Added example data for the tutorial to visualize isoforms

Snakefile

@@ -7,7 +7,7 @@ import shutil
 from matplotlib.backends.backend_pdf import PdfPages
 
 ##### Logistics
-configfile: "config_goeke.yaml"
+configfile: "config.yaml"
 workdir: config["outdir"]
 
 ReferenceFasta = config["genome_fasta"]
@@ -517,7 +517,7 @@ rule iupred2a_analysis:
     threads: 1
     group: "sequence_analysis"
     shell:
-        "echo {input.protein} >> {output.seq}; {input.iupred2a} -a {output.seq} long >> {output.idr}"
+        "tail +2 {input.protein} >> {output.seq}; {input.iupred2a} -a {output.seq} long >> {output.idr}"
 
 # Domain analysis
 rule interpro_scan:
@@ -585,23 +585,23 @@ rule functional_site_analysis:
         "perl {params.ps_scan} -d {params.prosite_dat} --pfscan {params.pf_scan} {input} > {output}"
 
 # Prion analysis
-rule prion_analysis:
-    input:
-        "Results/Genes_temp/{gene}/transcripts/{transcript}_protein.fa"
-    output:
-        "Results/Genes_temp/{gene}/transcripts/{transcript}_func_prion.txt"
-    params:
-        plaac = config["plaac_path"],
-        gene = "{gene}"
-    resources:
-        memory = 10,
-        time = 1,
-        tmpdir = 0
-    priority: 8
-    threads: 1
-    group: "sequence_analysis"
-    shell:
-        "java -jar {params.plaac} -i {input} -p all > {output}"
+# rule prion_analysis:
+#    input:
+#        "Results/Genes_temp/{gene}/transcripts/{transcript}_protein.fa"
+#    output:
+#        "Results/Genes_temp/{gene}/transcripts/{transcript}_func_prion.txt"
+#    params:
+#        plaac = config["plaac_path"],
+#        gene = "{gene}"
+#    resources:
+#        memory = 10,
+#        time = 1,
+#        tmpdir = 0
+#    priority: 8
+#    threads: 1
+#    group: "sequence_analysis"
+#    shell:
+#        "java -jar {params.plaac} -i {input} -p all > {output}"
 
 # Combine functional feature analysis on individual isoforms
 def functional_files():
@@ -611,7 +611,7 @@ def functional_files():
     if (config["pfam"]): files.append(rules.interpro_scan.output)
     if (config["porter"]): files.append(rules.porter_analysis.output)
     if (config["pfScan"]): files.append(rules.functional_site_analysis.output)
-    if (config["prion"]): files.append(rules.prion_analysis.output)
+#    if (config["prion"]): files.append(rules.prion_analysis.output)
     return files
 rule individual_transcript_analysis:
     input:

config.yaml

@@ -1,4 +1,5 @@
 ##### IsoTV Config File
+
 pipeline: "IsoTV"
 repo: "https://github.molgen.mpg.de/MayerGroup/IsoTV"
 
@@ -28,24 +29,7 @@ genome_annot: "/path/to/gencode.v32.primary_assembly.annotation.gtf"
 # samples with .bottom extension must be placed in the RawData folder
 # condition_replicate
 samples:
-    A549_1: "A549_r1_r3"
-    A549_2: "A549_r2_r1"
-    #A549_3: "A549_r3_r2"
-    A549_5: "A549_r5_r3"
-    HCT116_1: "HCT116_r1_r4"
-    HCT116_3: "HCT116_r3_r2"
-    HCT116_4: "HCT116_r4_r1"
-    HCT116_5: "HCT116_r5_r1"
-    HEPG2_1: "HEPG2_r1_r1"
-    HEPG2_4: "HEPG2_r4_r2"
-    HEPG2_5: "HEPG2_r5_r3"
-    K562_1: "K562_r1_r2"
-    K562_2: "K562_r2_r1"
-    K562_3: "K562_r3_r1"
-    K562_4: "K562_r4_r2"
-    MCF7_1: "MCF7_r1_r2"
-    MCF7_3: "MCF7_r3_r3"
-    MCF7_4: "MCF7_r4_r2"
+    condition1: "replicate1"
 
 threads: 16
 
@@ -111,7 +95,7 @@ gene_file: "/path/to/genes.tab"
 
 ### Output file and folder
 
-output_plots: "loveisgone.pdf"
+output_plots: "test.pdf"
 
