Journal: eLife

Article Title: Functional imaging of nine distinct neuronal populations under a miniscope in freely behaving animals

doi: 10.7554/eLife.110277

Figure Lengend Snippet: ( a ) Surgical paradigm. In a TetO-GCaMP6s × CaMKII-tTa mouse, 9 AAV retro viruses are injected into downstream brain regions and gradient-index (GRIN) lens implanted into the target region. ( b ) Simultaneous recording of GCaMP6s (top) and behavior (bottom) during a social memory task. Scale bar = 100 µm ( c ) GCaMP6s recordings are processed. Constrained non-negative matrix factorization (CNMF)-defined ROIs (top) and ΔF/F traces (bottom) are exported. Scale bar = 100 µm. ( d ) Mice are head fixed and FOV under the GRIN lens imaged using the multiplexed lambda method. ( e ) Transformations are determined using anatomical background images to co-register the two imaging platforms. The transformations are applied to CNMF-defined ROIs. Scale bar = 100 µm. ( f ) Multispectral data are collected for each ROI (top) and an average spectral fingerprint for all ROIs is generated (bottom). Mean ±1.5 SD. Scale bar = 100 µm. ( g ) A linear unmixing model is applied to determine the fluorophore contribution for each ROI. Scale bar = 100 µm. ( i ) Neural activity is sorted by cell type. Scale bars = 20 ΔF/F (vertical), 20 s (horizontal).

Article Snippet: We extracted spatial and temporal components of neuronal activity from miniscope videos using constrained non-negative matrix factorization for endoscopic data (CNMF-E) implemented through the Inscopix Data Processing Software.

Techniques: Injection, Imaging, Generated, Activity Assay

Journal: bioRxiv

Article Title: Identification and characterization of constrained non-exonic bases lacking predictive epigenomic and transcription factor binding annotations

doi: 10.1101/722876

Figure Lengend Snippet: An example genomic locus illustrating CNEP scores. The top line is the CNEP score track. In general, the CNEP score ranges between 0 and 1, but in this image the y-scale is capped at 0.5. Below the CNEP score track is the GENCODE gene annotation track, PhastCons element track, and then the PhastCons score track. Below the PhastCons score track is the Conservation Signature Score by CNEP (CSS-CNEP), followed by the UCSC Genome Browser ENCODE TF binding summary track (Txn Fac ChIP V2), and the ENCODE DNase I summary track (DNase Clusters V3). These tracks are then followed by chromatin state annotation across 127 samples based on a previously defined 25-state ChromHMM annotation based on imputed data . A color legend for the chromatin state annotations is also available in .

Article Snippet: We developed the Constrained Non-Exonic Predictor (CNEP) to make a probabilistic prediction based on features defined from large-scale epigenomics and TF binding data as to whether a base in the human genome will be in a constrained non-exonic element previously called from comparative genomics sequence analysis ( ).

Techniques: Binding Assay

Journal: bioRxiv

Article Title: Identification and characterization of constrained non-exonic bases lacking predictive epigenomic and transcription factor binding annotations

doi: 10.1101/722876

Figure Lengend Snippet: (a) The graph shows the cumulative distribution of the CNEP score genome-wide (green), in PhastCons constrained non-exonic (CNE) bases (red), and bases that are not in PhastCons constrained elements and also not in exons (notCNE) (blue). (b) A scatter plot with each point corresponding to one feature that CNEP uses. The x-axis shows the average CNEP score in bases that have the feature present, while the y-axis shows the expected CNEP score based on the feature’s overlap with constrained non-exonic bases. Only 48,364 features that cover at least 200kb are shown. The full table corresponding to these values can be found in Supplementary Table 2 . The diagonal line is the y=x line. The vertical line corresponds to the genome-wide average CNEP score. The horizontal line corresponds to the genome-wide expected average CNEP score. (c) A plot showing the average fraction of the 350 Roadmap DNase I experiments in which the base overlaps a called peak for each CNEP score value, rounded to the nearest 0.001, covering at least 1000 bases. In total, there was 1000 such values. (d) A plot showing the average fraction of bases assigned across the 127 epigenomes to each of 14-groups based on 25 ChromHMM chromatin states previously assigned the same color for each CNEP score value, rounded to the nearest 0.001. A color with the state abbreviations is displayed at the bottom of the panel. (e) A plot of the ROC curve for the CNEP score predicting PhastCons non-exonic bases. The area under this curve is 0.79. Also shown is the performance of individual features and several baseline or existing scores ( Methods ). (f) A plot of the precision-recall curve for the CNEP score identifying PhastCons non-exonic bases and the same individual features and baseline and existing scores as (e). ROC and precision-recall curves for other constrained element sets can be found in Supplementary Fig. 3 .

