Processing ion ampliseq cancer panel data using nextgene software introduction the ion ampliseq cancer panel uses highly multiplexed pcr in order to generate amplicons from 46 different cancer genes. The full gene pik3ca panel was prepared using the ion ampliseq library kit 2. We have developed an assay called the ion ampliseq phenotrivium panel, which combines three groups of markers. Multiplex pcr master mix 2x, qiagen, 5 l ion ampliseq cancer hotspot panel v2 primer pool.
Comparison of manual and automated ampliseq workflows in the. With emphasis on validation with relevant single molecular pathology tests and the oncomine focus assay. Take advantage of low input from as little as 1 ng and flexible input ranges from 1 to 100 ng. The sequencing reaction is performed using the ion pgm dx sequencing kit on the ion pgm dx sequencer, which measures hydrogen ions that are generated during the incorporation of nucleotides into the nascent strand. Eurofins mwg operon launches ion proton sequencing services. The brca panel is part of an integrated workflow that includes ampliseq for illumina polymerase chain reaction pcrbased library preparation, illumina sequencing by synthesis sbs nextgeneration sequencing ngs technology, and automated analysis. From predesigned to customizable dna and rna panels for nextgeneration sequencing ngs, all of our content is available within the ion ampliseq designer including indepth descriptions and gene lists for each panel. If preparing ion ampliseq exome libraries, see the protocols listed on page 7. This platform allows researchers to simultaneously interrogate gene expression of up to 1200 targets in a single tube, thus combining the sensitivity of. Providing the ability to work directly with low input levels of total rna derived from challenging samples such as formalinfixe. Sarscov2 research panel by a massively parallel sequencing mps assay. Processing paired end data from the ion pgm sequencer using. Powerlogic ion meters are compatible with modbus networks as both slaves and masters, and can communicate easily with ion enterprise, ion setup or third.
Use of the ion ampliseq cancer hotspot panel in clinical molecular pathology laboratories for analysis of solid tumours. Ion proton sequencer and ion reporter software, provides. Obtaining high quality transcriptome data from formalinfixed. In general, library preparation of fragmented dna, for use with the ion torrent sequencing platform, involves repair of 3. Deep knowledge of the genetic features of sarscov2 is essential to track the ongoing pandemic through different geographical areas and to design and develop early diagnostic procedures, therapeutic strategies, public health interventions, and vaccines. Added support for the ion ampliseq direct ffpe dna kit.
Quantseq 3 mrnaseq library prep kit for ion torrent lexogen. Comprehensive evaluation of ampliseq transcriptome, a novel. Comprehensive evaluation of ampliseq transcriptome, a. Evaluation of the ion ampliseq sarscov2 research panel. Qubit dsdna hs assay kit pn q32851 taqman rnase p detection reagents kit. Five independent janus protocols are used in the ion ampliseq library preparation workflow. Ion ampliseq library preparation on the ion chef system user. Template preparation of ampliseq libraries using the ion chef. Here, we report the results of testing the assays sensitivity and the predictions obtained for known reference samples. Ampliseq for illumina brca panel assess all exons of. The prepared library underwent template preparation with the ion onetouch 2 and ion. This custom design tool lets you modify any predesigned panel or.
Ion ampliseq cancer panel primer pool thermo fisher bioz. A total of eight libraries were constructed per run. Modicon modbus is a communications protocol widely used in process control industries such as manufacturing. This panel consists of two primer pools that generate 237 amplicons covering the entire sarscov2 genome and 5 human expression controls. Lets find out what this new technology can do for you. Clinical application of ampliseqtargeted sequencing. Although making a master mix reduces the number of dispensing steps, a dead volume is likely to occur. Archer fusionplex protocol for ion torrent hubspot. Comparison of manual and automated ampliseq workflows in. Use of the ion ampliseq cancer hotspot panel in clinical. Ion ampliseq direct ffpe dna kit ion torrent dual barcode kit 196 ioncode barcode adapters 84 kit ion xpress barcode adapters kits ion library equalizer kit ion ampliseq readytouse panels ion ampliseq madetoorder and community panels. Kit and ioncode barcode adapters following th e manufacturers protocol for custom ion ampliseq s np panels thermo fisher scientific.
Using life technologies proprietary approach, consisting of the ion ampliseq exome kit, the. Clinical application of a custom ampliseq library and ion torrent pgm sequencing to comprehensive mutation screening for deafness genes shinya nishio,1,2 yoshiharu hayashi,3 manabu watanabe,3 and shinichi usami1,2. Routine use of the ion torrent ampliseq tm cancer hotspot. Ion ampliseq library kit 2 thermo fisher scientific.
