After HCMV-BAC preparation, amplicons were purified using magnetic beads (Agencourt AMPure XP) and fragmented using the Ion Xpress Plus DNA Fragment Library Preparation kit (Life Technologies). Barcodes adapters were ligated to fragment ends and 250 bp fragments were collected. The library was PCR amplified, then sequenced on the Ion Proton with the Ion Sequencing kit (Life Technologies). Bases callings were performed with Torrent Suite Software version 5.0.2. Mutations were obtained using Torrent Variant Caller using Somatic variant frequency and AD169_ATCC as reference. Mutations were then filtered against reference (Wild-type HCMV-BAC) using vcftools version 0.1.13.
Ion proton
Manufactured by Thermo Fisher Scientific
Sourced in United States, Canada
The Ion Proton is a next-generation sequencing (NGS) system designed for high-throughput DNA sequencing. It utilizes semiconductor-based technology to perform sequencing-by-synthesis, enabling rapid and accurate DNA analysis. The Ion Proton system is capable of generating high-quality sequence data for a wide range of applications, including genome sequencing, transcriptome profiling, and targeted gene panels.
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Lab products found in correlation
Ion onetouch 2 system, by Thermo Fisher Scientific (7 mentions)
Fupa reagent, by Thermo Fisher Scientific (6 mentions)
Ampliseq hifi mix, by Thermo Fisher Scientific (5 mentions)
Ion onetouch es, by Thermo Fisher Scientific (5 mentions)
Ion plus fragment library kit, by Thermo Fisher Scientific (5 mentions)
Ion total rna seq kit v2, by Thermo Fisher Scientific (5 mentions)
Ion library quantification kit, by Thermo Fisher Scientific (4 mentions)
Ion chef, by Thermo Fisher Scientific (4 mentions)
Ion onetouch system, by Thermo Fisher Scientific (4 mentions)
Miseq platform, by Illumina (3 mentions)
Ion proton 200 sequencing kit, by Thermo Fisher Scientific (3 mentions)
Ion pi sequencing 200 kit v3, by Thermo Fisher Scientific (3 mentions)
Ion pi template ot2 200 kit v3, by Thermo Fisher Scientific (3 mentions)
Exome primer pools, by Thermo Fisher Scientific (3 mentions)
Torrent suite software v 5, by Thermo Fisher Scientific (3 mentions)
Clc genomics workbench, by Qiagen (3 mentions)
Ion pi chip kit v2 bc, by Thermo Fisher Scientific (2 mentions)
Tumor mutation hotspots panel version 2, by Thermo Fisher Scientific (2 mentions)
Ion proton, by Illumina (2 mentions)
Ion pi template ot2 200 kit, by Thermo Fisher Scientific (2 mentions)
106 protocols using ion proton
1
HCMV-BAC Sequencing and Variant Calling
2
NGS-Based Molecular Analysis of Thyroid FNA
3
Genetic Screening for Charcot-Marie-Tooth Disease
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For the second series, we performed mutation screening of 60 known or candidate CMT‐related genes using the Illumina Miseq platform (Illumina Inc., San Diego, CA, USA) following the protocol described previously by Maeda et al.20 Subsequently, we performed mutation screening of other known CMT and IPN genes, including the MME gene, using WES (Fig 2 ). For the third series, we performed targeted resequencing of 72 known or candidate CMT‐related genes, including the MME gene, using a custom Ion AmpliSeqM panel (Life Technologies, Carlsbad, CA) (Fig 2 and Supplementary Table 1). Briefly, a custom panel targeting 72 genes was designed using the Ion AmpliSeq Designer tool (http://www.ampliseq.com ). Library and template preparation was performed according to manufacturer's instructions. Sequencing was performed on the Ion Proton (Life Technologies) using the Ion PI Chip kit v2 BC. Sequence data were aligned and mapped to the reference sequence, and variants were called using the Torrent variant caller. Variants were transferred to the CLC Genomics Workbench 6 software program (CLC bio, Aarhus, Denmark) for annotation and filtering.
