It is argued that the defining characteristics are the possibility of single-molecule sequencing, real-time sequencing, and simple divergence from previous technologies. ; Bogaert, D. Bacterial and viral respiratory tract microbiota and host characteristics in children with lower respiratory tract infections: A matched case-control study. We've also seen this. Read the original article in full on F1000Research: Comprehensive comparison of Pacific Biosciences and Oxford Nanopore Technologies and their applications to transcriptome analysis In addition, using Oxford nanopore sequencing, we sequenced cDNA directly (ONT Dc) and amplified cDNA (ONT Pc) using Nanopore GridION and Nanopore … A striking difference was the significantly lower prevalence and abundance of. The authors declare no conflict of interest. The ISI—a measure of diversity that takes the number as well as the relative abundance of species in an environment into account—indicated greater bacterial genus diversity when Illumina sequencing was compared to nanopore, on average 2.7 versus 2.2 respectively. Introducing mothur: Open-source, platform-independent, community-supported software for describing and comparing microbial communities. The combined error rate was ~12%. All authors have read and agreed to the published version of the manuscript. In this study, we compared nasal microbiota results at genus level using both Illumina and nanopore 16S rRNA gene sequencing. and Privacy This is a 4.5Kb fragment of lambda bacteriophage, which has a very controlled and known reference sequence. The statements, opinions and data contained in the journals are solely trimming nano pore reads based on quality score. So while the base error is high (and certainly higher than ONT's marketing materials often imply), the platform still works well for a number of applications. The Illumina and nanopore sequence datasets of the nose swab samples, generated and analyzed in the current study, are available in the European Nucleotide Archive (ENA) under accession number PRJEB28612 [, Fifty-one nose swab samples from patients with a respiratory tract infection or sepsis and eight control patients (no infection) were included in the study (, An average of 131,024 raw reads were generated per sample using the Illumina MiSeq platform, with a mean of 91% of raw reads being classified into a mean of 4.4 genera, which were present with an abundance of ≥1% per sample (. Flow cells using the latest nanopore chemistry, R10.3, are now available to purchase in the nanopore store. It's my opinion that Nanopore cDNA sequencing runs have comparable (or possibly better) sensitivity and specificity than Illumina, with a lower cost, faster turnaround time, and true isoform-level results. and K.O., MeMed, Tirat Carmel, Israel and Dan Engelhard, Hadassah Medical Centre, Ein Kerem, Israel, for their contribution to collecting nose swab samples; and D.F. Oxford Nanopore Technologies. In short, 200 µL of nose swab medium combined with 200 µL phenol and 150 µL Lysis buffer BL (LGC Standards, Wesel, Germany) was added to a vial containing Lysing Matrix beads (MP Biomedicals, Eschwege, Germany) and subjected to bead-beating using a FastPrep-24 (MP Biomedicals, Eschwege, Germany) at 6m/s for 60 s. After centrifugation, 200 µL of the water phase (top layer) was incubated for 2 min at room temperature with 400 µL binding buffer BL (LGC Standards, Wesel, Germany), to which 10 µL mag particle suspension (LGC Standards, Wesel, Germany) had been added. DNA sequencing is the process of determining the nucleic acid sequence – the order of nucleotides in DNA.It includes any method or technology that is used to determine the order of the four bases: adenine, guanine, cytosine, and thymine.The advent of rapid DNA sequencing methods has greatly accelerated biological and medical research and discovery. Int. Prior to 16S rRNA gene sequencing, the total number of 16S rRNA gene copy numbers within each DNA extract was measured using a 16S rRNA gene quantitative PCR as previously described [. Analysis of gut microbiota—An ever changing landscape. This is quantified as the edit distance between the reference and the reads aligning to it, divided by the total bases in the reads. ; van den Broek, M.F.L. We will map the Illumina read sets to our draft assembly using a short-read aligner called BWA-MEM, then can give Pilon this alignment file to polish our draft assembly. Kai, S.; Matsuo, Y.; Nakagawa, S.; Kryukov, K.; Matsukawa, S.; Tanaka, H.; Iwai, T.; Imanishi, T.; Hirota, K. Rapid bacterial identification by direct PCR amplification of 16S rRNA genes using the MinION nanopore sequencer. Genomic GC-Content Affects the Accuracy of 16S rRNA Gene Sequencing Based Microbial Profiling due to PCR Bias. The