reference-guided assembly pilon 1.23  (Illumina Inc)

Journal: PLOS Biology

Article Title: Transcriptomic atlas throughout Coccidioides development reveals key phase-enriched transcripts of this important fungal pathogen

doi: 10.1371/journal.pbio.3003066

Figure Lengend Snippet: (A) Micrographs of fixed samples from each flask at the time of RNA harvest for one replicate of spherule growth in Converse, 39°C, 10% CO 2 . Subsequent samples were taken from the same flask over time. Endospore release was first observed on day 3 in wildtype. As expected, the ryp1∆ mutant did not form spherules under these conditions. Surprisingly, it did form smaller rounded structures of unclear significance (open arrowheads) in addition to hyphae and some chains of oblong cells (black arrows) which have not been reported previously in Coccidioides to our knowledge. (B) Quantification of the proportion of each morphology in cultures on days 4–6, n ≥ 20 fields of view counted for each sample. Underlying data can be found in . (C) Heatmap of transcript abundance over time in spherulation conditions. Transcripts that had at least 10 reads detected in at least three samples were included as mean-centered rows in this heatmap. Rows are clustered based on correlation across all columns. Log 2 (counts per million) indicated by yellow and blue shading. (D) Micrographs of fixed samples from each flask at the time of RNA harvest for one replicate of hyphal growth in Converse, 25°C. Subsequent samples were taken from the same flask over time. “Pellet” and “Hyphae” are the same biological samples prepared in different ways as described in E. Open arrows indicate hyphae forming initial arthroconidia. Black arrowheads indicate branching hyphae. Black arrows indicate chains of oblong cells similar to those observed for ryp1∆ in spherulation conditions. (E) Schematic of preparation of hyphal samples for microscopy. “Pellet” samples were placed in a 96-well plate with glass bottom and pelleted at 584 × g for 2 min prior to visualization. For “hyphal” samples, 5 µL of fixed samples containing small clumps of hyphae were placed on a slide with a coverslip prior to visualization. (F) Heatmap of transcript abundance over time in hyphal conditions, displayed in the same manner and with the same criteria for inclusion as in C.

Article Snippet: Since we had one isolate of the ryp1∆ mutant, we further audited that mutant as follows: PILON 1.23 [ ] was used to generate a reference-guided assembly of the ryp1∆ mutant genome from paired-end Illumina reads and the GCA_018416015.2 C. posadasii reference [ ].

Techniques: Mutagenesis, Microscopy

Journal: PLOS Biology

Article Title: Transcriptomic atlas throughout Coccidioides development reveals key phase-enriched transcripts of this important fungal pathogen

doi: 10.1371/journal.pbio.3003066

Figure Lengend Snippet: (A) Number of significantly differential transcripts between wildtype and the ryp1∆ mutant at each timepoint specified. Transcripts that are induced by RYP1 (higher in wildtype than ryp1∆ ) are in purple, and transcripts that are repressed by RYP1 (higher in ryp1∆ than wildtype) are in green. (B) As in A but only with timepoints where paired spherule and hyphal wildtype datasets are available to highlight morphology-dependent genes. Dark purple and dark green correspond to the number of RYP1 -dependent transcripts that are also morphology-dependent (significantly differential between wildtype spherules and wildtype hyphae) at that timepoint. (C) Scatterplot demonstrating the expression of all detected transcripts at day 3. On the x-axis, values are the ratio of wildtype spherule over wildtype hyphal transcript abundance transformed to log 2 (counts per million). On the y-axis, values are the ratio of transcript abundance in wildtype spherules over ryp1∆ in spherulation conditions, transformed to log 2 (counts per million). (D) Overlap between transcripts that are significantly differential between wildtype and the ryp1∆ mutant at all timepoints of spherulation (days 1–6, excluding the 8 h timepoint since spherules and hyphae had not appeared by then), transcripts that are differentially expressed between wildtype and the ryp1∆ mutant over all timepoints of hyphal formation (days 1, 2, 3, and 6), and transcripts that are morphology-dependent in wildtype at all comparable timepoints (days 1, 2, 3, and 6). (E) Number of significantly differential transcripts between wildtype spherules and hyphae at each timepoint specified. Transcripts with higher abundance in spherules than hyphae are yellow and transcripts with higher abundance in hyphae than spherules are blue. (F) As in E, now highlighting dark yellow and dark blue transcripts corresponding to the number of morphology-dependent transcripts that are also regulated by RYP1 at the corresponding spherule timepoint. (G) Same graph as F except the dark yellow and dark blue transcripts now correspond to the number of morphology-dependent transcripts that are regulated by RYP1 at the corresponding hyphal timepoint. (H) Overlap between transcripts that are significantly differential between wildtype spherules and hyphae at all comparable timepoints (days 1, 2, 3, and 6) and transcripts that are differentially expressed between the ryp1∆ mutant in spherulation and hyphal-inducing conditions at the same timepoints.

Article Snippet: Since we had one isolate of the ryp1∆ mutant, we further audited that mutant as follows: PILON 1.23 [ ] was used to generate a reference-guided assembly of the ryp1∆ mutant genome from paired-end Illumina reads and the GCA_018416015.2 C. posadasii reference [ ].

