For fragments originally of 200 bp and 400 bp, the prepare amplicon libraries. Utility of different massive parallel sequencing platforms. Sequencing template was generated using emulsion pcr on the ion onetouch 2 using the ion pgm template ot2 200 kit. Template preparation from the diluted library was performed using an ion pgm template ot2 200 kit ion torrent, life technologies. Mix thoroughly and insert the mixture in a filter cartridge and carefully top with emulsion oil provided. Immediately prior to preparing the ion one touch 2 ot2 amplification mix and loading the reaction filter as outlined in the ion pgm template ot2 200 user guide, a 20 pm dilution of each library was prepared in a lobind tube and libraries were then pooled in equal volumes for normalization as outlined in. Primary sequencing data analysis was performed on the torrent server v4. This protocol is a guide to using archer ngs library preparation kits. Barcoding was carried out by ionxpress barcode kit 4471250. Molecular diagnostics of gliomas using next generation. Here are the most requested quick start guides and manuals for our product line. The library was mixed with ion sphere particles isps and the subsequent emulsion pcr and enrichment were performed using the ion pgm tm template ot2 200 template kit and the ion. Sequencing of the libraries was performed on the ion torrent pgm using the manual ion pgm tm sequencing 200 kit v2 man0007273, revision 1. Validation of a semiconductor nextgeneration sequencing.
Ion pi template ot2 200 kit v3 user guide 9 1 product information kit contents and storage conditions ion pi template ot2 200 kit v3 important. Ion pi template ot2 200 kit v2 kansas state university. Genetic heterogeneity in patients with bartter syndrome type 1. Frontiers characterization of core microbiomes and.
Library and template preparations were performed consecutively with the ampliseq library kit 2. This kit supports the ion 314 chip v2 bc, ion 316 chip v2 bc, and the ion. Ion pi template ot2 200 kit v2 user guide 9 1 product information kit contents and storage conditions ion pi template ot2 200 kit v2 important. Sequencing run times on page 60 for a table listing the number of flows for different read lengths. Simultaneous whole mitochondrial genome sequencing with. Metagenomic sequencing was performed using ion torrent personal genomic machine pgm with 318 chips. Analytical validation of the nextgeneration sequencing. Agilent haloplex target enrichment and surecall data.
The sample libraries were subsequently sequenced using the ion torrent pgm sequencer following the ion pgm template ot2 200 kit life technologies ltd, paisley, uk. First genome sequencing and comparative analyses of. Use the ion pi hiq template and sequencing kits for improved variant calling accuracy. Library preparation, amplification and sequencing was performed using ion xpress plus fragment library kit for ab library builder system, ion pgm template ot2 200 kit and ion pgm sequencing 200 kit v2 life technologies europe bv, zug, switzerland according to the manufacturers instructions. What are possible reasons of low quality reads on ion. Q sequencing kit is compatible with the following template preparation kits. Prepare the emulsion pcr mix reagents provided in the kit in a pcr hood and add the pooled pcr products and the sphere particles provided on the bench. The emulsion was done with onetouch 2 and ion onetouch es devices by using the ion pgm template ot2 200 kit 4480974. The ot2 program script enabling the mixed workflow is discontinued in v5. The primary file for implementing the logic contained in the plugin. A barcoded target library of 200 bp was prepared for l182 and l31, which resulted in totals of 856,225 and 854,481 reads with mean read lengths of 181 bp and 167 bp, respectively. Wholegenome sequencing of two extensively drugresistant.
Conversion of the raw signal to base calls was performed on the torrent server using torrent suite software v3. Ion pgm system with reagent and wash bottles attached. Hpv genotyping of modified general primeramplicons is. Genotypes from the ysnps were used to identify manually each males yclade according to. Ion pi template ot2 200 kit v3 thermo fisher scientific.
Clonal evolution in myelodysplastic syndromes nature. The emulsion pcr was carried out using the ion pgm template ot2 200 kit life technologies as described in the appropriate user guide catalog number. Dilutions of all 10 libraries were then pooled together. Flows enter the appropriate number of flows for the read length e. Literature chips ion 314 chip v2 4482261 see your sequencing user guide for instructions. Templates were sequenced using the ion pgm sequencing 200 kit v2 on an ion 318v2 chip. Chapter 3 prepare templatepositive ion pgm hiq isps. Pros and cons of iontorrent next generation sequencing. Ion pgm hi q view ot2 kit thermo fisher scientific. However, if you are using the ion pgm template ot2 200 kit together with the ion pgm hiq sequencing kit, do not upgrade to torrent suite software v5. Template preparation, emulsion pcr, and ion sphere particle isp enrichment was performed using the ion pgm template ot2 200 kit life technologies, grand island, ny, usa according to the manufacturers instructions. The 200 bp libraries were enriched and prepared using the ion chef ion pgm ic 200 kit manual, man0007661, revision a.