Ion ampliseq readytouse, custom, or community panel. Pdf evaluation of the ion ampliseq sarscov2 research panel. Pdf evaluation of the ion ampliseq phenotrivium panel. The quantseq kit is a library preparation protocol designed to generate ion torrent compatible libraries of sequences close to the 3 end of the. Ampliseq for illumina library plus and indexes are optimized for use with illumina sequencers, and generate highly accurate data with an extensive menu of readytouse or made to order content. Target all exonic regions and flanking intronic sequences of brca1 and brca2. Target enrichment protocol resequencing of exomes fasteris. We help our customers accelerate life sciences research, solve complex analytical challenges, improve patient diagnostics, deliver medicines to. In the current study, we adopted and modi ed protocol from ion ampliseq. Dec 16, 2015 background whole transcriptome sequencing rnaseq represents a powerful approach for whole transcriptome gene expression analysis.
The ion ampliseq transcriptome human gene expression kit enables the simultaneous measurement of the expression levels of over 20,000 human refseq genes in a single assay. In between each protocol an off deck step such as thermocycling. The library was prepared using the ion ampliseq library kit 2. This approach offers many advantages over microarray or qpcr data such as faster turnaround and data analysis, sample multiplexing, lower rna inputs, and ability to use degraded or ffpederived samples. Ampliseq for illumina custom and community panels reference guide. Ampliseq for illumina library prep, index adapters, and. Ampliseq for illumina brca panel assess all exons of brca1. The ion ampliseq transcriptome human gene expression kit ampliseq was recently introduced by. Ion ampliseq dna and rna library preparation user guide for use with. Performance characteristics of the ampliseq cancer hotspot. Updated for ion ampliseq hd panel design and ordering.
Ion ampliseq dna and rna library preparation user guide pn. The ion ampliseq exome protocol with pcr based enrichment combines a short turnaround time and easy integrated bioinformatics to provide alignment of the prepared library to a reference where all indels and snp are reported. Dilution step tube 1 tube 2 tube 3 tube 4 sample into te buffer 5 l of stock into 495 l te 5 l of tube 1 into 495 l te 10 l of tube 2 into 90 l te 10 l of tube 3 into 90 l te dilution 100x 100x 10x 10x. Targeted sequencing solutions thermo fisher scientific. The ampliseq for illumina solution offers a highly multiplexed polymerase chain reaction pcrbased workflow for use with. Z sample input requirements comparison of enrichment and sequencing efficiency ontargeted reads% and uniformity of coverage percentage of pcr duplicate. Targeted amplification for enhanced detection of biothreat. Dna library preparation with 1 or 2pool panels using qpcr quantification. Development of an automated ampliseq library building. Ampliseq for illumina focus panel reference guide 000039456. The pdf report from my ion ampliseq cancer panel runs shows that for the. Dec 01, 2016 a total of 10 ng dna per sample were used for the target enrichment by a multiplex pcr and each dna pool was amplified with the ion ampliseq library kit in conjunction with the ion ampliseq custom primer pool protocols according to the manufacturer procedures life technologies, darmstadt, germany. Qiagen kit handbooks and user manuals are available at.
The ion ampliseq rna library kit supports all ion ampliseq rna readytouse panels, including the following. Ngs library was constructed using the ion ampliseq library kit 2. Lee a1, lee sh2, jung ck1, park g2, lee ky2, choi hj3, min ko4, kim tj5, lee ej5, lee ys6. Nextgeneration sequencing of human opioid receptor genes. Ion ampliseq cancer panel primer pool, supplied by thermo fisher, used in various techniques. Ion ampliseq dna and rna library preparation user guide. The ion ampliseq exome rdy kit enables the fastest and simplest exome enrichment, with oligo pools for ultrahighmultiplex pcr. Amplify target regions from dna or cdna with the ion ampliseq library kit and. Evaluation of the ion ampliseq sarscov2 research panel by.
Processing paired end data from the ion pgm sequencer. To read the fulltext of this research, you can request a copy directly from the authors. We have opened our protocols, datasets, and source code to the world to enable the. Compares different ampliseq workflows to investigate the possibility of using automated library building in forensic genetic case work. Our mission is to enable our customers to make the world healthier, cleaner and safer. Working with key cancer researchers and incorporating information from literature. Scalable multiplex pcr reactions the ion ampliseq 2. Application guide ion ampliseq library preparation on the ngs express perkinelmer 5 because there are several points at which the samples can be stored in the process, the overall library preparation was broken down into multiple smaller steps. To miniaturize this procedure for mosquito hts, we tested a modified protocol in reduced volume of reagent table 1. Ion ampliseq direct ffpe dna kit ion torrent dual barcode kit 196. Nov 01, 2017 library building was performed on the ion chef using the ion ampliseq kit for chef dl8 thermo fisher scientific according to the manufacturers protocol. Add the pcr master mix to each well of the plate to be used. Nucleic acid from a variety of sourcesincluding formalin.