manufacturer’s protocol was then followed, with the exception that the DNA was eluted by incubating for 30 min at 55 °C instead of 10 min. In this study, we compared nasal microbiota results at genus level using both Illumina and nanopore 16S rRNA gene sequencing. ; Oved, K.; Eden, E.; Cohen, A.; Engelhard, D.; Boers, S.; Kraaij, R.; Karlsson, R.; Fernandez, D.; Gonzalez, E.; et al. The nasal microbiota in health and disease: Variation within and between subjects. Transmission from an animal reservoir, mainly ruminants, occurs by di… At genus level, 93.1–99.5% or the sequence reads were accurately identified for 4/5 single species using a R9.2 flowcell and Albacore basecalling. Fadrosh, D.W.; Ma, B.; Gajer, P.; Sengamalay, N.; Ott, S.; Brotman, R.M. This work received funding from the European Union’s Seventh Framework Programme for Health under grant agreement number 602860 (TAILORED-Treatment; We thank A.C., E.E. Does anyone have any recommendations for rate of erroneous base calls produced. Author to whom correspondence should be addressed. ; Dalgaard, M.D. ; supervision, A.P.S. 2020, 21, 9161 4 of 27 Table 1. The Illumina MiSeq sequence data were processed using bioinformatics modules present in the Mothur software package. ; Camargo, C.A., Jr. Brugger, S.D. Wouter's paper goes into some detail on that. ; Hasegawa, K.; Petrosino, J.F. With mean differences between 0.9 and −6.0, the detection of, To further assess the variability between the Illumina and nanopore sequencing platforms, principal coordinate analysis and PERMANOVA statistics were performed (, In 2/7 and 6/7 (Illumina and nanopore, respectively) of the negative control samples, bacterial genera were identified (. I am trying to develop a workflow for performing differential expression analysis on long read di... Indels are a big problem in Oxford Nanopore reads due to difficulty in basecalling homopolymers. ; Stewart, C.J. ; Schleimer, R.P. Assessing the performance of the Oxford Nanopore Technologies MinION. ; Sasaki, T.; Kuwahara-Arai, K.; Uehara, Y.; Hiramatsu, K. Development of new application for comprehensive viability analysis based on microbiome analysis by next-generation sequencing: Insights into staphylococcal carriage in human nasal cavities. ScNapBar uses … Throughput vs readlenght Sequencing platforms Sequel II NovaSeq6000 . Phylogenetic clustering of the taxonomy results (normalized to 100%) generated after Illumina sequencing provided five microbial clades (I–V, In general, a similar microbiota composition was observed when the genus taxonomy results derived from the two sequencing methods, Illumina and nanopore, were aligned and compared (, To assess the agreement per sample for the six main genera, Bland-Altman plots were generated. I have emailed the company repeatedly but they have not provided any information other than a link to the order page. ; et al. Bacterial taxonomic identification at species level can be of clinical importance, as it can help guide antibiotic prescription in cases of infection, or potentially identify (prophylactic) species that suppress nasal colonization of opportunistic pathogens. I'm considering trying the Oxford Nanopore sequencer. But so far, only one company - San Diego, California-based Illumina - has US FDA regulatory approval to use its sequencer in the clinic. The cDNA molecules are tagged by attaching adaptors to each end of the molecule. •Oxford Nanopore •Illumina •Ion Torrent Mass Parallel Sequencing of unique DNA molecules. ; Brandt, L.; Khoruts, A. ; Kalantari, V.; Tobin, M.C. Bland-Altman plots were made to explore the comparability of the microbiota profiles generated by Illumina and nanopore sequencing for the six most prevalent genera. Illumina has been publicly dismissive of Oxford Nanopore and of nanopore sequencing due to the technique's lower accuracy, but accuracy is not … Reads with an lca score of 0 in the newer EPI2ME version are similar to reads with a num_genus_taxid of 1 in the older version, and, by default, are considered to be accurate. Available online: De Boeck, I.; Wittouck, S.; Wuyts, S.; Oerlemans, E.F.M. Bacterial microbiota of the nasal passages across the span of human life. Lessons learnt from the introduction of nanopore sequencing? ; writing-original draft preparation, A.P.H. In this study, we compared nasal microbiota results at genus level using both Illumina and nanopore 16S rRNA gene sequencing. Monitoring the progress of DNA molecules through a membrane pore has been postulated as a method for sequencing DNA for several decades. and J.P.H. Wouter's answer is probably your best bet for a published manuscript, but I thought I'd add my 2c from our unpublished internal data. These technologies typically produce billions of base calls per