4
Genomic characterization of Bacillus licheniformis
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Genomic DNA of Bacillus licheniformis GL174 was extracted using UltraClean® Microbial DNA Isolation Kit (MoBio, Solana Beach CA, USA) from 5 mL of an overnight culture. Afterwards, Genomic DNA was fragmented and sequenced using ION Proton (Life technologies©) sequencing technology. Genome assembly was performed with the Newbler program.
Sequencing data, assembly and gene prediction were submitted to a public database and are available at BioProject database (http://www.ncbi.nlm.nih.gov/bioproject/ ) with accession number PRJNA274883. The gene annotation process was performed using the annotation pipeline implemented in the BASys bacterial annotation system (https://www.basys.ca /) so that all the coding sequences were assigned to a COG (Cluster of Orthologs) functional class. In addition, the identified coding sequences were compared with the InterPro database (https://www.ebi.ac.uk/interpro/ ) for double annotation of the protein functions. Among all the identified protein functions, we isolated the sequences related to chemotaxis and motility, plant wall degrading enzymes and plant colonization, iron nutrition and metabolism, phosphate nutrition and metabolism, nitrogen uptake and metabolism, lipopeptides and other secondary metabolites biosynthesis and oxidative stress response.
Sequencing data, assembly and gene prediction were submitted to a public database and are available at BioProject database (
5
Mutational Analysis of Liver Metastases
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To investigate mutational pattern of different liver metastases, we examined biomarkers (single-nucleotide polymorphism, SNP) through genome-wide exploration using NGS. To preliminarily select genes to construct a custom panel for target capture sequencing, we performed WES for 10 triplets, each comprising primary colorectal tumor and normal colorectal mucosa and matched liver metastases. Genomic DNA from fresh tissue was samples sequenced on an Ion™ Proton (Life Technologies, Carlsbad, CA, USA) platform according to the manufacturer’s instructions. Normal colorectal mucosa was sequenced to exclude germline variants. The read alignments and variant analyses were performed according to the predefined workflow.
We constructed a custom panel of 96 genes selected based on driver mutations identified using WES and Tumor Mutation Hotspots Panel version 2 (Life Technologies, Carlsbad, CA, USA). Genomic DNA from FFPE tissue samples of patients in both cohorts was subsequently sequenced for SNPs using an Ion™ Torrent Personal Genome Machine (PGM) according to the manufacturer’s instructions. For a given gene loci, the fraction of mutant alleles was calculated by diving the number of mutant reads by the number of total reads. A 5% cutoff value was employed. A sample was considered wild-type for a given gene when all sequenced loci harbored <5% mutant alleles.
We constructed a custom panel of 96 genes selected based on driver mutations identified using WES and Tumor Mutation Hotspots Panel version 2 (Life Technologies, Carlsbad, CA, USA). Genomic DNA from FFPE tissue samples of patients in both cohorts was subsequently sequenced for SNPs using an Ion™ Torrent Personal Genome Machine (PGM) according to the manufacturer’s instructions. For a given gene loci, the fraction of mutant alleles was calculated by diving the number of mutant reads by the number of total reads. A 5% cutoff value was employed. A sample was considered wild-type for a given gene when all sequenced loci harbored <5% mutant alleles.
6
Genomic Sequencing and Variant Analysis
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Genomic DNA from the B371 and B448 isolates, as well as the two genomic DNA pools generated from the progeny in cross B371 x B448, were sequenced using the Ion Proton (Life Technologies) sequencer by Bioarray (Alicante, Spain). Reads (mean length 163-180 nt) were mapped on the ASM15095 v229 Botrytis cinerea B05.10 assembly1 using the Torrent Mapping Alignment Software (TMAP). Average coverage was 100× (with genome base coverage at 20× higher than 95% in all the samples). Torrent Variant Caller (TVC) software was used to obtain sequence variants and their frequencies, and SnpEff v4.2 software was implemented to annotate these variants and the impact of mutations in coding sequences.