 ### Processed file paths - required if not using ONT long read processing workflow
 
@@ -122,7 +106,7 @@ counts_data: "/path/to/Results/Quantification_nonred/all_counts_deseq2norm_all.t
 # Is data continuous
 continuous: FALSE
 
-### Tools paths and functional analysis
+### External tool paths and functional analysis
 
 aa: TRUE
 

config_example_pdk2.yaml

@@ -0,0 +1,155 @@
+##### IsoTV Config File
+
+pipeline: "IsoTV"
+repo: "https://github.molgen.mpg.de/MayerGroup/IsoTV"
+
+### Ouput directory
+
+outdir: "example"
+
+### General pipeline parameters:
+
+basecalling: FALSE
+preprocess: FALSE
+annotation: TRUE
+quantification: TRUE
+
+###### ONT long read processing config
+### Basecalling pipeline parameters
+
+guppy: "/path/to/Guppy324/bin/guppy_basecaller"
+flowcell: FLO-MIN106
+kit: SQK-DCS109
+
+### Reference Files
+genome_fasta: "/path/to/GRCh38.p12.primary_assembly.genome.fa"
+genome_annot: "/path/to/gencode.v32.primary_assembly.annotation.gtf"
+
+### Samples
+# samples with .bottom extension must be placed in the RawData folder
+# condition_replicate
+samples:
+    A549_1: "A549_r1_r3"
+    A549_2: "A549_r2_r1"
+    A549_5: "A549_r5_r3"
+    HCT116_1: "HCT116_r1_r4"
+    HCT116_3: "HCT116_r3_r2"
+    HCT116_4: "HCT116_r4_r1"
+    HCT116_5: "HCT116_r5_r1"
+    HEPG2_1: "HEPG2_r1_r1"
+    HEPG2_4: "HEPG2_r4_r2"
+    HEPG2_5: "HEPG2_r5_r3"
+    K562_1: "K562_r1_r2"
+    K562_2: "K562_r2_r1"
+    K562_3: "K562_r3_r1"
+    K562_4: "K562_r4_r2"
+    MCF7_1: "MCF7_r1_r2"
+    MCF7_3: "MCF7_r3_r3"
+    MCF7_4: "MCF7_r4_r2"
+
+threads: 16
+
+# Use pychopper results
+pychopper: TRUE
+
+# Use annotation to improve splice junction mapping (minimap2 --junc_bed parameter)
+minimap2_opts_junction: TRUE
+
+# Minimum read quality to keep:
+min_mean_q: 5
+
+# Stringency of porechop heuristic:
+porechop_heu_stringency: 0.25
+
+# Options passed to minimap2 during indexing:
+minimap2_index_opts: "-k14"
+
+# Extra options passed to minimap2:
+minimap2_opts: "-uf"  # required for stranded data e.g. when pychopper filtered
+
+# Minmum mapping quality:
+minimum_mapping_quality: 5
+
+# Options passed to spliced_bam2gff:
+spliced_bam2gff_opts: "-s"  # required for stranded data e.g. when pychopper filtered
+
+# -c parameter:
+minimum_cluster_size: 3
+
+# -p parameter:
+minimum_isoform_percent: 1
+
+# -d parameter:
+exon_boundary_tolerance: 10
+
+# -e parameter:
+terminal_exon_boundary_tolerance: 50
+
+# Extra options passed to minimap2 when mapping polished reads:
+minimap2_opts_polished: "-uf"   # required for stranded data e.g. when pychopper filtered
+
+# Options passed to spliced_bam2gff when converting alignments of polished reads:
+spliced_bam2gff_opts_pol: "-s"  # required for stranded data e.g. when pychopper filtered
+
+# Options passed to collapse_partials when collapsing fragmentation artifacts
+# Internal exon boundary tolerance:
+collapse_internal_tol: 5
+
+# Five prime boundary tolerance:
+collapse_five_tol: 500
+
+# Three prime boundary tolerance:
+collapse_three_tol: 50
+
+maximum_secondary: 200
+secondary_score_ratio: 1
+
+##### Feature Analysis Config
+### Input genestt
+
+gene_file: "data/genes.tab"
+
+### Output file and folder
+
+output_plots: "test.pdf"
+
+### Processed file paths - required if not using ONT long read processing workflow
+
+nanopore_gtf: "data/PDK2.nanopore.gtf"
+polished_reads: "data/PDK2.transcriptome.fas"
+counts_data: "data/PDK2.counts.txt"
+
+# Is data continuous
+continuous: FALSE
+
+### External tool paths and functional analysis
+
+aa: TRUE
+
+iupred2a_path: "/path/to/iupred2a/iupred2a.py"
+iupred2a: TRUE
+
+brewery_path: "/path/to/Brewery/Brewery.py"
+porter: TRUE
+
+interproScan_path: "/path/to/my_interproscan/interproscan-5.38-76.0/interproscan.sh"
+pfam: TRUE
+
+prositeScan_path: "/path/to/ps_scan/ps_scan.pl"
+pfScan_path: "/path/to/ps_scan/pfscan"
+prositeDat_path: "/path/to/prosite.dat"
+pfScan: TRUE
+
+#plaac_path: "/path/to/plaac.jar"
+#prion: FALSE
+
+minIsoTPM: 1
+maxIsoNum: 8
+minIsoPct: 10
+
+### Misc paths
+
+java : "/pkg/openjdk-11.0.3.2-0/profile"
+
+lock1: "/.../lock.txt"
+lock2: "/.../lock2.txt"

