From 5e47b2e478c0a42fbfdd608f922ebc4430d37978 Mon Sep 17 00:00:00 2001
From: Daniel Mapleson <d.mapleson@gmail.com>
Date: Sun, 25 Feb 2024 20:59:28 +0000
Subject: [PATCH] Normalisation and bug fixes (#39)

* fix(essentiality): essentiality analysis report now contains data

Previously we were using the .all.csv as input to the essentiality report file, however this expect the .tsv input from the insertion site analysis output.  This fix changes that.

Also we remove the .all.csv file from output generated by the R script as it doesn't serve any purpose.

Also updated the docs.

fixing stuff

* feat(normalisation): adding plot file normalisation to the compare pipeline

We add normalisation to the beginning of the plot compare pipeline.

fix no normalisation issue

---------

Co-authored-by: Sarah Bastkowski <sarah.bastkowski@quadram.ac.uk>
---
 README.md                                     |   3 +
 docs/pipeline_combine.png                     | Bin 38292 -> 40366 bytes
 docs/pipeline_combine.pu                      |   8 +-
 quatradis/comparison/cli.py                   |  16 +-
 quatradis/comparison/comparison.py            |  30 ++-
 quatradis/comparison/essentiality.py          |   3 +-
 quatradis/comparison/essentiality_analysis.py |  66 +++--
 quatradis/comparison/scatterplot.py           |   2 +-
 quatradis/comparison/split.py                 |   2 +-
 quatradis/comparison/tradis_comparison.R      |   2 +-
 quatradis/pipelines/compare.smk               |  81 +++---
 quatradis/pipelines/pipeline.py               | 255 +++++++++++++-----
 quatradis/tisp/cli.py                         |   7 +-
 quatradis/tisp/generator/from_values.py       |  10 +-
 quatradis/tisp/normalise.py                   |  17 +-
 quatradis/tisp/parser.py                      |   4 +-
 tests/py/comparison/scatterplot_test.py       |   2 +
 tests/py/tisp/normalise_test.py               |  58 +++-
 tests/py/util/mapper_test.py                  |  97 ++++++-
 tests/scripts/comparison_test.sh              |   4 +-
 tests/scripts/plot_test.sh                    |   4 +-
 tradis                                        |   4 +-
 22 files changed, 472 insertions(+), 203 deletions(-)

diff --git a/README.md b/README.md
index 31a8178..619dbb1 100644
--- a/README.md
+++ b/README.md
@@ -149,6 +149,9 @@ conda activate quatradis
 # Install mamba if you haven't already
 conda install -c conda-forge mamba
 
+# Install edgeR separately because this doesn't get imported correctly with quatradis.
+mamba install -c conda-forge -c bioconda bioconductor-edger
+
 # Install quatradis
 mamba install -c conda-forge -c bioconda quatradis
 ```
diff --git a/docs/pipeline_combine.png b/docs/pipeline_combine.png
index 03cc4de6d4ec72f773aedad2a4f64cbdef60e2a2..5c813077c02e7ed16182c884fdcc5ccc6243afa9 100644
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diff --git a/docs/pipeline_combine.pu b/docs/pipeline_combine.pu
index 477eb89..b0ccd9e 100644
--- a/docs/pipeline_combine.pu
+++ b/docs/pipeline_combine.pu
@@ -12,7 +12,7 @@ split again
 end split
 
 #lightblue:start tradis pipeline compare;
-
+:normalise plot files;
 split
 : create count file for
 original plot and original embl;
@@ -37,10 +37,8 @@ split
 each plot file;
 :essentiality analysis;
 split again
-:compare plot files
-create figures;
-split again
-: logfc comparison;
+:compare insertion sites
+create logfc and pval for genes;
 : create gene report;
 end split
 
diff --git a/quatradis/comparison/cli.py b/quatradis/comparison/cli.py
index b5499be..e97e384 100644
--- a/quatradis/comparison/cli.py
+++ b/quatradis/comparison/cli.py
@@ -3,7 +3,7 @@
 from quatradis.comparison.scatterplot import scatterplot_run
 from quatradis.comparison.presence_absence import presence_absence_run
 from quatradis.comparison.comparison import prep_essentiality_pipeline_output_for_comparison, run_comparisons, \
-    generate_logfc_plot
+    insertion_site_comparison
 from quatradis.comparison.gene_stats import gene_statistics
 from quatradis.comparison.plot_logfc import PlotLogOptions
 from quatradis.comparison.split import split_plot as sp
@@ -16,9 +16,9 @@ def add_subparser(subparsers):
     compare_parser_desc = "Comparative analysis and visualisation of TraDIS experiments (albatradis)"
     compare_parser = subparsers.add_parser("compare", help=compare_parser_desc)
     compare_subparsers = compare_parser.add_subparsers(title=compare_parser_desc)
-    create_parser("logfc_plot", compare_subparsers, logfc_plot, add_logfc_options,
-                  "Run logfc analysis for a specific plot file direction: forward, reverse, combined, original over all experiments.",
-                  usage="tradis compare logfc_plot [options] --condition_dirs <condition_plotfiles>+ --control_dirs <control_plotfiles>+ <EMBL_file> <analysis_type>")
+    create_parser("insertion_sites", compare_subparsers, insertion_site_comparison_cli, add_insertion_site_comparison_options,
+                  "Run an insertion site comparison for a specific plot file direction: forward, reverse, combined, original over all experiments to produce a file including edgeR output (logfc, p and q values)",
+                  usage="tradis compare insertion_sites [options] --condition_dirs <condition_plotfiles>+ --control_dirs <control_plotfiles>+ <EMBL_file> <analysis_type>")
     create_parser("presence_absence", compare_subparsers, presence_absence, add_pa_options,
                   "Take in gene report files and produce a heatmap",
                   usage="tradis compare presence_absence [options] <EMBLfile> <gene_reports>+")
@@ -42,7 +42,7 @@ def add_subparser(subparsers):
                   usage="tradis compare essentiality_analysis [options] --controls <control_gene_files> --conditions <condition_gene_files> --ess_controls <control_essential_gene_files> --ess_conditions <condition_essential_gene_files>")
 
 
-def add_logfc_options(parser):
+def add_insertion_site_comparison_options(parser):
     parser.add_argument('emblfile', help='Annotation file in EMBL format', type=str)
     parser.add_argument('analysis_type', help='Analysis type: forward, reverse, combined, original', type=str)
     parser.add_argument('--controls',
@@ -71,7 +71,7 @@ def add_logfc_options(parser):
                         required=True)
 
 
-def logfc_plot(args):
+def insertion_site_comparison_cli(args):
     if len(args.controls) != len(args.conditions):
         raise "Must have equal number of control and condition files to process"
 
@@ -92,8 +92,8 @@ def logfc_plot(args):
         report_decreased_insertions=not args.dont_report_decreased_insertions,
         minimum_block=args.minimum_block)
 
-    plotfile, scoresfile = generate_logfc_plot(args.analysis_type, args.controls, args.conditions,
-                                               args.emblfile, args.prefix, options, args.verbose)
+    plotfile, scoresfile = insertion_site_comparison(args.analysis_type, args.controls, args.conditions,
+                                                     args.emblfile, args.prefix, options, args.verbose)
 
     return plotfile, scoresfile
 
diff --git a/quatradis/comparison/comparison.py b/quatradis/comparison/comparison.py
index 2b3088b..999b0c0 100644
--- a/quatradis/comparison/comparison.py
+++ b/quatradis/comparison/comparison.py
@@ -1,11 +1,9 @@
-import subprocess
-from tempfile import mkstemp
 import os
-import csv
 import shutil
+import subprocess
+from tempfile import mkstemp
 
 from quatradis.comparison.plot_logfc import PlotLog
-from quatradis.comparison.essentiality import GeneEssentiality
 from quatradis.util.file_handle_helpers import ensure_output_dir_exists
 
 
@@ -45,20 +43,20 @@ def run_comparisons(controls_all, conditions_all, annotations, prefix, compariso