7
Differential Expression of lncRNAs in Cancer Tissue
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The RNA with integrity number >7.0 was optimum for cDNA library construction. The cDNA libraries were then processed for the Proton Sequencing process according to the commercial protocols. Next, Ion Proton (Life Technologies) was used to perform single-end and polyA-selection sequencing of the three pairs of samples. The MapSplice program (v2.1.6) was used to align clean reads to the human genome (version: GRCH37). We quantified the lncRNA expression as Reads Per Kilobase per Million mapped reads (RPKM) and lncRNAs with sum read counts <10 across all samples were abandoned. EBseq algorithm was used to identify differentially expressed lncRNAs between cancer tissues and adjacent non-cancerous tissues. Differences in RNA expression were regarded as significant if values of the false discovery rate were <0.05 and the fold change was >2. All differentially expressed lncRNAs are listed in Table S1 .
8
Targeted Sequencing of SNP Variants
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Individual primer pairs were designed using Batch Primer 3 (http://probes.pw.usda.gov/batchprimer3/ ) at default settings for generic primers with total amplicon size set as an optimum of 100bp with the amplified region containing the target SNP (S1 Table ). The primer sequences and genomic DNA were submitted to Floodlight Genomics (FG, Knoxville, TN) for targeted sequencing. Floodlight Genomics uses a Hi-Plex approach to amplify targets (80-100bp) in a multiplexed PCR reaction and then generates sample specific sequences using a next-generation sequencing device (e.g. Ion Proton or Illumina MiSeq). The targeted sequencing was done at cost as part of the FG Educational and Research Outreach Program and the final data was obtained using an Ion Proton (Life Technologies). Binned raw sequence data obtained from FG was then used to map the sample-specific sequences to a reduced representation reference genome containing only the target sequences. SNP genotypes were assigned to target sites with at least 10X sequence coverage. Sites with <15% alternate allele frequency were considered homozygous genotypes.
9
Comprehensive Transcriptome Profiling of Human Cells
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Total RNA was isolated from cells by using Trizol (Sigma, Gillingham, Dorest. UK). The reverse transcription to cDNA was performed with IonAmpliseqTM Transcriptome Human Gene Expression kit (Life Technologies). We then used the Human Gene Expression Core Panel primer set (Life Technologies) to prepare small amplicon gene expression libraries targeting 20,000 genes (95% of the RefSeq gene database). The cDNA amplicon libraries (125–300 bp) were ligated to adapters and amplified using IonXpress RNA-seq barcoded primers (5′). cDNA libraries were clonally amplified using an Ion PI template OT2 200 kit (Life technologies, USA) on an Ion OneTouch2 system (Life technologies) as per the manufacturer’s instructions. Samples were processed using the Ion Proton 200 sequencing kit and loaded onto a P1 chip and sequenced on an Ion Proton (Life technologies) using default parameters (single-end, forward sequencing). Base calling, adaptor trimming, barcode deconvolution, alignment and Ampliseq gene expression analysis was performed on Torrent Suite version 3.6 (Life technologies).
10
Ion Proton Whole Exome Sequencing Protocol
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Whole exome sequencing was achieved using the Ion Proton platform (AmpliSeq kit). Briefly, 100 ng DNA from each sample were collected and the extracted DNA is then amplified using AmpliSeq HiFi mix (Life Technologies, Carlsbad, CA, USA) for 10 cycles. The resultant PCR products were then pooled followed by primer digestion using FuPa reagent (Life Technologies, Carlsbad, CA, USA). A ligation step was then conducted using Ion P1 and Ion Xpress Barcode adapters. After that the libraries were purified and quantified using qPCR and the Ion Library Quantification Kit (Life Technologies, Carlsbad, CA, USA). The next step included emulsion of the libraries using Ion OneTouch System to attach the DNA fragments to the Ion Sphere particles. The final step in the library preparation included enrichment of the Ion Sphere particles using Ion OneTouch ES (Life Technologies, Carlsbad, CA, USA). Once the library became ready, they are loaded on the sequencing chip which is then inserted into the Ion Proton instrument (Life Technologies, Carlsbad, CA, USA) for sequencing.
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