example/data/PDK2.counts.txt

@@ -0,0 +1,3 @@
+class_code,A549_1,A549_2,A549_5,HCT116_1,HCT116_3,HCT116_4,HCT116_5,HEPG2_1,HEPG2_4,HEPG2_5,K562_1,K562_2,K562_3,K562_4,MCF7_1,MCF7_3,MCF7_4,transcript_id,gene_name,ref_transcript,gene_id,gene_type
+=,10.7123842660785,15.0113335568354,16.2651214801261,0,11.5434688558883,10.49952673775,10.1420512602935,0,7.86124895592787,10.4796284084991,2.44646283064557,0,2.19493190223773,1.57696537194305,3.82454516596614,10.8899378184551,11.490643684754,TCONS_00023419,PDK2,ENST00000505897.5,ENSG00000005882.12,protein_coding
+=,25.7097222385884,20.0151114091139,17.2816915726339,17.5114992178197,24.75990421263,27.2674276472911,23.6647862740183,8.05561597267535,19.6531223898197,16.1225052438448,1.46787769838734,0,2.19493190223773,0,15.2981806638645,27.8298410916074,33.2994163925523,TCONS_00023420,PDK2,ENST00000503176.6,ENSG00000005882.12,protein_coding

example/data/PDK2.nanopore.gtf

@@ -0,0 +1,17 @@
+chr17	pinfish	transcript	50095380	50106363	.	+	.	transcript_id "TCONS_00023419"; gene_id "XLOC_007312"; gene_name "PDK2"; oId "f3fe429f-0296-4cc4-a2cf-aa295cd23fbe|60"; cmp_ref "ENST00000505897.5"; class_code "="; tss_id "TSS13403";
+chr17	pinfish	exon	50095380	50095553	.	+	.	transcript_id "TCONS_00023419"; gene_id "XLOC_007312"; exon_number "1";
+chr17	pinfish	exon	50097423	50097564	.	+	.	transcript_id "TCONS_00023419"; gene_id "XLOC_007312"; exon_number "2";
+chr17	pinfish	exon	50105371	50105442	.	+	.	transcript_id "TCONS_00023419"; gene_id "XLOC_007312"; exon_number "3";
+chr17	pinfish	exon	50105885	50106363	.	+	.	transcript_id "TCONS_00023419"; gene_id "XLOC_007312"; exon_number "4";
+chr17	pinfish	transcript	50095387	50111369	.	+	.	transcript_id "TCONS_00023420"; gene_id "XLOC_007312"; gene_name "PDK2"; oId "bf7faaeb-3870-4b4a-8d23-854e564f9462|14"; cmp_ref "ENST00000503176.6"; class_code "="; tss_id "TSS13403";
+chr17	pinfish	exon	50095387	50095553	.	+	.	transcript_id "TCONS_00023420"; gene_id "XLOC_007312"; exon_number "1";
+chr17	pinfish	exon	50097423	50097564	.	+	.	transcript_id "TCONS_00023420"; gene_id "XLOC_007312"; exon_number "2";
+chr17	pinfish	exon	50105371	50105442	.	+	.	transcript_id "TCONS_00023420"; gene_id "XLOC_007312"; exon_number "3";
+chr17	pinfish	exon	50105885	50106069	.	+	.	transcript_id "TCONS_00023420"; gene_id "XLOC_007312"; exon_number "4";
+chr17	pinfish	exon	50106794	50106883	.	+	.	transcript_id "TCONS_00023420"; gene_id "XLOC_007312"; exon_number "5";
+chr17	pinfish	exon	50107076	50107153	.	+	.	transcript_id "TCONS_00023420"; gene_id "XLOC_007312"; exon_number "6";
+chr17	pinfish	exon	50108156	50108232	.	+	.	transcript_id "TCONS_00023420"; gene_id "XLOC_007312"; exon_number "7";
+chr17	pinfish	exon	50108319	50108417	.	+	.	transcript_id "TCONS_00023420"; gene_id "XLOC_007312"; exon_number "8";
+chr17	pinfish	exon	50108612	50108719	.	+	.	transcript_id "TCONS_00023420"; gene_id "XLOC_007312"; exon_number "9";
+chr17	pinfish	exon	50109287	50109400	.	+	.	transcript_id "TCONS_00023420"; gene_id "XLOC_007312"; exon_number "10";
+chr17	pinfish	exon	50109957	50111369	.	+	.	transcript_id "TCONS_00023420"; gene_id "XLOC_007312"; exon_number "11";