     if verbose:
         print("Running comparison between condition and control and write plot files and gene report.\n")
 
-    forward_plotfile, forward_scoresfile = generate_logfc_plot('forward', controls_all, conditions_all,
-                                                               annotations, prefix, comparison_options, verbose)
+    forward_plotfile, forward_scoresfile = insertion_site_comparison('forward', controls_all, conditions_all, annotations,
+                                                                     prefix, comparison_options, verbose)
     if verbose:
         print("Forward insertions have been compared.\n")
-    reverse_plotfile, reverse_scoresfile = generate_logfc_plot('reverse', controls_all, conditions_all, annotations,
-                                                               prefix, comparison_options, verbose)
+    reverse_plotfile, reverse_scoresfile = insertion_site_comparison('reverse', controls_all, conditions_all, annotations,
+                                                                     prefix, comparison_options, verbose)
     if verbose:
         print("Reverse insertions have been compared.\n")
-    combined_plotfile, combined_scoresfile = generate_logfc_plot('combined', controls_all, conditions_all, annotations,
-                                                                 prefix, comparison_options, verbose)
+    combined_plotfile, combined_scoresfile = insertion_site_comparison('combined', controls_all, conditions_all, annotations,
+                                                                       prefix, comparison_options, verbose)
     if verbose:
         print("Combined insertions have been compared.\n")
-    original_plotfile, original_scoresfile = generate_logfc_plot('original', controls_all, conditions_all, annotations,
-                                                                 prefix, comparison_options, verbose)
+    original_plotfile, original_scoresfile = insertion_site_comparison('original', controls_all, conditions_all, annotations,
+                                                                       prefix, comparison_options, verbose)
     if verbose:
         print("Original insertions have been compared.\n")
 
@@ -66,11 +64,15 @@ def run_comparisons(controls_all, conditions_all, annotations, prefix, compariso
            forward_scoresfile, reverse_scoresfile, combined_scoresfile, original_scoresfile
 
 
-def generate_logfc_plot(analysis_type, controls_all, conditions_all,
-                        annotations, prefix, options, verbose):
+def insertion_site_comparison(analysis_type, controls_all, conditions_all,
+                              annotations, prefix, options, verbose):
+
+    # Create CSV report file containing LogFC and PValue information for genes based on their insertion sites
     t = TradisComparisonRunner(conditions_all, controls_all, verbose, options.minimum_block,
                                analysis_type, prefix)
     t.run()
+
+    # Create plot sytle files containing the logfc and p and q values at every position in the reference
     p = PlotLog(t.get_report_file_path(), annotations, options,
                 output_plot_filename=os.path.join(prefix, analysis_type + ".logfc.plot"),
                 output_scores_filename=os.path.join(prefix, analysis_type + ".pqvals.plot"))
diff --git a/quatradis/comparison/essentiality.py b/quatradis/comparison/essentiality.py
index 356fcbb..7ec0086 100644
--- a/quatradis/comparison/essentiality.py
+++ b/quatradis/comparison/essentiality.py
@@ -74,8 +74,9 @@ def run(self):
             print(cmd)
         subprocess.check_output(cmd, shell=True)
 
+        os.remove(self.count_file + ".all.csv")
+
         if self.verbose:
-            print("all.csv\t" + self.count_file + ".all.csv")
             print("essen.csv\t" + self.count_file + ".essen.csv")
 
         return self
diff --git a/quatradis/comparison/essentiality_analysis.py b/quatradis/comparison/essentiality_analysis.py
index 1a8942d..9f97200 100644
--- a/quatradis/comparison/essentiality_analysis.py
+++ b/quatradis/comparison/essentiality_analysis.py
@@ -1,9 +1,9 @@
 import csv
 import os
 from dataclasses import dataclass
-from tempfile import mkstemp
 
 from quatradis.comparison.essentiality import GeneEssentiality
+from quatradis.util.file_handle_helpers import ensure_output_dir_exists
 
 
 @dataclass
@@ -13,10 +13,11 @@ class EssentialityInput:
     only_ess_control_files: list
     only_ess_condition_files: list
 
-def gene_names_from_essentiality_file(filename, verbose=False):
-    with open(filename, 'r') as fileh:
-        reader = csv.reader(fileh, delimiter=',', quotechar='"')
-        gene_names = [r[1] for r in reader if r[1] != 'gene_name']
+
+def gene_names_from_essentiality_file(filename):
+    with open(filename, "r") as fileh:
+        reader = csv.reader(fileh, delimiter=",", quotechar='"')
+        gene_names = [r[1] for r in reader if r[1] != "gene_name"]
         print("Number of all genes:" + str(len(gene_names)))
 
     return gene_names
@@ -26,19 +27,20 @@ def get_all_gene_names(control_files, condition_files):
     all_gene_names = set()
 
     for filename in condition_files:
-        with open(filename, 'r') as fileh:
-            reader = csv.reader(fileh, delimiter='\t', quotechar='"')
-            gene_names1 = [r[1] for r in reader if len(r) > 1 and r[1] != 'gene_name']
+        with open(filename, "r") as fileh:
+            reader = csv.reader(fileh, delimiter="\t", quotechar='"')
+            gene_names1 = [r[1] for r in reader if len(r) > 1 and r[1] != "gene_name"]
             all_gene_names = all_gene_names.union(set(gene_names1))
 
-    for f in control_files:
-        with open(filename, 'r') as fileh:
-            reader = csv.reader(fileh, delimiter='\t', quotechar='"')
-            gene_names2 = [r[1] for r in reader if len(r) > 1 and r[1] != 'gene_name']
+    for filename in control_files:
+        with open(filename, "r") as fileh:
+            reader = csv.reader(fileh, delimiter="\t", quotechar='"')
+            gene_names2 = [r[1] for r in reader if len(r) > 1 and r[1] != "gene_name"]
             all_gene_names = all_gene_names.union(set(gene_names2))
 
     return list(all_gene_names)
 
+
 def all_gene_essentiality(input: EssentialityInput, analysis_type, verbose=False):
 
     all_gene_names = get_all_gene_names(input.control_files, input.condition_files)
@@ -48,7 +50,7 @@ def all_gene_essentiality(input: EssentialityInput, analysis_type, verbose=False
     genes_ess = {g: GeneEssentiality() for g in all_gene_names}
     if analysis_type == "original":
         for f in input.only_ess_condition_files:
-            ess_gene_names = gene_names_from_essentiality_file(f, verbose)
+            ess_gene_names = gene_names_from_essentiality_file(f)
             if verbose:
                 print("ess_gene_names condition: " + str(len(ess_gene_names)))
                 print("genes_ess: " + str(len(genes_ess)))
@@ -57,7 +59,7 @@ def all_gene_essentiality(input: EssentialityInput, analysis_type, verbose=False
                     genes_ess[e].condition += 1
                 genes_ess[e].number_of_reps = len(input.only_ess_condition_files)
         for f in input.only_ess_control_files:
-            ess_gene_names = gene_names_from_essentiality_file(f, verbose)
+            ess_gene_names = gene_names_from_essentiality_file(f)
             if verbose:
                 print("ess_gene_names control: " + str(len(ess_gene_names)))
                 print("genes_ess: " + str(len(genes_ess)))
@@ -73,16 +75,19 @@ def all_gene_essentiality(input: EssentialityInput, analysis_type, verbose=False
 