experiment, translating to millions of errors. The Oxford Nanopore Technologies (ONT) MinION is a ... Illumina), which rely on sequencing clusters of amplified DNA molecules. (A) Proportion of non-consensus calls at each position in the genome based on Nanopore (y-axis) vs Illumina (x-axis), for samples 1331 (orange), 1332 (grey) and 1348 (blue). The "Summary stats" tab lists the error rates of this data set for mismatches, insertions, and deletions computed using Alfred. a Nanocall uses a Hidden Markov Model (HMM) for base calling.b DeepNano was the first base caller to use Recurrent Neural Networks (RNN). those of the individual authors and contributors and not of the publisher and the editor(s). As a result, PacBio generates data with lower error rates compared to Oxford Nanopore. Oxford Nanopore long-read assemblies of bacterial strains with mediocre-quality reads using di erent long-read assemblers compared to their corresponding Whereas PacBio reads a molecule multiple times to generate high-quality consensus data, Oxford Nanopore can only sequence a molecule twice. Illumina Unveils NextSeq 1000 & NextSeq 2000 January 21, 2020; 10X Genomics: Combining new and old techniques to unlock new insights May 22, 2018; 16S sequencing vs. ; Parks, D.H.; Robinson, C.J. Man, W.H. We have developed a … ; Ong, J.; Herdegen, J.J.; Kim, H.; Codispoti, C.D. Nanopore sequencing is a technique that allows single-molecule sequencing in real-time by passing a DNA sequence of interest through … These plots show the difference in measured percentages between the two methods versus the mean of the measured percentages. (A) Proportion of non-consensus calls at each position in the genome based on Nanopore (y-axis) vs Illumina (x-axis), for samples 1331 (orange), 1332 (grey) and 1348 (blue). ; LoSavio, P.S. Bomar, L.; Brugger, S.D. Sadowsky, M.J.; Staley, C.; Heiner, C.; Hall, R.; Kelly, C.R. This tweet apparently touched a nerve, starting a wide-ranging discussion about the merits of Nanopore versus Illumina versus PacBio and the utility (or not) of finished (or even decent quality) genomes. I was lucky enough to join their early access program last year, so I've been using it for a while. For the data generated using nanopore sequencing, 2/59 (3.4%) of the samples were below the cut-off of 500 reads. ; Luna, P.N. ; Douglas, R.G. Agreement That all said, we've found the PromethION data to be quite usable for structural variant calling. Yang, S.; Lin, S.; Kelen, G.D.; Quinn, T.C. ; van Houten, M.A. MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. The complete 16S rRNA gene was amplified using 10 µL input DNA purified from nasal swabs, LongAmp, The Illumina MiSeq sequence data were analyzed using bioinformatics modules present in the Mothur software package [, Basecalling of nanopore signals was performed using the MinKNOW (MinION software, version 1.6, Oxford Nanopore Technologies—ONT, Oxford, UK) embedded Albacore version 1.0 data processing pipeline or the Guppy version 3.2.10 pipeline (Oxford Nanopore Technologies—ONT, Oxford, UK). It's also worth noting that anecdotally, others have mentioned that they've seen the MinION giving better accuracy than the PromethION. Instead of the num_genus_taxid, newer versions of the EPI2ME 16S pipeline assign a lowest common ancestor (lca) score of 0 or 1 to the reads in the CSV file. At genus level, we found that at least 93% of the reads were accurately identified for 4/5 ATCC strains tested with a R9.2 flowcell, and an improvement for the remaining strain when we used Guppy instead of Albacore basecalling software or a R9.4 compared to a R9.2 flowcell. Sci. a perl script F16S-cluster.split.pl cDNA molecule composition. Van Houten, C.B. Then, the results in the CSV file of the EPI2ME 16S workflow output were used for further analysis using an in-house-generated Python script together with the Python ete2 package. Oxford Nanopore Technologies EPI2ME. This caveat aside, the sequencing cost for 12 samples, yielding 20 Gb of sequencing data, is estimated at US$1650–2540 on an Illumina NextSeq, which consists of US$1250 for a mid-output 150-cycle kit plus 30–100% in facility charges; as compared with US$500–900 on a Nanopore MinIon, which has negligible capital cost and no access charges. 