Project description:Methylated DNA immunoprecipitation sequencing (MeDIP-Seq) is a widely used approach to study DNA methylation genome-wide. Here, we present a novel MeDIP-Seq protocol compatible with the Ion Torrent semiconductor-based sequencing platform that is scalable and accurately identifies sites of differential DNA methylation. Additionally, we demonstrate that the high-throughput data derived from MeDIP-Seq on the Ion Torrent platform provides adequate coverage of CpG cytosines, the methylation states of which we validated at single-base resolution on the Infinium HumanMethylation450K Beadchip array. We applied this integrative approach to further investigate the role of DNA methylation in alternative splicing and to profile 5-mC and 5-hmC variants of DNA methylation in normal human brain tissue that we observed localize over distinct genomic regions. These applications of MeDIP-Seq on the Ion Torrent platform have broad utility and add to the current methodologies for profiling genome-wide DNA methylation states in normal and disease conditions. MeDIP-Seq on Ion Torrent Platform in HCT116 and Human Brain
Project description:We describe XmaI-RRBS method for rapid and affordable genome-wide DNA methylation analysis, with library preparation taking only four days and sequencing possible within four hours. Small sizes of the XmaI-RRBS libraries allow their multiplexing and sequencing on the benchtop high-throughput machines. Described here is the first RRBS protocol validated for the Ion Torrent Personal Genome Machine. DNA from MCF7 cell line and 6 normal breast samples (total 7 samples) were subjected to reduced representation bisulfite sequencing analysis (XmaI-RRBS) by using Ion Torrent platform.
Project description:The recent development of a semiconductor-based, non-optical DNA sequencing technology promises scalable, low-cost and rapid sequence data production. The technology has previously been applied mainly to genomic sequencing and targeted re-sequencing. Here, we demonstrate the utility of Ion Torrent semiconductor-based sequencing for sensitive, efficient and rapid chromatin immunoprecipitation followed by sequencing (ChIP-seq) through the application of sample preparation methods that are optimized for ChIP-seq on the Ion Torrent platform. We leverage this method for epigenetic profiling of tumor tissues. Examination of histone modifications in mouse dendentic cells stimulated with LPS, matched melanoma derived cell line, melanoma tumor tissue
Project description:The recent development of a semiconductor-based, non-optical DNA sequencing technology promises scalable, low-cost and rapid sequence data production. The technology has previously been applied mainly to genomic sequencing and targeted re-sequencing. Here, we demonstrate the utility of Ion Torrent semiconductor-based sequencing for sensitive, efficient and rapid chromatin immunoprecipitation followed by sequencing (ChIP-seq) through the application of sample preparation methods that are optimized for ChIP-seq on the Ion Torrent platform. We leverage this method for epigenetic profiling of tumor tissues.
Project description:The ligation step in RNA sequencing library generation is a known source of bias. We present the first comparison of the standard duplex adaptor protocol supplied by Life Technologies for use on the Ion Torrent PGM with an alternate single adaptor approach involving CircLigase (CircLig). We also investigate whether using the thermostable ligase Methanobacterium thermoautotrophicum RNA ligase K97A (Mth K97A) for the initial ligation step in the CircLigase protocol reduces bias. A pool of small RNA fragments of known composition was converted into a sequencing library using one of three protocols and sequenced on an Ion Torrent PGM. The single adaptor CircLigase-based approach significantly reduces, but does not eliminate, bias in Ion Torrent data. Using Mth K97A as part of the CircLig method does not further reduce bias.
Project description:MicroRNA sequencing using Ion Torrent Proton platform of the undamaged heart of the red spotted newt Notophthalmus viridescens. MicroRNAS were identified using MIRPIPE
Project description:We investigated a panel of 21 genes by parallel sequencing on the Ion Torrent Personal Genome Machine platform. We sequenced 65 CRCs that were treated with cetuximab or panitumumab ( 37 samples were responsive and 28 were resistant).
Project description:Next generation sequencing using the Ion Torrent PGM platform after PNLDC1 silencing in mouse embryonic stem cells E14 using esiRNA
Project description:Purpose: to reveal the small RNA profile of postnatal retina in Wistar WU albino rat by Ion-Torrent PGM sequencing technology. Methods : Wistar WU rats ranging in age from P1 to P21 were anesthetized by inhalation using Forane prior to sacrifice at the same hour to avoid circadian variation. Total RNA was extracted from the retinal tissues of various developmental stages using NucleoSpin miRNA kit (Macherey–Nagel, Düren, Germany) following manufacturer's instructions. The miRNA concentration was assessed using Qubit microRNA Assay Kit (ThermoFisher Scientific, MA, USA). The RNA integrity number (RIN), an algorithm for judging the integrity of RNA samples, were evaluated using Agilent 2100 Bioanalyzer (Agilent Technologies, USA) following the manufacturing instruction of the RNA 6000 Nano kit and RIN>7 was considered acceptable. MicroRNAs was also determined using the Agilent Small RNA Kit to have deeper view in the 10 to 40-nucleotide size range. Small RNA library construction from pooled retinal samples (N=3, in each age-group) were carried out according to the Ion Total RNA-Seq Kit v2 protocol (Revision E) with slight modification. Enzymatic reaction was performed in half reaction volume, while cleaning procedures were executed with the accurate final volume according to the protocol. The Ion 316 or 318 ™ Chip v2 was used for sequencing on the Ion Torrent PGM™ instrument according to the protocol of Ion PGM™ Hi-Q View Sequencing Kit. To assess reliability of miRNA workflow solution in 316 v2 chip, two independent P7 samples were assayed (biological replicates) and P21 sample were sequenced as technical replicates in every sequencing procedure. Some samples were also sequenced in 318 v2 chip (P3, P5, P10, P15 and P21) as biological replicate. Ion Torrent Suite Platform was used to trim the raw sequence data and remove any residual sequencing adapter fragments that remained on the 5′ or 3′ ends. Reads were mapped to the non-coding RNAs from ENSEMBL [Rnor_6.0 (GCA_000001895.4)] using TMAP algorithm. These aligned BAM (Binary Alignment Map) files were further processed in Galaxy Web-based platform (Afgan, 2018) via Cufflinks, Cuffmerge and Cuffdiff (Version 2.2.1.3) application (Trapnell, 2010). Further analysis and visualising of the datasets was carried out in R Studio Software environment. qRT–PCR validation was performed using TaqMan assays.