     return genes_ess
 
-def add_gene_essentiality_to_file(input_filename, output_filename, genes_ess, analysis_type):
+
+def add_gene_essentiality_to_file(
+    input_filename, output_filename, genes_ess, analysis_type
+):
     """
     We can add information on gene essentiality to the comparison output,
     but this will not reflect full set of essential genes as the output does not contain all genes
     In order to prevent confusion this is "switched" off, but could be used if uncommented
     """
-    with open(input_filename, 'r') as inputfh:
+    with open(input_filename, "r") as inputfh:
         output_content = []
 
-        reader = csv.reader(inputfh, delimiter=',', quotechar='"')
+        reader = csv.reader(inputfh, delimiter=",", quotechar='"')
         input_content = [r for r in reader]
         # if analysis_type == "original":
         #     print("Number of cells: " + len(input_content))
@@ -99,21 +104,34 @@ def add_gene_essentiality_to_file(input_filename, output_filename, genes_ess, an
 
         output_content = input_content
 
-        with open(output_filename, 'w') as outputfh:
+        with open(output_filename, "w") as outputfh:
             for line in output_content:
                 outputfh.write(",".join(line) + "\n")
 
 
-def essentiality_analysis(input: EssentialityInput, output_dir, analysis_type, output_filename=""):
+def essentiality_analysis(
+    input: EssentialityInput, output_dir, analysis_type, output_filename=""
+):
 
-    #out_csv = mkstemp()
+    # out_csv = mkstemp()
     genes_ess = all_gene_essentiality(input, analysis_type)
-    #add_gene_essentiality_to_file(out_csv, output_filename, genes_ess, analysis_type)
-    #os.remove(out_csv)
+    # add_gene_essentiality_to_file(out_csv, output_filename, genes_ess, analysis_type)
+    # os.remove(out_csv)
 
+    ensure_output_dir_exists(output_dir)
     ess = open(os.path.join(output_dir, "essentiality.csv"), "w+")
     ess.write("Gene,Essentiality,Control,Condition,Replicates\n")
     for e in genes_ess:
-        ess.write(",".join([e, str(genes_ess[e].status()), str(genes_ess[e].control),
-                            str(genes_ess[e].condition), str(genes_ess[e].number_of_reps)]) + "\n")
+        ess.write(
+            ",".join(
+                [
+                    e,
+                    str(genes_ess[e].status()),
+                    str(genes_ess[e].control),
+                    str(genes_ess[e].condition),
+                    str(genes_ess[e].number_of_reps),
+                ]
+            )
+            + "\n"
+        )
     ess.close()
diff --git a/quatradis/comparison/scatterplot.py b/quatradis/comparison/scatterplot.py
index f4b959b..9a0a9eb 100644
--- a/quatradis/comparison/scatterplot.py
+++ b/quatradis/comparison/scatterplot.py
@@ -48,7 +48,7 @@ def __init__(self, conditions, controls, window_size, prefix, normalise=False, v
         self.normalise = normalise
 
         if normalise:
-            n = NormalisePlots(self.conditions + self.controls, 0.0000001, verbose=self.verbose)
+            n = NormalisePlots(self.conditions + self.controls, 0.0000001, output_dir="normalised", verbose=self.verbose)
             plotfiles, max_reads = n.create_normalised_files()
             self.conditions = plotfiles[0:len(self.conditions)]
             self.controls = plotfiles[len(self.conditions):]
diff --git a/quatradis/comparison/split.py b/quatradis/comparison/split.py
index d44a903..cafe6d7 100644
--- a/quatradis/comparison/split.py
+++ b/quatradis/comparison/split.py
@@ -27,7 +27,7 @@ def get_combined_file_path(self):
         return self._construct_file_path("combined")
 
     def _create_split_plot_file(self, forward, reverse, filename):
-        p = PlotFromValuesGenerator(forward, reverse, filename, self.gzipped)
+        p = PlotFromValuesGenerator(forward, reverse, filename)
         p.construct_file()
 
     def create_all_files(self):
diff --git a/quatradis/comparison/tradis_comparison.R b/quatradis/comparison/tradis_comparison.R
index d677a02..7b3ab2d 100755
--- a/quatradis/comparison/tradis_comparison.R
+++ b/quatradis/comparison/tradis_comparison.R
@@ -119,4 +119,4 @@ abline(v=2, col="red")
 
 
 #write results
-write.table(diff,file=opt$output,append=FALSE, quote=TRUE, sep=",", row.names=FALSE, col.names=c("locus_tag","gene_name","function","logFC","logCPM","PValue","q.value"))
+write.table(diff,file=opt$output,append=FALSE, quote=FALSE, sep=",", row.names=FALSE, col.names=c("locus_tag","gene_name","function","logFC","logCPM","PValue","q.value"))
diff --git a/quatradis/pipelines/compare.smk b/quatradis/pipelines/compare.smk
index c977b44..d940219 100644
--- a/quatradis/pipelines/compare.smk
+++ b/quatradis/pipelines/compare.smk
@@ -1,21 +1,30 @@
 import os
 import shutil
 
+input_files=[]
+norm_files=[]
 plotnames=[]
 controlnames=[]
 conditionnames=[]
-plot_lut={}
+norm_lut={}
 for p in config["condition_files"]:
+    input_files.append(p)
     plotname = os.path.basename(p).split('.')[0]
     plotnames.append(plotname)
     conditionnames.append(plotname)
-    plot_lut[plotname]=p
+
 
 for p in config["control_files"]:
+    input_files.append(p)
     plotname = os.path.basename(p).split('.')[0]
     plotnames.append(plotname)
     controlnames.append(plotname)
-    plot_lut[plotname]=p
+
+for p in input_files:
+    plotname = os.path.basename(p).split('.')[0]
+    n=os.path.join(config["output_dir"], "analysis", plotname, "original.plot.gz")
+    norm_files.append(n)
+    norm_lut[plotname]=n
 
 ZCAT_CMD='gzcat'
 if not shutil.which('gzcat'):
@@ -24,14 +33,27 @@ if not shutil.which('gzcat'):
 	else:
 		raise Error("Couldn't find gzcat or zcat on your system.  Please install and try again.")
 
+def make_no_normalise_cmd():
+	cmds = []
+	for i in range(len(input_files)):
+		out_dir = os.path.join(config["output_dir"], "analysis", plotname)
+		cmds.append("mkdir -p " + out_dir)
+		cmds.append("if [[ $(file " + input_files[i] + " | grep ASCII | wc -l | cut -f 1) > 0 ]]; then gzip -c " + input_files[i] + " > " + norm_files[i] + "; else cp " + input_files[i] + " " + norm_files[i] + "; fi")
+
+	return "; ".join(cmds)
+
+def make_normalise_cmd():
+    return "tradis plot normalise -o " + os.path.join(config["output_dir"], "analysis") + " -n original.plot.gz " + \
+        ("--minimum_proportion_insertions=" + config["minimum_proportion_insertions"] if config["minimum_proportion_insertions"] else "") + \
+            " " + " ".join(input_files)
 
 rule finish:
     input:
         expand(os.path.join(config["output_dir"], "gene_report.tsv")),
         expand(os.path.join(config["output_dir"], "comparison", "{type}", "plot_absscatter.png"), type=["original", "forward", "reverse", "combined"]),
-        #expand(os.path.join(config["output_dir"],"comparison","{type}","{type}.compare.csv"), type=["original", "forward", "reverse", "combined"]),
-        expand(os.path.join(config["output_dir"],"comparison","{type}","essentiality.csv"), type=["original", "forward", "reverse", "combined"]),
-        expand(os.path.join(config["output_dir"],"analysis","{plot}","{type}.count.tsv.all.csv"), type=["original", "combined", "forward", "reverse"], plot=plotnames)
+        #expand(os.path.join(config["output_dir"], "comparison", "{type}", "{type}.compare.csv"), type=["original", "forward", "reverse", "combined"]),
+        expand(os.path.join(config["output_dir"], "comparison", "{type}", "essentiality.csv"), type=["original", "forward", "reverse", "combined"]),
+        expand(os.path.join(config["output_dir"], "analysis", "{plot}", "{type}.count.tsv.essen.csv"), type=["original", "combined", "forward", "reverse"], plot=plotnames)
     run:
         print("All done!")
