Journal: mBio

Article Title: Origin and Dynamics of Mycobacterium tuberculosis Subpopulations That Predictably Generate Drug Tolerance and Resistance

doi: 10.1128/mbio.02795-22

Figure Lengend Snippet: Transwell-tolerance-resistance (TTR) system with chromosomal barcoding for high-resolution time-kill studies. (A) TTR system setup. M. tuberculosis is seeded into a 24-transwell plate, and a robotic system performs regular medium exchanges providing controlled drug levels in each transwell culture. One-half of each well is directly plated onto drug-free and drug-containing media at defined time points, while the other half is washed free of drug, regrown to an OD 595 of 0.2, and then similarity plated. (B) Construction of a barcoded M. tuberculosis library starting with approximately 20,000 unique barcode sequences printed on a microarray which produced 4,401 unique barcodes in the subsequently generated plasmid library. (C) Venn diagram showing the number of different barcodes present in each day 0 culture for experiment 2 (as indicated by the color legend). (D) Frequency of each barcode (dots) in each culture (as indicated by the color legend, y axis). The x axis shows the distribution of all barcodes ranked by frequency. (E and F) Time-kill kinetic studies using barcoded M. tuberculosis cultures showing the number of CFU observed during experiment 2 (E) and experiment 3 (F) showing rifampicin-sensitive (blue dots) and rifampicin-resistant (red) CFU. (G) Barcode-level time-kill kinetics of experiment 2. Individual barcode count kill curves, adjusted for the number of CFU in each assay well (see Materials and Methods), denoted as mean barcode (BC)-defined CFU, were generated in all replicate culture wells at each time point, excluding wells that contained even one rifampicin-resistant CFU. Each line represents the trajectory of a unique barcode. The bold dotted line represents the mean value of total barcodes at each time point. All barcode reads above the 10-barcode count per well cutoff were included in the analysis; however, mean barcodes lower than 1 are reported (below the dotted line) when this is due to averaging barcode numbers across wells. AB, antibiotic.

Article Snippet: Barcode oligonucleotides were custom synthesized on a high-density microarray slide and eluted to generate a barcoded forward primer pool (MYcroarray; Biodiscovery, LLC, Ann Arbor, MI).

Techniques: Microarray, Produced, Generated, Plasmid Preparation

Journal: mBio

Article Title: Origin and Dynamics of Mycobacterium tuberculosis Subpopulations That Predictably Generate Drug Tolerance and Resistance

doi: 10.1128/mbio.02795-22

Figure Lengend Snippet: Features of directly plated (DP) and differentially detectable (DD) drug tolerance. (A and B) The number of barcodes recovered from DP wells that were present in <25%, 26 to 50%, 51% to 75%, or >75% of the wells at the indicated time point (see key in the figure) for experiment 2 (A) and experiment 3 (B). (C) Trend of the Spearman rank correlations between the number of wells in which a barcode is found at day 20 versus each earlier time point, calculated for all barcodes detectible at each indicated time point interval. (D and E) Correlation of barcode frequency between day 20 and day 0 (D) and between day 4 and day 0 (E). Each unique barcode that was present in at least one well at each time point is shown by a dot, and the frequency for each barcode represents a mean frequency of all wells at that time point. r is calculated by Spearman’s rank correlation. (F and G) Venn diagram comparing the number of different barcodes in experiment 2 versus experiment 3 that were present either in any well at day 0 (F) or in any well during the drug-tolerant phase (days 16 and 20) (G) ( P < 0.0001, one sample test for binomial proportion). (H) The number of unique barcodes identified in experiment 2 in each well (dots) and at each time point. Green dots show barcode numbers of DP cultures and orange dots show barcode numbers after regrowth plating (RP). (I) The number of unique DD barcodes (defined as barcodes that were only present in RP, but not DP wells) identified in experiment 2 in each well over time. Each indicated time is a RP time point, which corresponds to the experiment day on which rifampicin was washed from culture wells and allowed to be regrown prior to plating. * * , P < 0.01; *** * , P < 0.0001, using a two-tailed paired t test.

Article Snippet: Barcode oligonucleotides were custom synthesized on a high-density microarray slide and eluted to generate a barcoded forward primer pool (MYcroarray; Biodiscovery, LLC, Ann Arbor, MI).

