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Biotechnology & Immunology


Ultrapure Water as an Integral Component of Sample Preparation for Sequencing


Ultrapure water was successfully used in several steps for sample preparation (Figure 5). In particular, it was employed in molecular biological procedures, such as for PCR runs for sample preparation (Decoding and Coding PCRs, Figure 3) and for sequencing reactions, which were performed using the Ion PGMTM sequencer (Figure 6). High water quality with a resistivity of 18 MΩ x cm was required for sequencing with this instrument according to the instruction manual (see User Guide [11]), and reliable sequencing would otherwise not be guaranteed. The ultrapure water used for the experiments described in this study was free of detectable quantities of RNase and DNase (concentrated RNases and DNases below the detection limits of 20 ng/mL and 80 ng/mL, resp.) and was delivered in consistent, stable quality required for the experiments, with a very low TOC concentration (≤ 2 ppb) and a resistivity of 18.2 MΩ x cm that was compensated to 25°C.


Quantitative and Kinetic Analysis of Cellular Signalling Activities


A quantitative and kinetic analysis of downstream signalling activities shows a differential activation pattern, which is specifi c for each particular stimulus (Figure 7). Thus, in EGFld- treated cells, the immediate early gene (IEG) response was strongly activated (IEGs are genes that are activated transiently and rapidly in response to cellular stimuli via MAP kinase signalling cascades; exemplary IEGs are EGR1, EGR2, FOS, FOSB and JUN [12]), whereas signalling pathways related to immune stress responses (IL6, IL8 and NFkB) were not activated (Figure 7, columns 1–4). Cells treated with both EGFld and the inhibitor lapatinib showed a nearly complete inactivation of all signalling pathways measured (Figure 7, columns 5–8). In cells stimulated with PMA/serum, immune stress responses were markedly induced (Figure 7, columns 9–12), which, in contrast, were only partially reverted after treatment with lapatinib (Figure 7, columns 13–16) (cf. data sets for 2 hours after addition of lapatinib).


Ultrapure water was effectively used within multi-parametric assays and subsequent NGS analyses. In a sample experimental setup, it was demonstrated that cellular effects of compounds could be examined using the EXTassay multi-parametric profi ling platform by monitoring the activities of various signalling pathways. This will enable researchers to better understand the mechanisms of ON and OFF target effects of compounds within cells already during the early stages of drug development. Further, this profi ling approach may thus substantially lower attrition rates and costs in medical drug discovery.


Figure 7: Quantitative and kinetic analysis of cellular signalling activities using the specifi c assay technology (the binding sites for the transcription factors are designated AP1, SRE and DLG4; see text for explanations of the other signalling pathway reporters). Signalling activities induced by the specifi c stimulus EGF-like domain (EGFld) or by the broad stimulus phorbol-12myristate-13-acetate (PMA)/10% serum are inhibited to varying degrees by the inhibitor lapatinib. The column numbers are given in italics.


Discussion


The ultrapure water employed in the experiments described here can be readily used for the biochemical, molecular biological and cell biological applications described above, leading to meaningful results. This water was utilised, inter alia, for the critical steps of NGS sample preparation using PCR strategies and subsequent high- throughput sequencing runs (Figures 3 and 6). Using multi-parametric assays, robust responses of cellular signalling that were differentiated between a specifi c and a broad stimulus could be identifi ed (Figure 7).


Sequencing reactions in particular require high ultrapure water standards, which the Type 1 water used in these experiments meets with its exceptionally low TOC concentration and consistent quality.


In addition, ultrapure water obtained from the lab water system was free of detectable nuclease activity – this feature is important as just a trace of nuclease activity may substantially interfere with sensitive applications in molecular biology, such as PCR runs – and constantly provides a conductivity of 18.2 MΩ x cm.


Accurate sequencing with the Ion PGMTM machine can therefore be performed on samples prepared with the ultrapure water generated by the lab water system used. Such ion torrent sequencing requires constant water purity with a resistivity of 18.2 MΩ x cm. Unpublished data show that purifi ed reagent-grade water with a resistivity of less than 18.2 MΩ x cm is not suitable for biochemical and molecular biological applications, such as Western blot analyses and PCR runs. All quality parameters listed for ultrapure water must be fulfi lled in order to obtain reproducible results.


By contrast, use of purifi ed water with poorer quality parameters or slight impurities will lead to unsatisfactory results. Tap water should be completely avoided from the start as it contains many constituents including salts, organic compounds, nucleases, microorganisms and unbound DNA molecules. Moreover, tap water quality may considerably vary from region to region.


Summary


Figure 6: Quality control of sequencing using the Ion Torrent Personal Genome Machine® (PGMTM sequencer). A) The ion sphere particle (ISP) density shows the loading density of the bead-loaded IPSs according to a colour scale. B) The ISP summary contains information on the loading density (loading 87%, 9.84 million wells), the enrichment of sequence-loaded ISPs (95% enrichment) before sequencing, the number of clonal ISPs (97% clonal), the sum of all sequences obtained (7.26 million reads) and the number of the sequences that could be used for analysis (6 million reads blasted). C) Information on the average length of the sequences individually obtained (read length).


Multi-parametric assays allow comprehensive profiling of cellular signalling activities and are increasingly used in the early phases of drug discovery to rapidly and cost- effectively discriminate between desirable ON target and undesirable OFF target effects of compounds. The assays described in this study are based on genetic sensors coupled to molecular barcode reporters, and require that all materials utilised meet the highest quality standards. This was exemplarily demonstrated for ultrapure water that was successfully used for various experimental steps within multi-parametric assays.


Article fi rst published in Lab Asia 24.1 January/February


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