 
@@ -49,10 +71,21 @@ rule prepare_embl:
         prime_feature_size="--prime_feature_size=" + config["prime_feature_size"] if config["prime_feature_size"] else ""
     shell: "tradis compare prepare_embl --output={output} {params.minimum_threshold} {params.window_size} {params.window_interval} {params.prime_feature_size} --emblfile {input.embl} {input.plot}"
 
+rule normalise:
+    input:
+        input_files
+    output:
+        norm_files
+    message: "Normalising plot files"
+    log: os.path.join(config["output_dir"], "analysis", "normalise.log")
+    params:
+        cmd=make_normalise_cmd() if config["normalise"] else make_no_normalise_cmd()
+    shell: "{params.cmd}"
+
 
 rule split_plots:
     input:
-        p=lambda wildcards: plot_lut[wildcards.plot]
+        p=lambda wildcards: norm_lut[wildcards.plot]
     output:
         c=os.path.join(config["output_dir"], "analysis", "{plot}", "combined.plot.gz"),
         f=os.path.join(config["output_dir"], "analysis", "{plot}", "forward.plot.gz"),
@@ -74,39 +107,13 @@ rule count_plots:
     params:
         output_dir=os.path.join(config["output_dir"], "analysis", "{plot}"),
         suffix="count.tsv"
-    wildcard_constraints:
-        type="(?!original).*"
     shell: "tradis plot count -o {params.output_dir} -s {params.suffix} {input.embl} {input.p}"
 
 
-rule copy_original:
-    input:
-        lambda wildcards: plot_lut[wildcards.plot]
-    output:
-        os.path.join(config["output_dir"],"analysis","{plot}","original.plot.gz")
-    message: "Copying original plot file to target folder: {input}"
-    params:
-        output_dir=os.path.join(config["output_dir"],"analysis","{plot}")
-    shell: "if [[ $(file {input} | grep ASCII | wc -l | cut -f 1) > 0 ]]; then gzip -c {input} > {output}; else cp {input} {output}; fi"
-
-
-rule count_original_plots:
-	input:
-		p=rules.copy_original.output,
-		embl=config["annotations"]
-	output:
-		os.path.join(config["output_dir"], "analysis", "{plot}", "original.count.tsv")
-	message: "Analysing original plot and embl file {input.p}, {input.embl}"
-	params:
-		output_dir=os.path.join(config["output_dir"], "analysis", "{plot}")
-	shell: "tradis plot count -o {params.output_dir} -s count.tsv {input.embl} {input.p}"
-
-
 rule essentiality:
     input:
         c=os.path.join(config["output_dir"], "analysis", "{plot}", "{type}.count.tsv"),
     output:
-        all=os.path.join(config["output_dir"], "analysis", "{plot}", "{type}.count.tsv.all.csv"),
         ess=os.path.join(config["output_dir"], "analysis", "{plot}", "{type}.count.tsv.essen.csv")
     log: os.path.join(config["output_dir"], "analysis", "{plot}", "{type}.count.tsv.essen.log")
     message: "Determining gene essentiality for {input}"
@@ -128,7 +135,7 @@ rule plot:
     shell: "tradis compare figures {params.window_size} {params.prefix} {params.controls} {params.conditions} > /dev/null 2>&1"
 
 
-rule logfc:
+rule insertion_site_comparison:
     input:
         controls=lambda wildcards: expand(os.path.join(config["output_dir"], "analysis", "{control}", wildcards.tt + ".count.tsv"), control=controlnames),
         conditions=lambda wildcards: expand(os.path.join(config["output_dir"], "analysis", "{condition}", wildcards.tt + ".count.tsv"), condition=conditionnames),
@@ -143,7 +150,7 @@ rule logfc:
         output_dir="--prefix=" + os.path.join(config["output_dir"], "comparison", "{tt}"),
         zcat=ZCAT_CMD
     message: "Calculating logfc for {output}"
-    shell: "SEQLENGTH=$({params.zcat} {params.combined} | wc -l); tradis compare logfc_plot {input.embl} {params.tt} {params.output_dir} -g ${{SEQLENGTH}} --verbose --controls {input.controls} --conditions {input.conditions} > {log} 2>&1"
+    shell: "SEQLENGTH=$({params.zcat} {params.combined} | wc -l); tradis compare insertion_sites {input.embl} {params.tt} {params.output_dir} -g ${{SEQLENGTH}} --verbose --controls {input.controls} --conditions {input.conditions} > {log} 2>&1"
 
 
 rule gene_stats:
@@ -165,8 +172,8 @@ rule gene_stats:
 
 rule essentiality_analysis:
     input:
-        controls = lambda wildcards: expand(os.path.join(config["output_dir"],"analysis","{control}",wildcards.type + ".count.tsv.all.csv"),control=controlnames),
-        conditions = lambda wildcards: expand(os.path.join(config["output_dir"],"analysis","{condition}",wildcards.type + ".count.tsv.all.csv"),condition=conditionnames),
+        controls = lambda wildcards: expand(os.path.join(config["output_dir"],"analysis","{control}",wildcards.type + ".count.tsv"),control=controlnames),
+        conditions = lambda wildcards: expand(os.path.join(config["output_dir"],"analysis","{condition}",wildcards.type + ".count.tsv"),condition=conditionnames),
         ess_controls= lambda wildcards: expand(os.path.join(config["output_dir"],"analysis","{control}",wildcards.type + ".count.tsv.essen.csv"),control=controlnames),
         ess_conditions=lambda wildcards: expand(os.path.join(config["output_dir"],"analysis","{condition}",wildcards.type + ".count.tsv.essen.csv"),condition=conditionnames)
     output:
diff --git a/quatradis/pipelines/pipeline.py b/quatradis/pipelines/pipeline.py
index 2ef30dd..856dba9 100644
--- a/quatradis/pipelines/pipeline.py
+++ b/quatradis/pipelines/pipeline.py
@@ -8,42 +8,90 @@
 
 
 def add_subparser(subparsers):
-    pipeline_parser_desc = "TraDIS pipelines that stitch together other tools in this package."
+    pipeline_parser_desc = (
+        "TraDIS pipelines that stitch together other tools in this package."
+    )
     pipeline_parser = subparsers.add_parser("pipeline", help=pipeline_parser_desc)
     pipeline_subparsers = pipeline_parser.add_subparsers(title=pipeline_parser_desc)
 
-    create_parser("create_plots", pipeline_subparsers, create_plots_pipeline, create_plots_options,
-                  "Creates transponson insertion site plot files for multiple fastqs in parallel where possible using snakemake.",
-                  description='''This pipeline uses snakemake, therefore it is possible to customise how this operates to distribute the workload
-    across a cluster using a snakemake config file.''',
-                  usage="tradis pipeline create_plots [options] <fastq list file> <reference>")
-
-    create_parser("compare", pipeline_subparsers, compare_pipeline, compare_options,
-                  "Calculates gene essentiality for a set of transposon insertion site plots files ",
-                  description='''This pipeline uses snakemake, therefore it is possible to customise how this operates to distribute the workload
-    across a cluster using a snakemake config file.''',
-                  usage="tradis pipeline compare [options]")
+    create_parser(
+        "create_plots",
+        pipeline_subparsers,
+        create_plots_pipeline,
+        create_plots_options,
+        "Creates transponson insertion site plot files for multiple fastqs in parallel where possible using snakemake.",
+        description="""This pipeline uses snakemake, therefore it is possible to customise how this operates to distribute the workload
+    across a cluster using a snakemake config file.""",
+        usage="tradis pipeline create_plots [options] <fastq list file> <reference>",
+    )
+
+    create_parser(
+        "compare",
+        pipeline_subparsers,
+        compare_pipeline,
+        compare_options,
+        "Calculates gene essentiality for a set of transposon insertion site plots files ",
+        description="""This pipeline uses snakemake, therefore it is possible to customise how this operates to distribute the workload
+    across a cluster using a snakemake config file.""",
+        usage="tradis pipeline compare [options]",
+    )
 
 
 def create_plots_options(parser):
-    parser.add_argument('fastqs', type=str,
-                        help='Either a line separated text file containing a list of fastq formatted reads for processing or a single fastq file (can be gzipped).')