Techniques: Two Tailed Test

Journal: mBio

Article Title: Origin and Dynamics of Mycobacterium tuberculosis Subpopulations That Predictably Generate Drug Tolerance and Resistance

doi: 10.1128/mbio.02795-22

Figure Lengend Snippet: Drug-tolerant populations that generate rifampicin-resistant mutants. (A) Dot plot showing resistance versus susceptible culture results for each well in experiment 2 plated at the indicated time points. Wells containing only drug-susceptible CFU are shown in blue, and those with any drug-resistant CFU are shown in red. DP indicates directly plated wells, and RP indicates regrowth plated wells with the day indicated corresponding to the experiment day on which rifampicin was washed from culture wells and allowed to be regrown prior to plating. (B) The number of unique barcodes identified in each well cultured on rifampicin following DP (red circles) versus RP (red squares) ( P = 0.008, Fisher’s exact test). (C) Individual kill curves were generated using deep sequence data of the 9 rifampicin resistance-associated barcodes identified by DP. All barcode reads above the 10-barcode count per well cutoff were included in the analysis; however, mean barcodes lower than 1 are reported (below the dotted line) when this is due to averaging barcode numbers across wells. (D) The number of unique drug resistance-associated barcodes in each DP well where no drug resistance was detected (green circle) and where resistance was detected by rifampicin plating (red circles) compared with the number of barcodes from these same wells that were not resistance associated (blue diamonds). (E) The number of unique drug resistance-associated barcodes in each DD well where no drug resistance was detected (green circle) and where resistance was detected by rifampicin plating (red circles) compared with the number of barcodes from these same wells that were not resistance associated (purple diamonds). (F) Barcode plot showing the relationship between each unique barcode detected at day 0 in DP cultures, the barcodes that were still detectable by DP during the day 16 and 20 drug tolerant phase, and the barcodes that were detected by DP in rifampicin-resistant CFU. Lines on the same vertical axis represent the same barcode. Solid red lines indicated barcodes found in drug-resistant CFU which were also detectable as resistance-associated barcodes in drug-tolerant or day 0 barcodes. Dotted red lines indicate barcodes in drug-resistant CFU that were not detected in either drug-tolerant or day 0 barcodes. The order in which barcodes are positioned across the x axis on day 0 is arbitrary. (G) Diversity and conservation of barcodes detected in drug-resistant CFU. All barcodes detected on day 0 are distributed across the top row of the panel (green lines) according to their mean relative frequency at day 0 (from low to high frequency). All of the barcodes identified in resistant CFU are assigned a specific color and position that is aligned in the figure according to their mean frequency in drug susceptible day 0 wells. Both DP and RP cultures from specific time points are marked by a different color background. Barcodes found in resistant DP or FP cultures are indicated. Color and position denote a unique barcode. When resistance barcodes were detected in independent cultures, they are shown in the same color and x axis position. The y axis denotes cultures named with the day of treatment and replicate number. (H) Venn diagram showing the resistance-associated barcodes detected in experiment 2 and experiment 3.

Article Snippet: Barcode oligonucleotides were custom synthesized on a high-density microarray slide and eluted to generate a barcoded forward primer pool (MYcroarray; Biodiscovery, LLC, Ann Arbor, MI).

Techniques: Cell Culture, Generated, Sequencing

Journal: bioRxiv

Article Title: A screen of 1,049 schizophrenia and 30 Alzheimer’s-associated variants for regulatory potential

doi: 10.1101/447557

Figure Lengend Snippet: The x-axis shows different activity thresholds for defining the set of positive controls, using data in the K562 cell line. The set of negative controls is fixed. For each set of positive controls, we show the difference with the negative controls (Y axis) and the confidence intervals for the difference. In batch 1, moderately active positive control oligos (K562 mean activity greater than ∼ 0) show significantly higher activity than the negative controls. In batch 2, only the most highly active positive control oligos show significantly higher activity than negative controls.

Article Snippet: The final pool of 11,750 uniquely barcoded oligonucleotides (see supplementary table 4) was ordered from the Broad Technology Labs (Broad Institute, Cambridge MA) The library we received from the BROAD institute was amplified, cloned and transfected as described ( ; ) with slight modifications.

Techniques: Activity Assay, Positive Control