Article Snippet: We developed the Constrained Non-Exonic Predictor (CNEP) to make a probabilistic prediction based on features defined from large-scale epigenomics and TF binding data as to whether a base in the human genome will be in a constrained non-exonic element previously called from comparative genomics sequence analysis ( ).

Techniques: Genome Wide

Journal: bioRxiv

Article Title: Identification and characterization of constrained non-exonic bases lacking predictive epigenomic and transcription factor binding annotations

doi: 10.1101/722876

Figure Lengend Snippet: (a) Heatmap representation of conservation state parameters of the ConsHMM conservation state model defined in Ref. 24. Rows correspond to different conservation states. The states were previously clustered into eight groups based on these parameters and colored accordingly. The left half indicates for each state the probability of each species having a nucleotide aligning the human reference genome, regardless of whether it matches the human reference. The right half indicates for each state the probability of each species having a nucleotide matching the human reference genome. Individual columns correspond to species, the names of which are available in Ref. 24. The major groups of species are colored and labeled. Color scale for the heatmap is shown at the bottom. (b) The first column reports the genome % of each state excluding chrY. The second column contains the AUC of the CNEP score for predicting CNE bases in each state, where for this and the remaining columns the constrained elements are from PhastCons. CNE bases that are not in the target conservation state are excluded when computing the AUC. The next column reports the AUC when exons are first extended by 200bp. The next three columns contain the fold enrichment for CNE bases, Low_CNE bases, and the ratio of the enrichment of Low_CNE bases to CNE bases. The next three columns contain the fold enrichment for notCNE bases, High_notCNE bases, and the ratio of the enrichment of High_notCNE bases to notCNE bases. The last column shows the average CSS-CNEP score in CNE bases in the state. Adjacent pairs of columns on a red-white color scale are on the same color scale. The other columns are on a column specific color scale. The bottom row gives the base % of the genome for the four sets. Results based on all the constrained element sets can be found in Supplementary Fig. 6. (c) Plot showing the AUC values for each ROC curve for predicting PhastCons CNE bases in specific ConsHMM conservation states shown in Supplementary Fig. 7a . The AUC values are displayed from left to right in decreasing value and positioned along the x-axis based on the cumulative fraction of PhastCons CNE bases they cover. The points are color-coded based on the conservation state coloring shown in (a). States with the highest AUC values are labeled. Similar plots, but for additional constrained element sets can be found in Supplementary Fig. 7 and based on excluding bases within 200bp of exons from the positives can be found in Supplementary Fig. 8. (d) Precision-recall analysis for predicting PhastCons Low_CNE bases among CNE bases using additional comparative genomics information. In this analysis, Low_CNE bases are positive bases and High_CNE bases are negative bases. The predictions based on the CSS-CNEP score as well as the PhastCons, PhyloP, and GERP++ constraint scores are shown based on ranking from lowest to highest value. Also shown for the PhastCons, PhyloP, and GERP++ scores are precision-recall curves, based on dividing a score into four hundred bins and ordering the bins based on increasing enrichment on a training set containing separate positions than used for the evaluation ( Methods ). The plot also shows the cumulative precision recall of the conservation states when ordered based on enrichment for Low_CNE bases in the training data. Additionally, a single point is shown for each of the other three constrained element sets corresponding to predictions based on bases not covered by them. Similar plots for additional constrained elements can be found in Supplementary Fig. 9 .

Article Snippet: We developed the Constrained Non-Exonic Predictor (CNEP) to make a probabilistic prediction based on features defined from large-scale epigenomics and TF binding data as to whether a base in the human genome will be in a constrained non-exonic element previously called from comparative genomics sequence analysis ( ).