-    parser.add_argument('reference', type=str,
-                        help='The fasta formatted reference for processing.')
-    parser.add_argument('-o', '--output_dir', default="results",
-                        help='The output directory to use for all output files (default: results)')
-    parser.add_argument('-n', '--threads', type=int, default=1,
-                        help='number of threads to use when mapping and sorting (default: 1)')
-    parser.add_argument('-a', '--aligner', default="bwa",
-                        help='mapping tool to use (bwa, smalt, minimap2) (default: bwa)')
-    parser.add_argument('-m', '--mapping_score', type=int, default=30,
-                        help='mapping quality must be greater than X (Default: 30)')
-    parser.add_argument('-t', '--tag', type=str, default="",
-                        help='the tag to remove from fastq input (default: "")')
-    parser.add_argument('-mm', '--mismatch', type=int, default=0,
-                        help='number of mismatches allowed when matching tag (default: 0)')
-    parser.add_argument('-sp', '--snakemake_profile', type=str,
-                        help='If provided, pass this directory onto snakemake.  Assumes there is a file called "config.yaml" in that directory.')
+    parser.add_argument(
+        "fastqs",
+        type=str,
+        help="Either a line separated text file containing a list of fastq formatted reads for processing or a single fastq file (can be gzipped).",
+    )
+    parser.add_argument(
+        "reference", type=str, help="The fasta formatted reference for processing."
+    )
+    parser.add_argument(
+        "-o",
+        "--output_dir",
+        default="results",
+        help="The output directory to use for all output files (default: results)",
+    )
+    parser.add_argument(
+        "-n",
+        "--threads",
+        type=int,
+        default=1,
+        help="number of threads to use when mapping and sorting (default: 1)",
+    )
+    parser.add_argument(
+        "-a",
+        "--aligner",
+        default="bwa",
+        help="mapping tool to use (bwa, smalt, minimap2) (default: bwa)",
+    )
+    parser.add_argument(
+        "-m",
+        "--mapping_score",
+        type=int,
+        default=30,
+        help="mapping quality must be greater than X (Default: 30)",
+    )
+    parser.add_argument(
+        "-t",
+        "--tag",
+        type=str,
+        default="",
+        help='the tag to remove from fastq input (default: "")',
+    )
+    parser.add_argument(
+        "-mm",
+        "--mismatch",
+        type=int,
+        default=0,
+        help="number of mismatches allowed when matching tag (default: 0)",
+    )
+    parser.add_argument(
+        "-sp",
+        "--snakemake_profile",
+        type=str,
+        help='If provided, pass this directory onto snakemake.  Assumes there is a file called "config.yaml" in that directory.',
+    )
 
 
 def create_plots_pipeline(args):
@@ -55,15 +103,15 @@ def create_plots_pipeline(args):
 
     fastqs = [args.fastqs]
     if not fhh.is_fastq(args.fastqs):
-        with open(args.fastqs, 'r') as fql:
+        with open(args.fastqs, "r") as fql:
             fastqs = [x.strip() for x in fql.readlines() if x]
 
     snakemake_config = os.path.join(args.output_dir, "create_plots_config.yaml")
     fastq_dir, fq_fn = os.path.split(args.fastqs)
-    with open(snakemake_config, 'w') as ofql:
+    with open(snakemake_config, "w") as ofql:
         ofql.write(create_yaml_option("output_dir", args.output_dir))
         ofql.write(create_yaml_option("reference", args.reference))
-        ofql.write("fastq_dir: \"" + fastq_dir + "\"\n")
+        ofql.write('fastq_dir: "' + fastq_dir + '"\n')
         ofql.write("fastqs:\n")
         for x in fastqs:
             ofql.write("- " + x + "\n")
@@ -75,30 +123,86 @@ def create_plots_pipeline(args):
 
     pipeline = find_pipeline_file("create_plots.smk")
 
-    start_snakemake(pipeline, snakemake_config, threads=args.threads,
-                                snakemake_profile=args.snakemake_profile, verbose=args.verbose)
+    start_snakemake(
+        pipeline,
+        snakemake_config,
+        threads=args.threads,
+        snakemake_profile=args.snakemake_profile,
+        verbose=args.verbose,
+    )
 
 
 def compare_options(parser):
-    parser.add_argument('--condition_files', type=str, nargs='+',
-                        help='A set of condition plot files to process (can be gzipped)')
-    parser.add_argument('--control_files', type=str, nargs='+',
-                        help='A set of control plot files to process (can be gzipped)')
-    parser.add_argument('-o', '--output_dir', default="results",
-                        help='The output directory to use for all output files (default: results)')
-    parser.add_argument('-n', '--threads', type=int, default=1,
-                        help='number of threads to use when processing (default: 1)')
-    parser.add_argument('--annotations', '-a',
-                        help='If provided genes in this EMBL annotations file will expanded based on data in the plotfile.',
-                        type=str, default=None)
-    parser.add_argument('--minimum_threshold', '-m',
-                        help='Only include insert sites with this number or greater insertions', type=int, default=5)
-    parser.add_argument('--prime_feature_size', '-z',
-                        help='Feature size when adding 5/3 prime block when --use_annotation', type=int, default=198)
-    parser.add_argument('--window_interval', '-l', help='Window interval', type=int, default=25)
-    parser.add_argument('--window_size', '-w', help='Window size', type=int, default=100)
-    parser.add_argument('-sp', '--snakemake_profile', type=str,
-                        help='If provided, pass this directory onto snakemake.  Assumes there is a file called "config.yaml" in that directory.')