example/data/PDK2.transcriptome.fas

@@ -0,0 +1,4 @@
+>TCONS_00023419
+GGTGCGCGAGCGCTGCCCGCGCGGGGACCACAACCAAAGTCGCGGCCGCCGCAGCCATGCGCTGGGTGTGGGCGCTGCTGAAGAATGCGTCCCTGGCAGGGGCGCCCAAGTACATAGAGCACTTCAGCAAGTTCTCCCCGTCCCCGCTGTCCATGAAGCAGTTTCTGGACTTCGGATCCAGCAATGCCTGTGAGAAAACCTCCTTCACCTTCCTCAGGCAGGAGCTGCCTGTGCGCCTGGCCAACATCATGAAAGAGATCAACCTGCTTCCCGACCGAGTGCTGAGCACACCCTCCGTGCAGCTGGTGCAGAGCTGGTATGTCCAGAGCCTCCTGGACATCATGGAGTTCCTGGACAAGGATCCCGAGGACCATCGCACCCTGAGCCAGTTCACTGACGCCCTGGTCACCATCCGGAACCGGCACAACGACGTGGTGCCCACCATGGCACAAGGCGTGCTTGAGTACAAGGACACCTACGGCGATGACCCCGTCTCCAACCAGAACATCCAGTACTTCCTGGACCGCTTCTACCTCAGCCGCATCTCCATCCGCATGCTCATCAACCAGCACAGTGGGTGCCGGCCACAGCGGCGGGGAGCGGGCGGTGGGGGGGGCGGTGCTGGGGCCCAGGGCCGGGCTGCTGAGGGGACCTAGACCACTCTTCAGAACCCCACAAAGGGAGTCTTTGAATAGTTACTCCAGTAACTATGGAGTTAATGGCTCCAACATGGAAAAATAAAATTTTTCTTTCTCATTGATTTTCCATTTCAAAACGTTTTGTTTTCAGTGTTTGCAAAATGTAAAATTATGTCACATCTTTAAAAGAATGTTTAATTTAGTATTTATAAAAACTCTCATTATGTTC
+>TCONS_00023420
+GAGCGCTGCCCGCGCGGGGACCACAACCAAAGTCGCGGCCGCCGCAGCCATGCGCTGGGTGTGGGCGCTGCTGAAGAATGCGTCCCTGGCAGGGGCGCCCAAGTACATAGAGCACTTCAGCAAGTTCTCCCCGTCCCCGCTGTCCATGAAGCAGTTTCTGGACTTCGGATCCAGCAATGCCTGTGAGAAAACCTCCTTCACCTTCCTCAGGCAGGAGCTGCCTGTGCGCCTGGCCAACATCATGAAAGAGATCAACCTGCTTCCCGACCGAGTGCTGAGCACACCCTCCGTGCAGCTGGTGCAGAGCTGGTATGTCCAGAGCCTCCTGGACATCATGGAGTTCCTGGACAAGGATCCCGAGGACCATCGCACCCTGAGCCAGTTCACTGACGCCCTGGTCACCATCCGGAACCGGCACAACGACGTGGTGCCCACCATGGCACAAGGCGTGCTTGAGTACAAGGACACCTACGGCGATGACCCCGTCTCCAACCAGAACATCCAGTACTTCCTGGACCGCTTCTACCTCAGCCGCATCTCCATCCGCATGCTCATCAACCAGCACACCCTCATCTTTGATGGCAGCACCAACCCAGCCCATCCCAAACACATCGGCAGCATCGACCCCAACTGCAACGTCTCTGAGGTGGTCAAAGATGCCTACGACATGGCTAAGCTCCTGTGTGACAAGTATTACATGGCCTCACCTGACCTGGAGATCCAGGAGATCAATGCAGCCAACTCCAAACAGCCGATTCACATGGTCTACGTCCCCTCCCACCTCTACCACATGCTCTTTGAGCTCTTCAAGAATGCCATGAGGGCGACTGTGGAAAGCCATGAGTCCAGCCTCATTCTCCCACCCATCAAGGTCATGGTGGCCTTGGGTGAGGAAGATCTGTCCATCAAGATGAGTGACCGAGGTGGGGGTGTTCCCTTGAGGAAGATTGAGCGACTCTTCAGCTACATGTACTCCACAGCACCCACCCCCCAGCCTGGCACCGGGGGAACGCCGCTGGCTGGCTTTGGTTATGGGCTCCCCATTTCCCGCCTCTACGCCAAGTACTTCCAGGGAGACCTGCAGCTCTTCTCCATGGAAGGCTTTGGGACCGATGCTGTCATCTATCTCAAGGCCCTGTCCACGGACTCGGTGGAGCGCCTGCCTGTCTACAACAAGTCAGCCTGGCGCCACTACCAGACCATCCAGGAGGCCGGCGACTGGTGTGTGCCCAGCACGGAGCCCAAGAACACGTCCACGTACCGCGTCAGCTAAGGGCCGCCGTGCATCTGCACCTGAGAGGACGGACTGCCGCCTCTGGGTCCCCCCACCGTGGTGCCCCTCACCATCCTCCTGGGGGAGCAGGGGGTGGGTTCTCCCTGATGACCAGGTTCTGTCTCTATGGAAGTCACTGCGGTGATAGGTCTGTGATGGTCCCTAAGTGCCAGTCCATCTCTGTGGAGACCCCTCGGTGGCCTCCCTATCTCTGTGGGCGATGCCTGAGGGTTAGGGATGTCTCCACCCTGATGGGGTGTCCCAGAGACATTTTCCCATGGCAGTCCTCCTCTCTGAGACCAGGGCTGTCACTTTTCTGCCAGGGGTACTGGGTCCCCCTCAGCACCCTCCACAGCACAGGCCTTCCAAGTGGATGTCCCGTTGCCTTATTCCCCCAGCCCACAAAGGCACCCTGGCCTTGGCCTGCTGAAGTGTTAGGAAGAGGGTGGGTGCCCTCCAGACCTGGGGACTGAGTGGGGAAAGGAGTTACACCCGTGAGTGGGGAATGAGGCTGGTCCTGCAGCCTCTCCCTCCGCTCAGGGCTTGAAGGTCGGTGGCGGAGGGGGTGGCTCTCACAGGGCCCAACTCTAAAGTGGAAGAACCTTGTTAGACCGAGAGCTTGCCATCCAGCCAAGCTGCTCGAGGCCCTGCAGTGGCCTTGGCAATGTCTGTGCCACCTCCTGAGCCCTCCCAGCATGTCCTCACATGCTCATGCCCACCCGCTCCTCCACAAGCCTAGTCCATCCTGCCTGAGCTCCAGCCCCCAGCCCCCACTGTGCCCAGACATGTGTGCTCAGGGTGGCTTTCTCCCTAGGACCTTCTGTGTATATAGTTAGTTTTATAACCCTGAATGCCCCCACCCTTCCCCTAAGCACACAGGGGTTAAAGCTGTGTGTCCCTCCCAGTGGCTGTGGCAGTGACAGTGACACCCACACCCACAGTAAAGAGGAGACTGAATGAGACTGGCCTGGCTGCATCCCTGGGGGAGGGGACCCACACGTGCGCACGTACACACGCACACTGCGGTGCCCTGTGACCACCACATGACGCACCGGGGACGTCCGGCCCAGCTCAGGACTGTGAGTAAAACACTGGGCAAAGCCATCTGTTCAGAACGCCGGATGGCCCAGCACCTGGTGACAGACCTGTTGTCCCACCATACTCAGGGCCCAGCAGAGTCCGGCCAACCCAACAACTGGAGCAGACAGAAATTCTGGTCACTGGCACATCATAAGGATTTATTGAAATAAATTGAGAACTGCCTCCCCTTCA

example/data/genes.tab

@@ -0,0 +1 @@
+PDK2

scripts/individual_transcript_analysis.py

@@ -17,7 +17,7 @@
 
     elif ("iupred2a" in filename):
         output.append("#####IUPred2A Analysis")
-        lines = lines[7:]
+        lines = lines[6:]
         for position in lines:
             output.append(position.strip())
 

scripts/visualization.py

@@ -204,10 +204,10 @@ def plotSequenceAnalysis(transcripts_plot, colors, longest_length_protein, fig,
         buffer_phos = 0.075
         buffer_den = 0.5
 