Techniques: Labeling

Journal: bioRxiv

Article Title: Identification and characterization of constrained non-exonic bases lacking predictive epigenomic and transcription factor binding annotations

doi: 10.1101/722876

Figure Lengend Snippet: (a) The plot shows for PhastCons High_CNE, CNE, Low_CNE, High_notCNE, notCNE, and Low_CNE bases the proportional site frequency spectrum based on a set of 105 unrelated individuals in the YRI population in terms of # SNPs per base pair eligible for a SNP to be called, normalized for the number of sites with a variant in each set ( Methods ). The last column includes all SNPs with minor allele count greater than 10. (b) Similar plot to (a), except showing the absolute site frequency spectrum per base pair eligible for a SNP to be called normalized by estimated mutation rates. Corresponding plots for additional constrained elements can be found in Supplementary Fig. 10 and plots controlling for difference in background selection can be found in Supplementary Fig. 11 . Plots at higher thresholds of the CNEP score for notCNE bases can be found in Supplementary Fig. 12. (c) The plot shows the difference of the distribution of motif enrichments relative to the distribution for a randomized set of the motifs for the PhastCons High_CNE, CNE, Low_CNE, High_notCNE, notCNE, and Low_CNE bases. The x-axis is the rank position of the motif among the 1,646 motifs. The y-axis is the difference between the log 2 fold enrichment based on the actual motif calls and the median log 2 fold enrichment from three randomized versions at the same rank position ( Methods ). Similar plots for other constrained elements can be found in Supplementary Fig. 13 and at other thresholds for defining notCNE high bases in Supplementary Fig. 14. (d) Scatter plot of individual motif enrichments. The x and y axes corresponds to the log 2 fold enrichments in PhastCons Low_CNE and High_CNE bases respectively. The blue lines separate the three regions used for the GO enrichment analysis, High_CNE strongly preferred, High_CNE moderately preferred, and Low_CNE preferred, where at least one of the Low_CNE or the High_CNE log 2 enrichment is greater than or equal to 0.5 ( Supplementary Table 6 ). The gray line is the y=x line where both Low_CNE and High_CNE log 2 enrichments are less than 0.5. Similar plots based on other thresholds of the CNEP score can be found in Supplementary Fig. 15 . Selected motifs are labeled. (e) The distribution of enrichments for DNase I Hypersensitive Sites (DHS) from 156 experiments in mouse, where the sites are mapped to human and enrichments are computed relative to enrichments for a randomized DHS, for PhastCons High_CNE, CNE, Low_CNE, High_notCNE, notCNE, and Low_CNE bases ( Methods ). Similar plots for other constrained elements can be found in Supplementary Fig. 16. (f) A bar graph corresponding to the enrichments shown in (e) for Low_CNE bases. Bars are colored to indicate if the experiment is of whole brain or cerebrum (red), embryonic day 11.5 (dark blue), or neither (gray). Similar plots for other constrained elements can be found in Supplementary Fig. 17 . A table of the enrichment values can be found in Supplementary Table 7 .

Article Snippet: We developed the Constrained Non-Exonic Predictor (CNEP) to make a probabilistic prediction based on features defined from large-scale epigenomics and TF binding data as to whether a base in the human genome will be in a constrained non-exonic element previously called from comparative genomics sequence analysis ( ).

Techniques: Variant Assay, Mutagenesis, Selection, Labeling

Journal: bioRxiv

Article Title: Identification and characterization of constrained non-exonic bases lacking predictive epigenomic and transcription factor binding annotations

doi: 10.1101/722876

Figure Lengend Snippet: Scatter plot where each point corresponds to a dataset, the x-axis is the number of bases it covers, and the y-axis is the prediction underestimate value when using the CNEP score for prediction values. Selected datasets with a high combination of base coverage and underestimate values are labeled or placed in a box if they correspond to a DNase I hypersensitive experiment of embryonic brain, spinal cord, or eye. The color of the box corresponds to brain, spinal cord, or eye as indicated in the legend. The color and shape of the points are based on whether the point corresponds to a ChIP-atlas, ENCODE portal, or ReMap 2018 dataset or the set of new GENCODE exons between v19 and v28. Only datasets with a positive underestimate value are shown. Datasets covering more than 200 million base pairs are not shown, but all had an underestimate value of less than 0.01. Three datasets that had an underestimate value greater than 0.13 are not shown, but all covered less than 9,000 base pairs. Versions of these plots based on the input features to CNEP and based on shuffled versions of the additional datasets can be found in Supplementary Fig. 19 .

Article Snippet: We developed the Constrained Non-Exonic Predictor (CNEP) to make a probabilistic prediction based on features defined from large-scale epigenomics and TF binding data as to whether a base in the human genome will be in a constrained non-exonic element previously called from comparative genomics sequence analysis ( ).

Techniques: Labeling