+    parser.add_argument(
+        "--condition_files",
+        type=str,
+        nargs="+",
+        help="A set of condition plot files to process (can be gzipped)",
+    )
+    parser.add_argument(
+        "--control_files",
+        type=str,
+        nargs="+",
+        help="A set of control plot files to process (can be gzipped)",
+    )
+    parser.add_argument(
+        "-o",
+        "--output_dir",
+        default="results",
+        help="The output directory to use for all output files (default: results)",
+    )
+    parser.add_argument(
+        "--disable_normalisation",
+        action='store_true',
+        default=False,
+        help="Don't normalise the plots prior to comparison (default: false)",
+    )
+    parser.add_argument(
+        "-n",
+        "--threads",
+        type=int,
+        default=1,
+        help="number of threads to use when processing (default: 1)",
+    )
+    parser.add_argument(
+        "--annotations",
+        "-a",
+        help="If provided genes in this EMBL annotations file will expanded based on data in the plotfile.",
+        type=str,
+        default=None,
+    )
+    parser.add_argument(
+        "--minimum_threshold",
+        "-m",
+        help="Only include insert sites with this number or greater insertions",
+        type=int,
+        default=5,
+    )
+    parser.add_argument(
+        "--minimum_proportion_insertions",
+        help="If the proportion of insertions is too low compared to control, dont call decreased insertions below this level",
+        type=float,
+        default=0.1,
+    )
+    parser.add_argument(
+        "--prime_feature_size",
+        "-z",
+        help="Feature size when adding 5/3 prime block when --use_annotation",
+        type=int,
+        default=198,
+    )
+    parser.add_argument(
+        "--window_interval", "-l", help="Window interval", type=int, default=25
+    )
+    parser.add_argument(
+        "--window_size", "-w", help="Window size", type=int, default=100
+    )
+    parser.add_argument(
+        "-sp",
+        "--snakemake_profile",
+        type=str,
+        help='If provided, pass this directory onto snakemake.  Assumes there is a file called "config.yaml" in that directory.',
+    )
 
 
 def compare_pipeline(args):
@@ -110,12 +214,14 @@ def compare_pipeline(args):
         raise ValueError("Must have equal number of control and condition files")
 
     if len(args.control_files) <= 1:
-        raise ValueError("Must have 2 or more replicates of control and condition files")
+        raise ValueError(
+            "Must have 2 or more replicates of control and condition files"
+        )
 
     fhh.ensure_output_dir_exists(args.output_dir)
 
     snakemake_config = os.path.join(args.output_dir, "analysis_config.yaml")
-    with open(snakemake_config, 'w') as ofql:
+    with open(snakemake_config, "w") as ofql:
         ofql.write(create_yaml_option("output_dir", args.output_dir))
         ofql.write("condition_files:\n")
         for x in args.condition_files:
@@ -123,29 +229,44 @@ def compare_pipeline(args):
         ofql.write("control_files:\n")
         for x in args.control_files:
             ofql.write("- " + x + "\n")
+        ofql.write(create_yaml_option("normalise", not args.disable_normalisation, bool=True))
         ofql.write(create_yaml_option("threads", args.threads, num=True))
         ofql.write(create_yaml_option("annotations", args.annotations))
         ofql.write(create_yaml_option("minimum_threshold", args.minimum_threshold))
         ofql.write(create_yaml_option("prime_feature_size", args.prime_feature_size))
         ofql.write(create_yaml_option("window_interval", args.window_interval))
         ofql.write(create_yaml_option("window_size", args.window_size))
+        ofql.write(
+            create_yaml_option(
+                "minimum_proportion_insertions", args.minimum_proportion_insertions
+            )
+        )
 
     pipeline = find_pipeline_file("compare.smk")
 
-    start_snakemake(pipeline, snakemake_config, threads=args.threads, snakemake_profile=args.snakemake_profile,
-                    verbose=args.verbose)
+    start_snakemake(
+        pipeline,
+        snakemake_config,
+        threads=args.threads,
+        snakemake_profile=args.snakemake_profile,
+        verbose=args.verbose,
+    )
 
 
-def start_snakemake(snakefile, snakemake_config, threads=1, snakemake_profile=None, verbose=False):
+def start_snakemake(
+    snakefile, snakemake_config, threads=1, snakemake_profile=None, verbose=False
+):
     if verbose:
         print("Using snakemake config files at: " + ", ".join([snakemake_config]))
         print("Starting snakemake pipeline")
 
-    cmd_list = ["snakemake",
-                "--snakefile=" + snakefile,
-                "--configfile=" + snakemake_config,
-                "--cores=" + str(threads),
-                "--printshellcmds"]
+    cmd_list = [
+        "snakemake",
+        "--snakefile=" + snakefile,
+        "--configfile=" + snakemake_config,
+        "--cores=" + str(threads),
+        "--printshellcmds",
+    ]
 
     if snakemake_profile:
         cmd_list.append("--profile=" + snakemake_profile)
@@ -187,11 +308,13 @@ def find_pipeline_file(pipeline_file):
     raise RuntimeError("Could not find nextflow pipeline file.")
 
 
-def create_yaml_option(option, value, num=False):
+def create_yaml_option(option, value, num=False, bool=False):
     opt = option + ": "
     if num:
         opt += str(value)
+    elif bool:
+        opt += "true" if value else "false"
     else:
-        opt += "\"" + str(value) + "\""
+        opt += '"' + str(value) + '"'
     opt += "\n"
     return opt
diff --git a/quatradis/tisp/cli.py b/quatradis/tisp/cli.py
index 2ded00b..a547fbb 100644
--- a/quatradis/tisp/cli.py
+++ b/quatradis/tisp/cli.py
@@ -8,6 +8,7 @@
 from quatradis.tisp.analyse import count_insert_sites
 from quatradis.tisp.normalise import NormalisePlots
 
+
 def add_subparser(subparsers):
     plot_parser_desc = "TraDIS plot file tools"
     plot_parser = subparsers.add_parser("plot", help=plot_parser_desc)
@@ -103,9 +104,13 @@ def normalise_plot_options(parser):
     parser.add_argument('--minimum_proportion_insertions', '-d',
                         help='If the proportion of insertions is too low compared to control, dont call decreased insertions below this level',
                         type=float, default=0.1)
+    parser.add_argument('-o', '--output_dir', type=str, default="",
+                        help='The directory in which to put all output files (default: current working directory)')
+    parser.add_argument('-n', '--output_filename', type=str, default="normalised.plot.gz",
+                        help='The name for the normalised output files.  Normalised files will be distinguished by directory')
 
 
 def normalise_plots(args):
-    n = NormalisePlots(args.plot_files, args.minimum_proportion_insertions, output_temp_files=False)
+    n = NormalisePlots(args.plot_files, args.minimum_proportion_insertions, output_dir=args.output_dir, output_filename=args.output_filename, verbose=args.verbose)
     plotfiles, max_plot_reads = n.create_normalised_files()
     n.decreased_insertion_reporting(max_plot_reads)
\ No newline at end of file
diff --git a/quatradis/tisp/generator/from_values.py b/quatradis/tisp/generator/from_values.py
index 897341a..7f497b4 100644
--- a/quatradis/tisp/generator/from_values.py
+++ b/quatradis/tisp/generator/from_values.py
@@ -1,6 +1,10 @@
+import os
+
 import numpy
 import pyximport
 
+from quatradis.util.file_handle_helpers import ensure_output_dir_exists
+
 pyximport.install()
 
 from quatradis.tisp.write import write_plot_file, write_gzipped_plot_file
@@ -10,12 +14,10 @@ class PlotFromValuesGenerator:
     """
     Takes in arrays for forward and reverse integers and creates a new file
     """
-
-    def __init__(self, forward, reverse, filename, gzipped=False):
+    def __init__(self, forward, reverse, filename):
         self.forward = forward
         self.reverse = reverse