-        lg = fig.add_subplot(gs[curr_row_panel:curr_row_panel+5, 0])
+        lg = fig.add_subplot(gs[curr_row_panel:curr_row_panel+total_features, 0])
         lg.set_xlim((-0.11,0.11))
 
-        ax = fig.add_subplot(gs[curr_row_panel:curr_row_panel+5, 1:])
+        ax = fig.add_subplot(gs[curr_row_panel:curr_row_panel+total_features, 1:])
         ax.set_xlim((-0.5,longest_length_protein + 1))
         ax.patch.set_facecolor(transcript_color)
         ax.patch.set_alpha(0.15)
@@ -380,7 +380,7 @@ def plotSequenceAnalysis(transcripts_plot, colors, longest_length_protein, fig,
         ax.spines['left'].set_visible(False)
         #ax.spines['bottom'].set_visible(False)
         ax.set_yticks([], [])
-        curr_row_panel += total_features
+        curr_row_panel += total_features + 1
 
 def plotLegend(plt,fig,gs,total_gs):
     ax = fig.add_subplot(gs[total_gs-2:total_gs, 0:5])
@@ -399,10 +399,10 @@ def plotLegend(plt,fig,gs,total_gs):
     ss_abbvs = ["Alpha Helix","Beta Sheet","Other"]
     for i in range(0,len(ss_abbvs)):
         if (ss_abbvs[i] == "Other"):
-            plt.gca().add_patch(Rectangle((3,float(i*2)/5),3,1.0/5,edgecolor="black",facecolor=colors_ss3[i]))
+            plt.gca().add_patch(Rectangle((3,float(i*2 + 4)/5),3,1.0/5,edgecolor="black",facecolor=colors_ss3[i]))
         else:
-            plt.gca().add_patch(Rectangle((3,float(i*2)/5),3,1.0/5,edgecolor=colors_ss3[i],facecolor=colors_ss3[i]))
-        ax.text(7,float(i*2)/5 + 0.25,ss_abbvs[i],horizontalalignment = "left",verticalalignment = "top")
+            plt.gca().add_patch(Rectangle((3,float(i*2 + 4)/5),3,1.0/5,edgecolor=colors_ss3[i],facecolor=colors_ss3[i]))
+        ax.text(7,float(i*2 + 4)/5 + 0.25,ss_abbvs[i],horizontalalignment = "left",verticalalignment = "top")
     ax.annotate("Secondary Structure",(6.75,2), fontsize = 14, color = "black", ha = "center", va = "center", weight='bold')
     ax.axis("off")
 
@@ -506,6 +506,7 @@ def plotLegend(plt,fig,gs,total_gs):
 
 total_gs = 1
 transcripts_plot = sorted(list(transcripts.keys()))
+
 if (snakemake.config["quantification"]):
     df_temp = data[data["gene_name"] == gene]
     data_gene = df_temp[samples].sum().to_frame().transpose()
@@ -526,7 +527,7 @@ def plotLegend(plt,fig,gs,total_gs):
     for transcript_id in transcripts_delete:
         transcripts_plot.remove(transcript_id)
     total_gs += len(transcripts_plot) + 1
-total_gs += total_features * len(transcripts_plot) + 2
+total_gs += (total_features + 1) * len(transcripts_plot) + 2
 fig = plt.figure(figsize = (21,total_gs))
 gs = fig.add_gridspec(total_gs, 15)
 
@@ -565,7 +566,7 @@ def plotLegend(plt,fig,gs,total_gs):
     else:
         plotSequenceAnalysis(transcripts_plot, colors, longest_length, fig, gs, total_gs)
 
-    total_gs += total_features * len(transcripts_plot) + 2
+    total_gs += (total_features + 1) * len(transcripts_plot) + 2
     plotLegend(plt,fig,gs,total_gs)
 
 plt.subplots_adjust(wspace=0.05, hspace=0.05)