Ion torrent s5 system

Genomic DNA of BC, OC and PC patients were collected using buccal swabs and extracted through MagPurix instrument and Forensic DNA Extraction Kit (Zinexts Life Science Corp.- CodZP01001) according to the manufacturer’s protocol. NGS was executed by the Ion Torrent S5 system (Thermo Fisher Scientific, Waltham, MA, United States) after automatic library preparation using Ion Chef (Thermo Fisher Scientific, Waltham, MA, United States). Ion Chef consists of fragmentation and adapter ligation onto the PCR products, clonal amplification. The DNA libraries were quantified with Real-Time Step One PCR System (Thermo Fisher Scientific, Waltham, MA, United States) and the prepared samples were loaded onto an Ion 530™ chip by Ion Chef (Thermo Fisher Scientific, Waltham, MA, United States). Ion S5™ Plus (Thermo Fisher Scientific, Waltham, MA, United States) instrument was used for the sequencing. Specific plugins as “SampleId” and “Coverageanalysis” were used for NGS data analysis on the Torrent Suite 5.14.0 platform. The uniformity of base coverage was over 98% in all batches, and base coverage was over ×20 at all target regions. This NGS method cannot detect variations outside the +/−10 nucleotide coding sequence.

For whole viral genome sequencing, total RNA was reverse transcribed using Invitrogen SuperScript VILO^TM cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA). One hundred and seven samples were analyzed in eight different sequencing runs using the Ion Torrent S5 system (Thermo Fisher Scientific, Waltham, MA, USA) after library preparation, consisting of fragmentation and adapter ligation onto the PCR products and clonal amplification. cDNA libraries were then prepared using the Ion AmpliSeq SARS-CoV-2 Research Panel (Thermo Fisher Scientific, Waltham, MA, USA). After quantification of cDNA libraries with Real-Time Step One PCR System (Thermo Fisher Scientific, Waltham, MA, USA), the prepared samples of ion sphere particles (ISP) were loaded onto an Ion 520™ chip with the Ion Chef (Thermo Fisher Scientific, Waltham, MA, USA). Sequencing was performed using the Ion S5™ sequencing reagents (Thermo Fisher Scientific, Waltham, MA, USA). The Torrent Suite 5.14.0 platform and specific plugins were used for NGS data analysis. All analysed sequences showed an alignment accuracy of over 96% and a base coverage over 20× (Figure 1). The pangolin software was used for the assignment of SARS-CoV-2 lineages. All sequences were then submitted as FASTA files on gisaid.org, which provides open access to genomic data on SARS-CoV-2.

Samples treated with two ANE doses were harvested 24h and 48h after treatment for RNA-Seq analysis together with controls.
Two leaf disks were collected around the mid-vein of the distal leaflets of the most recently fully expanded leaf below the nearest inflorescence, from four different plants for each experimental condition. Messenger RNA was directly isolated from frozen and powdered leaf disk pools using the Dynabeads mRNA Direct Micro Kit (Thermo Fisher Scientific, Carlsbad, CA) following the manufacturer’s instruction. The concentration and quality of mRNA were assessed by an Agilent 4150 TapeStation system (Agilent Technologies, USA). Sequencing libraries were prepared from a range of 10-50 ng of poly(A) RNA using Ion Total RNA-Seq Kit v2 (Thermo Fisher Scientific) following the manufacturer’s protocol. The final double-stranded barcoded cDNA libraries were eluted in 15 µl of nuclease-free water. The concentration and size distribution were quantified through D1000 screen Tape (Agilent Tapestation 1500), normalized to get a molar concentration of 100pM, pooled, and sequenced using three Ion 540™ Chips on the Ion Torrent S5 System (Thermo Fisher Scientific).

Next-generation sequencing of the DNA isolated from the tumor tissue and organoids was carried out with the Oncomine Comprehensive Assay V3 (Thermo Fisher Scientific, United States) based on the Ion Torrent S5 System (Thermo Fisher Scientific, United States). Snap-frozen tumor tissue and organoids were used for isolation of DNA. The alignment was carried out on the basis of the GRCh37 genomic assembly. Single nucleotide variants (SNV) and small insertions and deletions (indel) as well as larger genome rearrangements were identified by Atlas Oncology Diagnostics (Ivanov et al., 2019 (link)).

Total RNAs were extracted, purified, and eluted from Formalin-Fixed Paraffin-Embedded (FFPE) tissue sections by the truXTRAC™ FFPE RNA Kit (Covaris, Inc., Woburn, MA) and then quantified using the Quant-iT RNA HS Assay Kit (Thermo Fisher Scientific, Waltham, MA). RNA-Seq libraries were generated using the Ion AmpliSeq™ targeted sequencing system (Thermo Fisher Scientific, Waltham, MA). A total of 395 genes were quantified, including 10 housekeeping (HK) genes as controls. After reverse transcription into complementary DNA (cDNA), the barcode adapter was ligated to the partially digested amplicon. Equimolar libraries were pooled after purification and normalization and subjected to the Ion Chef™ system (Thermo Fisher Scientific) for enrichment and template preparation. Afterwards, 200 bp sequencing was performed to obtain mapped reads (approximately 2-3 million per sample) using the Ion Torrent S5 system (Thermo Fisher Scientific). Next-generation sequencing (NGS) data were subsequently analyzed using Ion Torrent Suite software version 5.2.0 (Thermo Fisher Scientific) for NGS read alignment, reference mapping, variant calling, and data management. NGS read quality control and quality assurance were conducted using standardized criteria [15 (link)]. Reads of HK genes were used for gene expression normalization. Finally, normalized reads per million (nRPM) were log2-transformed.

DNA from the fresh-frozen pulverized tumor tissue was isolated using a DNEasy Blood and Tissue Kit (Qiagen, Venlo, The Netherlands). Targeted next-generation sequencing with a mean coverage of 500X coverage was performed on the Ion Torrent S5 system (ThermoFischer Scientific) using a custom next-generation sequencing (NGS) panel based on the Ion Ampliseq™ Cancer Hotspot Panel targeting mutational hotspots of 64 cancer-related genes. Variants with an allele frequency of at least 5% were reported. Variant call files were generated and analysis was performed using Alissa (Agilent Technologies Alissa Interpret v5.1.7).