         self.filename = filename
-        self.gzipped = gzipped
 
         self.forward_length = len(self.forward)
         self.reverse_length = len(self.reverse)
@@ -29,7 +31,7 @@ def construct_file(self):
         if len(self.reverse) == 0:
             self.reverse = numpy.zeros(p_len, dtype=float)
 
-        if self.gzipped:
+        if self.filename.endswith('.gz'):
             write_gzipped_plot_file(self.filename, self.forward, self.reverse, p_len)
         else:
             write_plot_file(self.filename, self.forward, self.reverse, p_len)
diff --git a/quatradis/tisp/normalise.py b/quatradis/tisp/normalise.py
index 4d2b780..01d097a 100644
--- a/quatradis/tisp/normalise.py
+++ b/quatradis/tisp/normalise.py
@@ -1,7 +1,6 @@
-
-from tempfile import mkstemp
+import os
 import numpy
-
+from quatradis.util.file_handle_helpers import ensure_output_dir_exists
 from quatradis.tisp.parser import PlotParser
 from quatradis.tisp.generator.from_values import PlotFromValuesGenerator
 
@@ -9,9 +8,10 @@
 class NormalisePlots:
     '''Given a set of plot files, find the one with the highest number of reads and normalise all the rest into new files'''
 
-    def __init__(self, plotfiles, minimum_proportion_insertions, output_temp_files=True, verbose=False):
+    def __init__(self, plotfiles, minimum_proportion_insertions, output_dir=".", output_filename="normalised.plot_gz", verbose=False):
         self.plotfiles = plotfiles
-        self.output_temp_files=output_temp_files
+        self.output_dir = output_dir
+        self.output_filename = output_filename
         self.verbose = verbose
         self.minimum_proportion_insertions = minimum_proportion_insertions
         self.plot_objs = self.read_plots()
@@ -20,9 +20,10 @@ def create_normalised_files(self):
         plot_objs, max_plot_reads = self.normalise()
         output_files = []
         for p in self.plotfiles:
-            output_filename = p + ".norm"
-            if self.output_temp_files:
-                fd, output_filename = mkstemp()
+            plotname = os.path.basename(p).split('.')[0]
+            out_dir = os.path.join(self.output_dir, plotname)
+            output_filename = os.path.join(out_dir, self.output_filename)
+            ensure_output_dir_exists(out_dir)
             pg = PlotFromValuesGenerator(plot_objs[p].forward, plot_objs[p].reverse, output_filename)
             pg.construct_file()
             output_files.append(output_filename)
diff --git a/quatradis/tisp/parser.py b/quatradis/tisp/parser.py
index 13b1684..a7a6f3c 100644
--- a/quatradis/tisp/parser.py
+++ b/quatradis/tisp/parser.py
@@ -140,7 +140,7 @@ def get_plot_file_length(plot_file, working_dir):
 
     def read(self):
         handle = PlotParser.create_file_handle(self.filename)
-        insert_site_array = pandas.read_csv(handle, delim_whitespace=True, dtype=float, engine='c',
+        insert_site_array = pandas.read_csv(handle, sep='\s+', dtype=float, engine='c',
                                             header=None).values
 
         self.genome_length = len(insert_site_array)
@@ -176,7 +176,7 @@ def __init__(self, filename, minimum_threshold=0):
         self.split_lines()
 
     def split_lines(self):
-        insert_site_array = pandas.read_csv(self.filename, delim_whitespace=True, dtype=float, engine='c',
+        insert_site_array = pandas.read_csv(self.filename, sep='\s+', dtype=float, engine='c',
                                             header=None).values
 
         self.genome_length = len(insert_site_array)
diff --git a/tests/py/comparison/scatterplot_test.py b/tests/py/comparison/scatterplot_test.py
index 9a6ba1a..174a390 100644
--- a/tests/py/comparison/scatterplot_test.py
+++ b/tests/py/comparison/scatterplot_test.py
@@ -1,3 +1,4 @@
+import shutil
 import unittest
 import os
 from quatradis.comparison.scatterplot import ScatterPlot
@@ -24,3 +25,4 @@ def test_valid(self):
         os.remove("scattertest_absscatter.png")
         os.remove("scattertest_linear.png")
         os.remove("scattertest_scatter.png")
+        shutil.rmtree("normalised")
diff --git a/tests/py/tisp/normalise_test.py b/tests/py/tisp/normalise_test.py
index 8284549..8243fde 100644
--- a/tests/py/tisp/normalise_test.py
+++ b/tests/py/tisp/normalise_test.py
@@ -1,40 +1,76 @@
 import filecmp
 import os
+import shutil
 import unittest
 
 from quatradis.tisp.normalise import NormalisePlots
 
 
-class ErrorReadingFile(Exception): pass
+class ErrorReadingFile(Exception):
+    pass
 
 
-class InvalidFileFormat(Exception): pass
+class InvalidFileFormat(Exception):
+    pass
 
 
-data_dir = os.path.join('data', 'tisp', 'normalise')
-plotparser_dir = os.path.join('data', 'tisp', 'parser')
+data_dir = os.path.join("data", "tisp", "normalise")
+plotparser_dir = os.path.join("data", "tisp", "parser")
 
 
 class TestNormalisePlots(unittest.TestCase):
 
     def test_big_differences(self):
-        p = NormalisePlots([os.path.join(data_dir, 'sample1'), os.path.join(data_dir, 'sample2')], 0.1)
+        p = NormalisePlots(
+            [os.path.join(data_dir, "sample1"), os.path.join(data_dir, "sample2")],
+            0.1,
+            output_dir="normalised",
+        )
         output_files, max_reads = p.create_normalised_files()
         self.assertEqual(2, len(output_files))
-        self.assertTrue(filecmp.cmp(os.path.join(data_dir, 'sample2'), output_files[1]))
-        self.assertTrue(filecmp.cmp(os.path.join(data_dir, 'expected_sample1'), output_files[0]))
+        self.assertTrue(filecmp.cmp(os.path.join(data_dir, "sample2"), output_files[1]))
+        self.assertTrue(
+            filecmp.cmp(os.path.join(data_dir, "expected_sample1"), output_files[0])
+        )
         self.assertTrue(p.decreased_insertion_reporting())
+        shutil.rmtree("normalised")
 
     def test_ignore_decreased_insertions(self):
-        p = NormalisePlots([os.path.join(data_dir, 'lowinsertions'), os.path.join(data_dir, 'highinsertions')], 0.1)
+        p = NormalisePlots(
+            [
+                os.path.join(data_dir, "lowinsertions"),
+                os.path.join(data_dir, "highinsertions"),
+            ],
+            0.1,
+            output_dir="normalised"
+        )
         self.assertFalse(p.decreased_insertion_reporting())
 
     def test_ignore_decreased_insertions_insert_sites(self):
-        p = NormalisePlots([os.path.join(data_dir, 'fewinsertions'), os.path.join(data_dir, 'manyinsertions')], 0.1)
+        p = NormalisePlots(
+            [
+                os.path.join(data_dir, "fewinsertions"),
+                os.path.join(data_dir, "manyinsertions"),
+            ],
+            0.1,
+            output_dir="normalised",
+        )
         self.assertFalse(p.decreased_insertion_reporting())
 
     def test_large_file_zipped(self):
-        p = NormalisePlots([os.path.join(plotparser_dir, 'Control2.out.CP009273.insert_site_plot.gz'),
-                            os.path.join(plotparser_dir, 'Chloramrep2-MICpool.out.CP009273.insert_site_plot.gz')], 0.1)
+        p = NormalisePlots(
+            [
+                os.path.join(
+                    plotparser_dir, "Control2.out.CP009273.insert_site_plot.gz"
+                ),
+                os.path.join(
+                    plotparser_dir,
+                    "Chloramrep2-MICpool.out.CP009273.insert_site_plot.gz",
+                ),
+            ],
+            0.1,
+            output_dir="normalised",
+        )
         output_files, max_reads = p.create_normalised_files()
         self.assertTrue(p.decreased_insertion_reporting())
+        shutil.rmtree("normalised")
diff --git a/tests/py/util/mapper_test.py b/tests/py/util/mapper_test.py
index f29cacc..ca932df 100644
--- a/tests/py/util/mapper_test.py
+++ b/tests/py/util/mapper_test.py
@@ -12,6 +12,7 @@
 
 data_dir = os.path.join("data", "util", "mapper")
 
+
 class MapperTest(unittest.TestCase):
     """
     These unit tests are for the mapper functions
@@ -22,29 +23,87 @@ def test_calc_read_len(self):
         self.assertEqual(44, read_len)
 
     def test_smalt_index_cmd(self):