Ion torrent technology has pioneered an entirely new approach to. A093 sequencing the novel coronavirus using the ion. Lowdna input protocol and a singleday ion ampliseq workflow. The new rapid approach was applied to two different. The ngs for dna analysis was conducted using a custom 23 gene ion ampliseq panel which was based on the ion ampliseq colon and lung cancer research panel v2 with an additional amplicon in the met gene to include the exon 14 skipping mutations and two amplicons of exons 2 and 3 of the h ras gene. Addedsupportforamplisequd indexesforillumina, ampliseq cd indexessetbforillumina,ampliseqcd indexessetcforillumina, andampliseqcd indexessetdforillumina. Fragment libraries were constructed by following the suggested ion pgm protocol and 6 l of sample. Submit your question at ion ampliseq chemistry is like your favorite swiss army knife. Ion ampliseq library preparation on the ion chef system user guide for use with. Novel cutting edge applications of ion ampliseq rna. Novel cutting edge applications of ion ampliseq rna technology.
Prepare dna with the ion ampliseq direct ffpe dna kit. Ion chip capacity table for ion ampliseq dna libraries updated c. This novel method allows for targeted sequencing of amplified. G c a t genes t a c g g c a t article evaluation of the ion ampliseq sarscov2 research panel by massive parallel sequencing federica alessandrini 1, sara caucci 2, valerio onofri 3, filomena melchionda 1, adriano tagliabracci 1, patrizia bagnarelli 2, laura di sante 2, chiara turchi 1, and stefano menzo 2 1 legal medicine unit, department of biomedical sciences and public health. When using the ion torrent pairedend protocol, the overlapping reads can be merged into highquality single reads in order to improve sequence accuracy.
Ion ampliseq library preparation, template generation, and chip. Tumor molecular profiling of nsclc patients using next. The library preparation is performed with ion ampliseq exome rdy kit. Ion ampliseq transcriptome human gene expression kit. After ligation, adaptordna constructs are purified and size selected, and amplified via polymerase chain reaction pcr. We describe protocols and first results of the ion ampliseq sarscov2 research panel by a massively parallel sequencing mps assay. Ion ampliseq dna and rna library preparation user guide pub. Miniaturization technologies for costeffective ampliseq. Pdf evaluation of the ion ampliseq sarscov2 research. Our protocol is compatible with all ion ampliseq panels and generates eight balanced libraries on the ion chef instrument in under 8 hours, with less than 1 hour handson time. Evaluation of ultralow input rna sequencing for the study. Ion ampliseq comprehensive cancer panel facilitates broad oncology surveys using only 40 ng of dna with 16,000 primer pairs multiplexed into just 4 pools. Demand panels ion ampliseq readytouse panels ion ampliseq madetoorder and. Automation protocol for information about preparing ion ampliseq libraries on the tecan freedom.
Genes free fulltext evaluation of the ion ampliseq sars. As outlined in table 2, several protocol changes were implemented targeting both the sample preparation and the ion ampliseq kit protocol. Clinical application of a custom ampliseq library and ion. Ion ampliseq kit for chef dl8 ion ampliseq chefready kits oncomine research assay chefready kits ion ampliseq direct ffpe dna kit ion ampliseq on. This can reduce the amount of filtering that is needed and increase the percentage of. The ampliseq for illumina sarscov2 community panel, in combination with ampliseq for illumina library prep, index, and accessories, is a targeted rnacdna amplicon assay for epidemiological research of the sarscov2 virus research use only. Sarscov2 genome sequence from morocco, obtained using ion. Genes free fulltext evaluation of the ion ampliseq. For use with ion ampliseq custom primer pools and ion ampliseq readytouse panels. This guide contains everything a firsttime user needs, including an overview of the ampliseq for illumina custom and community panels protocol, tips and techniques, protocol steps, kit contents, and usersupplied consumables. Life technologies recently released the first ion ampliseq offering for rna which included a library construction kit as well as an integrated design system on ampliseq.
For ultimate flexibility, the soontobereleased ion ampliseq designer webbased software will enable scientists to upload custom human content for assay designs. Target regions were amplified via three multiplex reactions 3 primer pools. The ampliseq for illumina solution offers a highly multiplexed polymerase chain reaction pcrbased workflow for use with targets ranging from a few to hundreds in a single run. Processing ion ampliseq cancer panel data using nextgene. With the new fast flow protocol on the ion s5 xl sequencer, we achieved sequencing and torrent suite analysis in as low as 55 minutes with 200 flows. These amplicons are sequenced on the ion pgm, allowing for rapid turnaround and low cost. This protocol is a guide to using archer ngs library preparation. The ion ampliseq sarscov2 research panel proved to be a flexible methodfor viral sequencing from a variety of sample types and viral loads, leading to a better understanding of the epidemiological impact of the virus as well as to help therapeutic development. Ion ampliseq and genomic dna gdna libraries for assaying 104, 105, and 106 dilutions. Ampliseq for illumina is a suite of ampliseq chemistry products that are compatible with illumina nextgeneration sequencing platforms.
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