 16 μl of ExoSAP-IT reagent in an ice bath. The mixture was then incubated at 37°C for 15 min to degrade remaining primers and dNTPs, followed by incubation at 80°C for 15 min to inactivate the ExoSAP-IT reagent. Upon obtaining the sequencing data, the primer sequences were trimmed by cutadapt, (29 ) and the abundance of sequences from each pool and enrichment between rounds were calculated using FASTAptamer. (30 (link))