-        index_cmd, exitcode = mapper.index_reference(os.path.join(data_dir, "smallref.fa"), "test.ref", 44, mapper="smalt", dry_run=True)
-        self.assertEqual("smalt index -k 13 -s 4 test.ref " + os.path.join(data_dir, "smallref.fa") + " > /dev/null 2>&1", index_cmd)
+        index_cmd, exitcode = mapper.index_reference(
+            os.path.join(data_dir, "smallref.fa"),
+            "test.ref",
+            44,
+            mapper="smalt",
+            dry_run=True,
+        )
+        self.assertEqual(
+            "smalt index -k 13 -s 4 test.ref "
+            + os.path.join(data_dir, "smallref.fa")
+            + " > /dev/null 2>&1",
+            index_cmd,
+        )
         self.assertEqual(0, exitcode)
 
     def test_smalt_align_cmd(self):
-        align_cmd, exitcode = mapper.map_reads(os.path.join(data_dir, "test.fastq"), os.path.join(data_dir, "smallref.fa"), "test.ref", "mapped.out", 44, mapper="smalt", dry_run=True)
-        self.assertEqual("smalt map -x -n 1 test.ref " + os.path.join(data_dir, "test.fastq") + " 1> mapped.out 2> mapped.out.stderr", align_cmd)
+        align_cmd, exitcode = mapper.map_reads(
+            os.path.join(data_dir, "test.fastq"),
+            os.path.join(data_dir, "smallref.fa"),
+            "test.ref",
+            "mapped.out",
+            44,
+            mapper="smalt",
+            dry_run=True,
+        )
+        self.assertEqual(
+            "smalt map -x -n 1 test.ref "
+            + os.path.join(data_dir, "test.fastq")
+            + " 1> mapped.out 2> mapped.out.stderr",
+            align_cmd,
+        )
         self.assertEqual(0, exitcode)
 
     def test_bwa_index_cmd(self):
-        index_cmd, exitcode = mapper.index_reference(os.path.join(data_dir, "smallref.fa"), "test.ref", 44, mapper="bwa", dry_run=True)
-        self.assertEqual("bwa index " + os.path.join(data_dir, "smallref.fa") + " > /dev/null 2>&1", index_cmd)
+        index_cmd, exitcode = mapper.index_reference(
+            os.path.join(data_dir, "smallref.fa"),
+            "test.ref",
+            44,
+            mapper="bwa",
+            dry_run=True,
+        )
+        self.assertEqual(
+            "bwa index " + os.path.join(data_dir, "smallref.fa") + " > /dev/null 2>&1",
+            index_cmd,
+        )
         self.assertEqual(0, exitcode)
 
     def test_bwa_align_cmd(self):
-        align_cmd, exitcode = mapper.map_reads(os.path.join(data_dir, "test.fastq"), os.path.join(data_dir, "smallref.fa"), "test.ref", "mapped.out", 44, mapper="bwa", dry_run=True)
-        self.assertEqual("bwa mem -k 13 -t 1 " + os.path.join(data_dir, "smallref.fa") + " " + os.path.join(data_dir, "test.fastq") + " 1> mapped.out 2> mapped.out.stderr", align_cmd)
+        align_cmd, exitcode = mapper.map_reads(
+            os.path.join(data_dir, "test.fastq"),
+            os.path.join(data_dir, "smallref.fa"),
+            "test.ref",
+            "mapped.out",
+            44,
+            mapper="bwa",
+            dry_run=True,
+        )
+        self.assertEqual(
+            "bwa mem -k 13 -t 1 "
+            + os.path.join(data_dir, "smallref.fa")
+            + " "
+            + os.path.join(data_dir, "test.fastq")
+            + " 1> mapped.out 2> mapped.out.stderr",
+            align_cmd,
+        )
         self.assertEqual(0, exitcode)
 
     # This test needs smalt installed
     def test_smalt(self):
-        mapper.index_and_align(os.path.join(data_dir, "test.fastq"), os.path.join(data_dir, "smallref.fa"), "test.ref", "mapped.out", mapper="smalt")
-        os.system("grep -v ^@ mapped.out > mapped.nohead.out && grep -v ^@ " + os.path.join(data_dir, "expected.smalt.sam") + " > expected.smalt.nohead.sam")
+        mapper.index_and_align(
+            os.path.join(data_dir, "test.fastq"),
+            os.path.join(data_dir, "smallref.fa"),
+            "test.ref",
+            "mapped.out",
+            mapper="smalt",
+        )
+        os.system(
+            "grep -v ^@ mapped.out > mapped.nohead.out && grep -v ^@ "
+            + os.path.join(data_dir, "expected.smalt.sam")
+            + " > expected.smalt.nohead.sam"
+        )
         self.assertTrue(filecmp.cmp("mapped.nohead.out", "expected.smalt.nohead.sam"))
         os.remove("test.ref.sma")
         os.remove("test.ref.smi")
@@ -53,8 +112,18 @@ def test_smalt(self):
 
     # This test need bwa installed
     def test_bwa(self):
-        mapper.index_and_align(os.path.join(data_dir, "test.fastq"), os.path.join(data_dir, "smallref.fa"), "test.ref", "mapped.out", mapper="bwa")
-        os.system("grep -v ^@ mapped.out > mapped.nohead.out && grep -v ^@ " + os.path.join(data_dir, "expected.bwa.sam") + " > expected.bwa.nohead.sam")
+        mapper.index_and_align(
+            os.path.join(data_dir, "test.fastq"),
+            os.path.join(data_dir, "smallref.fa"),
+            "test.ref",
+            "mapped.out",
+            mapper="bwa",
+        )
+        os.system(
+            "grep -v ^@ mapped.out > mapped.nohead.out && grep -v ^@ "
+            + os.path.join(data_dir, "expected.bwa.sam")
+            + " > expected.bwa.nohead.sam"
+        )
         self.assertTrue(filecmp.cmp("mapped.nohead.out", "expected.bwa.nohead.sam"))
         os.system("rm mapped.*")
         os.remove("expected.bwa.nohead.sam")
@@ -68,7 +137,9 @@ def test_sam2bam(self):
         os.system("rm *nice.bam*")
 
     def test_sam2bam_multithread(self):
-        mapper.sam2bam(os.path.join(data_dir, "expected.bwa.sam"), "nice.bam", threads=2)
+        mapper.sam2bam(
+            os.path.join(data_dir, "expected.bwa.sam"), "nice.bam", threads=2
+        )
         self.assertTrue(os.path.exists("nice.bam"))
         self.assertTrue(os.path.exists("nice.bam.bai"))
         self.assertTrue(os.path.exists("nice.bam.bamcheck"))
diff --git a/tests/scripts/comparison_test.sh b/tests/scripts/comparison_test.sh
index bc2168e..254cf44 100755
--- a/tests/scripts/comparison_test.sh
+++ b/tests/scripts/comparison_test.sh
@@ -11,7 +11,7 @@ echo -n "Checking 'tradis compare' help messages ... "
 ./tradis compare --help > /dev/null
 ./tradis compare figures --help  > /dev/null
 ./tradis compare presence_absence --help  > /dev/null
-./tradis compare logfc_plot --help  > /dev/null
+./tradis compare insertion_sites --help  > /dev/null
 ./tradis compare gene_report --help  > /dev/null
 ./tradis compare split --help  > /dev/null
 ./tradis compare essentiality --help  > /dev/null
@@ -46,7 +46,7 @@ echo "ok"
 echo -n "Checking 'tradis compare essentiality' ... "
 ESS_DATA_DIR=$DATA_DIR/comparison/essentiality
 ./tradis compare essentiality $ESS_DATA_DIR/combined.count.tsv
-ESS_OUT=$ESS_DATA_DIR/combined.count.tsv.all.csv
+ESS_OUT=$ESS_DATA_DIR/combined.count.tsv.essen.csv
 if [ ! -f "$ESS_OUT" ]; then
     echo "tradis compare essentiality test failed.  $ESS_OUT does not exist."
     exit 1
diff --git a/tests/scripts/plot_test.sh b/tests/scripts/plot_test.sh
index 1c20b9f..f5f4c36 100755
--- a/tests/scripts/plot_test.sh
+++ b/tests/scripts/plot_test.sh
@@ -37,6 +37,6 @@ rm -r temp_test
 echo "ok"
 
 echo -n "Checking 'tradis plot normalise' ... "
-./tradis plot normalise $DATA_DIR/normalise/fewinsertions $DATA_DIR/normalise/manyinsertions > /dev/null 2>&1
-rm $DATA_DIR/normalise/*.norm
+./tradis plot normalise --output_dir temp_test $DATA_DIR/normalise/fewinsertions $DATA_DIR/normalise/manyinsertions > /dev/null 2>&1
+rm -r temp_test
 echo "ok"
\ No newline at end of file
diff --git a/tradis b/tradis
index a8133b3..076e2be 100755
--- a/tradis
+++ b/tradis
@@ -3,7 +3,7 @@
 import argparse
 import sys
 
-import pkg_resources  # part of setuptools
+import importlib.metadata
 
 import quatradis.util.cli as qutils
 import quatradis.pipelines.pipeline as pipelines
@@ -11,7 +11,7 @@ import quatradis.tisp.cli as tisp
 import quatradis.comparison.cli as ca
 
 # Establish the software version from the package resources
-version = pkg_resources.require("quatradis")[0].version
+version = importlib.metadata.version("quatradis")
 
 def call_func(parser, args):
     """