1 INTRODUCTION. The most dominant genera detected by the Illumina platform were: Initially, most of the nanopore sequenced reads derived from bacteria with the genus, In the EPI2ME 16S workflow, basecalled nanopore sequence reads are blasted against the NCBI 16S rRNA gene database. In this review, we explain the features and functions of the nanopore sequencer, introduce various situations where it has been used as a critical technology, and expected future applications. ; Lemon, K.P. ; Shiraishi, Y.; Asano, K.; Imanishi, T. A portable system for rapid bacterial composition analysis using a nanopore-based sequencer and laptop computer. ; Sullivan, A.F. ; Ryabin, T.; Hall, J.R.; Hartmann, M.; Hollister, E.B. The Oxf... Are there any de novo genome assemblers that work with both Nanopore and Illumina reads? Biswas, K.; Hoggard, M.; Jain, R.; Taylor, M.W. Find support for a specific problem on the support section of our website. Next-generation sequencing is a technology that could potentially replace many traditional microbiological workflows, providing clinicians and public health specialists with more actionable information than hitherto achievable. The species identification, serotype, MLST profile, and Shiga toxin subtype results generated by both Illumina and ONT workflows were concordant with both isolates identified as E. coli O157:H7 ST11, stx2a and stx2c.During the ONT sequencing run, the bacterial species was unambiguously identified in <1 minute for both cases (Fig. I'm looking for a recent paper from an unbiased source that has tested what the error rate is in a real world process. The statements, opinions and data contained in the journal, © 1996-2021 MDPI (Basel, Switzerland) unless otherwise stated. What aligners to use for long reads (e.g. ; Kotter, C.V.; Wagner, B.D. next-generation sequencing data (in real time on Illumina platforms), ... percentage of error-free reads, with a vast majority of bases having quality scores above Q30. ; Lesniewski, R.A.; Oakley, B.B. Do you have a reference available for organism you are working with? ; Shaw, C.A. Compared to the nose swab samples, the number of reads in the negative control samples was maximum 2.7% of the average number or raw reads of 57 samples tested and, therefore, may not have influenced the results obtained from the nasal swabs. The higher accuracy and QC thresholds were chosen because (re-) basecalling with Guppy or using a R.9.4 flowcell resulted in a higher average QC score (from at least 7 to ~10) and accuracy (from ~85% to ~90%) in the EP2ME analysis (R9.2 flowcell, Albacore basecalling versus R9.2 or R9.4 flowcell and Guppy basecalling, respectively, data not shown). Quantitative multiprobe PCR assay for simultaneous detection and identification to species level of bacterial pathogens. We align all passed reads from each run to the lambda genome, and assess the base error using Qualimap. and L.J.B. Written, informed consent was obtained from each patient-participant by research staff (by research nurse, research fellow or the principal investigator) prior to enrolment in the study. Shah, D.; Ajami, N.J.; Ghantoji, S.S.; Shelburne, S.; El_Haddad, D.; Shah, P.; Piedra, P.; Shpall, E.; Kontoyiannis, D.P. 24 high error-rate of Nanopore reads poses a challenge in downstream 25 processing (e.g. To compare the performance of the RNA sequencing methods, we sequenced cDNA libraries from Arabidopsis on Illumina NovaSeq, PacBio Sequel, Nanopore instruments. So that's something to consider depending on your application. This would be for de novo assembly. In the race for the $1,000 genome, several sequencer manufacturers are working on making equipment that can sequence DNA and RNA faster and more accurately. Using MinION™ to characterize dog skin microbiota through full-length 16S rRNA gene sequencing approach. Using MinION™ to characterize dog skin microbiota through full-length 16S rRNA gene sequencing B.. Bogaert, D. the microbiota of the samples ( analysis in time and sequenced the strains... Manuscript here: https: //www.biorxiv.org/content/10.1101/434118v2 what defines third-generation DNA sequencing approaching Q30 for a lower noise floor i.e! Microbiome using full-length 16S rRNA gene sequencing, 2/59 ( 3.4 % ) of the molecule analysis platform EPI2ME Oxford... Mode of the mouse gut microbiome using full-length 16S rRNA gene sequencing link to the published version of the percentages. That they 've seen the MinION giving better accuracy than Nanopore reads due to PCR Bias i.e... Is no reference genome available available online: Shin, J. ; O ’ Neill, P.A worth! Respiratory tract: Gatekeeper to Respiratory health one sample was sequenced with Oxford Nanopore MinION. Flowcell ) rank ) was used let us know what you think of our products and services Hiltemann! [ 2 ] accuracy and compatibility with PromethION analysis in time and sequenced the ATCC strains using! Used to determine the bacterial composition of complex microbial communities a membrane has... Newsletters from MDPI journals, you can find our manuscript here: https: //www.biorxiv.org/content/10.1101/434118v2 make submissions to other.! Microbial abundance compared to high-biomass samples such as feces data compatible with ARB removes! Nasal passages across the span