Figure 1. Single-molecule analysis through microfluidic enrichment of rolling circle amplification products. (A) Sample preparation and target detection.

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Figure 1. Single-molecule analysis through microfluidic enrichment of rolling circle amplification products. (A) Sample preparation and target detection by generation of circular templates: (1) Molecular barcoding of DNA through ligation of barcoded target-specific padlock probes, (2) selective circularization of digested genomic DNA fragments through sequence end-complementary selector probes. 1.3 and 2.3.: Circularized molecules are clonally amplified through RCA, generating micron-sized DNA coils that can be fluorescently labelled through (1.4) hybridization of barcode complementary fluorescent probes, or (2.4) base-specific ligation of sequencing by ligation (SBL) interrogation probes in solution. Universal anchor probes with AlexaFluo750 label (illustrated in grey) guides identification of RCPs; SBL interrogation probes carry base-specific fluorescent labels that, after specific ligation to the anchor probe, permit nucleotide identification. (B) Assembled RCP enrichment microfluidic chip is placed directly under conventional low magnification fluorescent microscope objectives. (C) Exploded view of the multilayer PDMS microfluidic chip outlining microfluidic channels and the embedded membrane (detailed information about the assembly can be found in Supplementary Figure S1). (D) Close-up scheme of the interfaced flow cell with embedded membrane that captures RCPs when solutions are flowed through the chip. To the right: Schematic illustration of how RCPs are captured in the nano-porous membrane mesh by a combination of size exclusion and adsorption within the membrane fibres surface. (E) Selected FOV regions within images of a solution of RCPs analysed on a microscope slide without enrichment (top) and on a membrane after enrichment (bottom). From: Sensitive and inexpensive digital DNA analysis by microfluidic enrichment of rolling circle amplified single-molecules Nucleic Acids Res. 2017;45(8):e59. doi:10.1093/nar/gkw1324 Nucleic Acids Res | © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

Figure 2. Single-molecule counting performance Figure 2. Single-molecule counting performance. (A) Quantification range of microfluidic RCA products (RCP) enrichment (red) compared to no enrichment (black). The counted number of RCP molecules on Y axis is plotted against the expected number of RCP molecules on the X axis, estimated as described in Supplementary Note S1. Error bars ±1 st. dev. of the mean, n = 2. (B–D) Measured RCP counts are plotted against the expected RCP counts in a log-dilution series using (B) enrichment, (C) no enrichment and (D) state-of-the-art RCP quantification in flow (Aquila 400, Q-linea). Black solid lines indicate linear fit to the linear response data points. Slopes and R<sup>2</sup> values of linear fits are plotted in the figures. Red dashed lines indicate the maximum dose response. From: Sensitive and inexpensive digital DNA analysis by microfluidic enrichment of rolling circle amplified single-molecules Nucleic Acids Res. 2017;45(8):e59. doi:10.1093/nar/gkw1324 Nucleic Acids Res | © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

Figure 3. Enriched RCPs at high concentration can be resolved via confocal microscopy. (A) 3D rendered confocal Z stack of 1 pM RCPs labelled with either Cy3 or FITC, and enriched on a NC membrane. (B) A detailed 3D rendered region inside the membrane with Cy3 and FITC labelled RCPs. (C) Co-localization measurement of enriched RCPs and expected numbers of co-localization by chance. Artificial images with objects with random distributions and the same number, size and intensity of the measured RCPs were generated and co-localization were measured with the same parameters (Error bars ±1 st. dev. of the mean, n = 5 for random images, n = 3 for measured RCP images). (D) Distribution of RCP localization inside the membrane as a function of different flow rates used during enrichment. From: Sensitive and inexpensive digital DNA analysis by microfluidic enrichment of rolling circle amplified single-molecules Nucleic Acids Res. 2017;45(8):e59. doi:10.1093/nar/gkw1324 Nucleic Acids Res | © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

Figure 4. Multiplex quantification of pathogenic bacterial DNA and associated antibiotic resistance markers. (A) Dilutions of S. aureus genomic DNA were subjected to padlock probe ligation, RCA, specific RCP labelling and microfluidic RCP enrichment analysis (Error bars ±1 st. dev. of the mean, n = 2). Dashed line illustrates the limit of detection at 61 RCPs (calculated as negative control + 3 st. dev. from the mean). Inset graph shows linear fit of log expected versus log measured molecule count (R<sup>2</sup> = 0.99). (B) Representative region of an image after enrichment of processed sample mix containing E. coli/oxa-48 (EcO), S. aureus/mecA (Sa) and P. aeruginosa (Pa). Fluorescent colour code representing the different padlock probe DNA targets is depicted under the image. (C) Multiplex pathogen and antibiotic marker detection. RCP counts per sample and padlock probe DNA targets are illustrated as a heat map. Samples with ‘B’ correspond to mixes with added background DNA. For raw RCP counts and additional negative controls see Supplementary Table S6. From: Sensitive and inexpensive digital DNA analysis by microfluidic enrichment of rolling circle amplified single-molecules Nucleic Acids Res. 2017;45(8):e59. doi:10.1093/nar/gkw1324 Nucleic Acids Res | © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

Figure 5. Microfluidic enrichment analysis purifies RCPs interrogated by in solution SBL and enables accurate single nucloetide variation (SNV) counting. (A) KRAS oncogene codon 12 wild type and mutant synthetic DNA fragments were selectively circularized through selector probes, amplified through RCA and SNVs are interrogated by in solution single base SBL. (B) Improvement of SNR ratio after purification through RCP enrichment on PC membranes (Error bars ±1 st. dev. of the mean, n = 10 for RCPs and background pixels. (C) Enriched RCPs of equimolar mixtures (1:1) of wild type (Cy3 label) and mutant (Cy5 label) DNA fragments. (D) Subset of image in C showing fluorescent anchor primer (Alexa750) in white (top) and combined image of the four fluorescent channels (bottom) representing the interrogated base-specific staining for base A (Cy5), C (Texas Red), G (Cy3) and T (Alexa488). (E) SNV analysis of individual RCPs through in-house developed image processing script with base calling algorithm is plotted, with KRAS mutant RCPs as red objects and KRAS wild-type RCPs as yellow objects. (F) RCPs from KRAS wild type and mutant fragments were mixed in different ratios: 1:1, 10:1, 100:1, 1000:1, enriched and imaged on PC membranes. Expected ratio of SNVs for different ratios of mutant/wild-type mixes is plotted versus the measured ratio. From: Sensitive and inexpensive digital DNA analysis by microfluidic enrichment of rolling circle amplified single-molecules Nucleic Acids Res. 2017;45(8):e59. doi:10.1093/nar/gkw1324 Nucleic Acids Res | © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com