Whether it is due to the number of concurrent video streams or the combined bitrate of all video streams being too high, in such scenarios the ionr200 may run at maximum capacity and it may not be enough. Pgm template ot2 400 kit manual man0007219, revision 3. Ion pgm template ot2 200 kit user guide thermo fisher scientific. Ion pgm hiq ot2 kit 400, depending on the readlength. The ion pgm hiq view ot2 kit can be used with up to 400 baseread average insert libraries of any type prepared using any available ion. L of normalized library pools, and the spheres enrichment was performed on the ion onetouch. Nextgeneration sequencing of 16s ribosomal rna gene. The kit is designed to deliver the highest quality template for ion proton nextgeneration sequencing using the ion onetouch 2 system. The ion pgm template ot2 200 kit includes reagents required for preparing 10 reactions of templatepositive ion pgm template ot2 200 ion sphere particles isps on the ion onetouch 2 system. Validation of differentially methylated micrornas identified from an. Use only the supplied screw caps in the ion pi template ot2 200 kit v2 to store the reagents. Review the run report from the torrent browser and confirm. The ion pgm template ia 500 kit is configured to support your sequencing needs for applications requiring up to 500 bp read lengths. Generation of sequencing libraries was performed as described in the manual ion pgm tm template ot2 200 kit man0007220, revision 3.
Next gen sequencing lab manual 2016 apr 4 8, 2016 prepared by david ng material has been edited from sections of the following manuals. Blind study evaluation illustrates utility of the ion pgm system for. Highthroughput microdissection for nextgeneration sequencing. Multiplex of oncogene targetspecific panels technical.
Following library enrichment ion onetouch es, the library was processed for sequencing using the ion torrent 200 bp sequencing v2 chemistry and the barcoded. The ion pgm template ot2 200 kit can be used with up to 200 basepair average insert libraries of any type prepared using any available ion library kit. Unenriched libraries were qualitycontrolled using ion sphere quality control measurement on a qubit instrument. Evaluation of the precision id identity panel for the ion. For safety and biohazard guidelines, refer to the safety section in the ion pgm template ot2 200 kit user guide pub. Ion pgmemplate ot2 200 kit t thermo fisher scientific. As an integral component of the semiconductor sequencing workflow, the ion pgm hiq view ot2 kit is desig. Sequencing was performed using the ion 314 chip with the ion pgm sequencing 200 kit on the ion pgm system. Emulsion pcr and subsequent enrichment were performed using the ion onetouch template kit v2. Samples were sequenced on an ion torrent pgm using an ion 314 chip v2 according to the ion pgm sequencing 400 kit protocol publication no. Sequencing was performed with ion pgm 200 sequencing kit 4474004 on ion torrent pgm 316 chip. The ion pgm hiq view ot2 kit enables accurate and reproducible template preparation for libraries up to 400 base pairs using the ion onetouch 2 system and is compatible with the ion pgm system. The templated ion sphere particles were enriched on the ion onetouch es instrument.
This user guide should not be used with the ion xpress template kit part no. Ion pgm template ot2 200 kit thermo fisher scientific. Sequencing was performed on the ion pgm system using the ion pgm sequencing 200 kit v2 reagents and 314 ion chips following the manufacturers protocol. Rare genetic variants in cx3cr1 and their contribution to. When combined with the ion pgm hiq view sequencing kit, the ion pgm template ia 500 kit offers an ultrarapid sequencing workflow. Templating emulsion pcr was accomplished using the ion pgm template ot2 400 kit life technologies, cat. Template kit select the ion pgm template ot2 200 kit sequencing kit select the ion pgm sequencing 200 kit v2.
Incidental finding of severe hypertriglyceridemia in. Box contents attach the dust cover to the hinges, pressing down to lock them into place. Purpose this user guide describes how to use the ion onetouch 2 system to prepare enriched, template positive ion pgm hiq ion sphere particles with up to 400 basepair average insert libraries on the ion personal genome machine pgm system. Ion 316 chip v2 4488145 ion 318 chip v2 4488146 ion template preparation kits ion pgm template ia 500 kit1 a24622 ion. For ion torrent sequencing run with the library prepared in the previous steps, refer to the ion pgm template ot2 200 kit user. The normalized barcoded libraries were pooled in a 10plex or 32plex manner, clonally ampli. Molecular classification of anaplastic oligodendroglioma. Ion pgm template ot2 200 kit and the ion onetouch 2 system for templating and enrichment prior to chip loading. Do not use the kit with the ion onetouch system system. Up to 12 barcoded samples were multiplexed on ion 318 v2, or ion 316 v2 chips. Ion chef ion pgm ic 200 kit manual, man0007661, revision a.
Earlier i used v2 kit and its manual explicitly says 10 pm in 100 ul volume its the same concentration and volume but described in different words. Ion pi template ot2 200 kit v2 user guide thermo fisher. Frontiers the histidine decarboxylase gene cluster of. Pcr and enrichment were performed on the ion onetouch 2 system life technologies using the ion pgm 200 template ot2 200 kit or the ion pi tm template ot2 200 kit v3 man0007220 rev. Revealing the challenges of low template dna analysis with. The ion pi hiq ot2 200 kit contains template preparation reagents for sequencing 8 samples. Phylogenetic analyses of melanoma reveal complex patterns. Briefly, sequencing template preparation for 316 chips run was performed on ion torrent onetouch system i using emulsion pcr to amplify the fragments and bind them to sequencing beads following the ion pgm template ot2 200 kit protocol. Do not mix reactions or disposables including plates, solutions, and kit reagents from other template preparation kits. The ion pgm hiq view ot2 kit includes the reagents required for preparing 8 reactions of template positive ion pgm hiq view ion sphere particles isps on the ion onetouch 2 system. Pcrs were completed using the str 10plex pcr kit from ion torrent thermo fisher. The ion pgm hiq view ot2 kit includes the reagents required for preparing 8 reactions of templatepositive ion pgm hiq view ion sphere particles isps on the ion onetouch 2 system.