Techniques: Mutagenesis, Expressing, Transformation Assay

Journal: PLOS Biology

Article Title: Transcriptomic atlas throughout Coccidioides development reveals key phase-enriched transcripts of this important fungal pathogen

doi: 10.1371/journal.pbio.3003066

Figure Lengend Snippet: (A) Traces demonstrating chromosomal location of fold enrichment of ChIP signal/input in spherules (yellow) and hyphae (blue) at the designated timepoints relative to the annotated SOWgp gene. (B) As in A but demonstrating ChIP signal/input relative to the annotated RYP1 gene. (C) As in A but demonstrating ChIP signal/input relative to D8B26_005359, D8B26_005360, and D8B26_005361 genes (genes indicated by gray arrows). (D) Barplot demonstrating the proportion of S- RYP1 -dependent genes or H- RYP1 -dependent genes (as defined in 3D) whose promoters have Ryp1 binding by ChIP-Seq. (E) UpSet plot demonstrating the size of each individual set of genes whose promoters are bound by Ryp1 at each designated spherule/hyphal timepoint (bottom left) and size of overlap between each of these sets (magnitude on top, overlapping sets demonstrated by connected black circles on bottom). Each gene can only be assigned to one unique category. (F) Overlap of spherule-specific peaks (genes whose promoter is bound in spherule timepoints only, without Ryp1 binding in the corresponding hyphal timepoint or ryp1∆ mutant subjected to the same conditions as wildtype) on days 1, 2, and 4. (G) Motif enriched in DNA sequences of Ryp1 ChIP-Seq peaks found consistently in both days 2 and 4 spherule and hyphal datasets (498 sites, p = 4.7e-064 on day 4 and 193 sites, p = 3.7e-046 on day 2). (H) Percent of genes in each subset (from 3A, 3D) whose promoter regions have at least one hit for the Ryp1 binding motif in 4G. Promoters are defined as the sequence upstream of the coding sequence (CDS) start until the next upstream CDS is encountered, or 10 kb maximum. Fully RYP1 -dependent genes are those that are significantly differential between wildtype and ryp1∆ in all spherule timepoints (days 1–6) and all hyphal timepoints (days 1, 2, 3, and 6). * : p < 0.05, **: p < 0.005, by Fisher exact test. (I) Motif enriched in DNA sequences of Ryp1 ChIP-Seq peaks found uniquely in days 2 and 4 spherule datasets (>1,000 sites, p = 1.3e-044 on day 2, 215 sites, p = 4.5e-046 on day 4). (J) As in H but now for Ryp1 binding motif in I.

Article Snippet: Since we had one isolate of the ryp1∆ mutant, we further audited that mutant as follows: PILON 1.23 [ ] was used to generate a reference-guided assembly of the ryp1∆ mutant genome from paired-end Illumina reads and the GCA_018416015.2 C. posadasii reference [ ].

Techniques: Binding Assay, ChIP-sequencing, Mutagenesis, Sequencing

Journal: PLOS Biology

Article Title: Transcriptomic atlas throughout Coccidioides development reveals key phase-enriched transcripts of this important fungal pathogen

doi: 10.1371/journal.pbio.3003066

Figure Lengend Snippet: (A) Schematic of the location of the six genes in the spore-related cluster and position of hygromycin cassette integration. (B) Plots of log 2 (counts per million) over spherulation from data in for each gene in the cluster. DIT1 / DIT2 / DTR1 names from S. cerevisiae orthologs and “Cutinase” name based on Pfam hit to that domain. (C) Traces demonstrating chromosomal location of fold enrichment of Ryp1 ChIP signal/input in spherules (yellow) and hyphae (blue) at the designated timepoints relative to D8B26_005436 – D8B26_005438 genes. (D) Representative TEM images of wildtype and each DitCluster∆ mutant. The scale is the same for all images. (E) Quantification of arthroconidia cell wall width measurements of TEM images for wildtype and each DitCluster∆ mutant. ***: p < 0.0001, by unpaired t test. Underlying data can be found in .

Article Snippet: Since we had one isolate of the ryp1∆ mutant, we further audited that mutant as follows: PILON 1.23 [ ] was used to generate a reference-guided assembly of the ryp1∆ mutant genome from paired-end Illumina reads and the GCA_018416015.2 C. posadasii reference [ ].

Techniques: Mutagenesis

Journal: PLOS Biology

Article Title: Transcriptomic atlas throughout Coccidioides development reveals key phase-enriched transcripts of this important fungal pathogen

doi: 10.1371/journal.pbio.3003066

Figure Lengend Snippet: Our data uncover regulatory modules in Coccidioides development. The majority of Coccidioides transcription factors (TFs), depicted as triangles in the figure, show enhanced expression in spherules compared to hyphae. These TFs, along with Ryp1 (green hexagon) and a second regulator whose motif we report here, guide the expression of endospore-associated transcripts and secreted effectors, including proteases. Ryp1 also controls a core regulon that is expressed in both spherules and hyphae via a canonical Ryp1 motif. Finally, in arthroconidia, Ryp1 impacts the transcriptome, but likely through indirect regulation of one or more additional key regulators.

Article Snippet: Since we had one isolate of the ryp1∆ mutant, we further audited that mutant as follows: PILON 1.23 [ ] was used to generate a reference-guided assembly of the ryp1∆ mutant genome from paired-end Illumina reads and the GCA_018416015.2 C. posadasii reference [ ].

Techniques: Expressing