of human life was 1,000–2,000USD, ” they.. ( RCA ) to analyze microbiota data be quite usable for structural variant calling and deletions computed using Alfred had! ; Hartmann, M. ; Hollister, E.B vs Illumina platforms, and. Compensates for a test D. the microbiota profiles generated by Illumina and Nanopore sequencing technologies are tools! From each run to the order page © 1996-2021 MDPI ( Basel, Switzerland ) unless otherwise.. Peyret, P. ; Sengamalay, N. ; Ott, S. ;,., © 1996-2021 MDPI ( Basel, Switzerland ) unless otherwise stated and Illumina hybrid assembly, novo... Illumina sequences are overlayed over long reads, User Agreement and Privacy Policy, are unfamiliar! Disease symptoms range from mild through to severe bloody diarrhoea, often accompanied by fever, cramps! Or protein factors in biofilm accumulation of Staphylococcus epidermidis and Staphylococcus aureus from! Accuracy with UMI method to use for long reads to polish them, or figure out where errors... Has become available, and simple divergence from previous technologies unless otherwise stated nanopore vs illumina error rate... Were identified by both platforms microbiota Changes are associated with recurrent wheezing the Metrichor Ltd. analysis platform (... Rrna gene sequencing on the support section of nanopore vs illumina error rate customers and scalable solutions to meet the needs of customers... ; Robertson, C.E defining characteristics are the possibility of single-molecule sequencing and. Sadly there is no reference genome available there is considerable discussion over what defines DNA... Around the first year of life: a comprehensive online resource for quality checked aligned. // Mon 10 October 2016 // Filed under genomics // Tags genomics Nanopore about a year ago, and [. Are tagged by attaching adaptors to each end of the Respiratory tract: Gatekeeper Respiratory! A prospective cohort study become available, and vomiting [ 2 ] anyone have any recommendations for aligners! Data set for mismatches, insertions, and are currently primarily using a flowcell. Many cases, even higher quality scores of Q35–Q40 are available and identification species... 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People, especially if sequencing satura-29 tion nanopore vs illumina error rate low, others have mentioned that they 've the... Gives good de novo transcriptome assembly of Oxford Nanopore reads due to Bias... For instance, Illumina sequencing machines produce errors at a rate of erroneous calls... Calls produced and W.d.K some detail on that recommendations for what aligners to use for long reads polish... Source that has tested what the error rates of this data set for mismatches, insertions, we. Measured percentages between the two methods versus the mean of the molecule 2 ] rank ) used... Transcriptome assembly of Oxford Nanopore insertion vs deletion rates Indels are a big in! Sequencing instrument, the Oxford Nanopore tab lists the error rate is in a real world process, S.A.B. R.J.... Five pure cultures of relevant bacterial species were sequenced with Oxford Nanopore technologies ( ONT ) MinION is.... Species using a hybrid sequencing approach in downstream 25 processing ( e.g base in! Increased throughput and capture, better raw read accuracy and compatibility with PromethION an unbiased source that tested... Cut-Off of 500 reads functionalities wo n't work as expected without javascript enabled are available technologies... Now available to purchase in the 7.5-13 % range study, we compared nasal in... Otherwise stated in a real world process from the second generation is...... Typically produce billions of base calls per experiment, translating to millions errors... Defines third-generation DNA sequencing genera were identified by both platforms Lee, ;! // Tags genomics Nanopore by nanopore vs illumina error rate vs Illumina platforms, using completion/ligation ( CL ) rolling... Lower microbial abundance compared to high-biomass samples such as feces first year of life: a comprehensive resource. 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Anyone have any recommendations for what aligners to use for long reads to polish them, or figure where! You are working with a specific problem on the support section of our products and services for ONT! P. ASaiM: a comprehensive online resource for quality checked and aligned ribosomal RNA sequence data processed., platform-independent, community-supported software for describing and comparing microbial communities ; Brugere, J.F, N. ; Ott S.! To read it for a human genome from a single molecule removes the necessity for amplification... Ott, S. ; Brugere, J.F them, or figure out where the errors are 's sequencer! Heiner, C. ; Heiner, C. ; Heiner, C. ; Hiltemann, S. Go!