Workflow of detection and verification of SGRs in <i>S. typhimurium</i>.

<p>The SGRs detection and verification procedures in this work are as followings: (i) bacterial cells were grown in a chemostat for five days; (ii) samples were collected at each day and 454 pyrosequencing was performed on genomic DNA prepared from three samples collected at day one, two and three; (iii) reads with ‘split mapping’ signature were mined from the three datasets and further subjected to the confirmatory screening based on the three listed criteria; (iv) A substantial fraction of putative rearrangements were selected for experimental verification using padlock probe hybridization and/or PCR.</p

Detection of VEGF by PLA digital ASMD readout.

<p>Blue dots: detected signal; Diamond symbol: CV%.</p

Junction microhomology analysis.

<p>The distribution of overlapping microhomologies at junctions was compared between the three datasets (gen48, gen144 and gen240) and one simulated dataset using 20 million in silico chimeric reads. The observed junction microhomology distribution in the datasets gen48, gen144 and gen 240 were represented by triangles and the simulated distributions were represented by boxplot. (A) Comparison between the dataset gen48 and the simulated dataset (181 rearrangements). (B) Comparison between the dataset gen144 and the simulated dataset (120 rearrangements). (C) Comparison between the dataset gen240 and the simulated dataset (42 rearrangements).</p

Deduced frequencies of expected true SGRs.

<p>The frequencies were calculated as the number of expected true SGRs divided by the sequencing coverage for the three datasets gen48, gen144 and gen240, respectively. The distributions of the three rearrangement events (deletion, duplication and inversion) were compared between each pair of the three datasets using chi-square two-sample test.</p

Summary of stepwise detection and verification of genome rearrangements.

<p>(A) After initial screening based on the ‘split mapping’ signatures (B) After removal of artifacts and quality score analysis (C) After confirmatory screening based on the three criteria (D) After Padlock Probe and PCR verification (expected true rearrangements).</p

Schematic illustration of protein detection by using PLA with digital ASMD readout.

<p>Schematic illustration of protein detection by using PLA with digital ASMD readout.</p

(A) Standard curves for IL6 detection by PLA followed by digital ASMD readout (blue dots) or realtime PCR based readout (red dots).

<p>Error bar = ±1SD. (B) Comparison of the CV% between the two readout strategies (blue dots: digital ASMD readout; red dots: realtime PCR readout).</p

Size distribution of putative deletions.

<p>The size distribution of putative deletions with sizes less than 50 kb was examined for the three datasets, gen48, gen144 and gen240.</p

Comparison between confocal microscopy and the dedicated instrument for detection of RCPs.

<p>The quantitative response of the same dilution series of EC DNA was measured using the dedicated instrument as well as the confocal setup used in Jarvius <i>et al</i> 2006. Filled symbols: dedicated instrument, open symbols: Zeiss 510 Meta confocal microscope.</p

Sequences of the oligonucleotides used in the study. Underlined nucleotides are locked nucleic acids [17].

<p>Nucleotides in italic style are 2′OMe-RNA. The NRP is furthermore labeled by biotin in the 3′end.</p