Merge remote-tracking branch 'origin/master' into staging

.gitignore

@@ -22,3 +22,5 @@ nytprof.out
 __pycache__
 build/
 dist/
+.DS_Store
+docs/.DS_Store

docs/conf.py

@@ -69,7 +69,7 @@
 # The short X.Y version.
 version = '2.0'
 # The full version, including alpha/beta/rc tags.
-release = '2.0.5'
+release = '2.0.6'
 
 # The language for content autogenerated by Sphinx. Refer to documentation
 # for a list of supported languages.

docs/index.rst

@@ -60,16 +60,9 @@ New in release |release|
 
 This is a bugfix release. It fixes the following problem(s):
 
-- Some users have reported "Cannot open file" errors when running
-  NetMHCstabpan. This release adds a retry when this error in encountered.
-- This release adds stricter checking to pVACbind for unsupported amino acids.
-  Sequences containing an unsupported amino acid will be skipped. The
-  following amino acids are supported: ``A``, ``R``, ``N``, ``D``, ``C``, ``E``,
-  ``Q``, ``G``, ``H``, ``I``, ``L``, ``K``, ``M``, ``F``, ``P``, ``S``, ``T``,
-  ``W``, ``Y``, ``V``.
-- Some VEP predictions for supported variant types might not contain any
-  protein position information, rendering pVACseq unable to parse such
-  annotations. Annotations without protein position information will now be skipped.
+- When running pVAcseq with a proximal variants VCF, proximal DNPs affecting
+  multiple amino acids were not handled correctly and would result in an error.
+  This issue has now been fixed.
 
 New in version |version|
 ------------------------

docs/releases/2_0.rst

@@ -160,3 +160,11 @@ This is a bugfix release. It fixes the following problem(s):
   protein position information, rendering pVACseq unable to parse such
   annotations. Annotations without protein position information will now be skipped.
 
+Version 2.0.6
+-------------
+
+This is a bugfix release. It fixes the following problem(s):
+
+- When running pVAcseq with a proximal variants VCF, proximal DNPs affecting
+  multiple amino acids were not handled correctly and would result in an error.
+  This issue has now been fixed.

pvactools/lib/fasta_generator.py

@@ -111,20 +111,42 @@ def add_proximal_variants(self, somatic_variant_index, wildtype_subsequence, mut
                     codon_changes = [ item['codon_change'] for item in filtered_lines ]
                     proximal_variant_mutant_amino_acid = ProximalVariant.combine_conflicting_variants(codon_changes)
 
-                proximal_variant_position = int(protein_position) - 1 - mutation_offset
-                if proximal_variant_position <= 0 or proximal_variant_position >= len(wildtype_subsequence):
+                if '-' in protein_position:
+                    proximal_variant_start_position = int(protein_position.split('-')[0]) - 1 - mutation_offset
+                else:
+                    proximal_variant_start_position = int(protein_position) - 1 - mutation_offset
+                proximal_variant_end_position = proximal_variant_start_position + len(proximal_variant_mutant_amino_acid)
+
+                if proximal_variant_end_position <= 0 or proximal_variant_start_position >= len(wildtype_subsequence):
                     continue
                 if len(proximal_variant_wildtype_amino_acid) != len(proximal_variant_mutant_amino_acid):
                     print("Nearby variant is not a missense mutation. Skipping.")
                     continue
-                if wildtype_subsequence[proximal_variant_position] != proximal_variant_wildtype_amino_acid:
+
+                if proximal_variant_end_position > len(wildtype_subsequence):
+                    #the DNP extends past the end of the wildtype_subsequence
+                    missing_amino_acids_count = proximal_variant_end_position - len(wildtype_subsequence)
+                    #remove proximal variant amino acids after wildtype subsquence end
+                    proximal_variant_wildtype_amino_acid = proximal_variant_wildtype_amino_acid[:len(proximal_variant_wildtype_amino_acid) - missing_amino_acids_count]
+                    proximal_variant_mutant_amino_acid = proximal_variant_mutant_amino_acid[:len(proximal_variant_mutant_amino_acid) - missing_amino_acids_count]
+
+                if proximal_variant_start_position < 0:
+                    #DNP starts before the beginning of the wildtype subsequence
+                    missing_amino_acids_count = abs(proximal_variant_start_position)
+                    proximal_variant_start_position = 0
+                    #remove proximal variant amino acids before wildtype subsequence start
+                    proximal_variant_wildtype_amino_acid = proximal_variant_wildtype_amino_acid[missing_amino_acids_count:]
+                    proximal_variant_mutant_amino_acid = proximal_variant_mutant_amino_acid[missing_amino_acids_count:]
+
+                if wildtype_subsequence[proximal_variant_start_position:proximal_variant_end_position] != proximal_variant_wildtype_amino_acid:
                     sys.exit(
                         "Error when processing proximal variant.\n" +
                         "The wildtype amino acid for variant %s with substring %s is different than expected.\n" % (somatic_variant_index, wildtype_subsequence) +
-                        "Actual wildtype amino acid: %s\n" % wildtype_subsequence[proximal_variant_position] +
+                        "Actual wildtype amino acid: %s\n" % wildtype_subsequence[proximal_variant_start_position:proximal_variant_end_position] +
                         "Wildtype amino acid of the proximal_variant: %s" % proximal_variant_wildtype_amino_acid
                     )
-                wildtype_subsequence_with_proximal_variants = wildtype_subsequence_with_proximal_variants[:proximal_variant_position] + proximal_variant_mutant_amino_acid + wildtype_subsequence_with_proximal_variants[proximal_variant_position+1:]
+
+                wildtype_subsequence_with_proximal_variants = wildtype_subsequence_with_proximal_variants[:proximal_variant_start_position] + proximal_variant_mutant_amino_acid + wildtype_subsequence_with_proximal_variants[proximal_variant_end_position:]
         return wildtype_subsequence_with_proximal_variants
 
     def execute(self):

tests/test_data/fasta_generator/proximal_dnp/input.tsv

@@ -0,0 +1,11 @@
+chromosome_name	start	stop	reference	variant	gene_name	transcript_name	transcript_support_level	amino_acid_change	codon_change	ensembl_gene_id	hgvsc	hgvsp	wildtype_amino_acid_sequence	frameshift_amino_acid_sequence	fusion_amino_acid_sequence	variant_type	protein_position	transcript_expression	gene_expression	normal_depth	normal_vaf	tdna_depth	tdna_vaf	trna_depth	trna_vaf	index	protein_length_change
+chr16	28593449	28593450	AG	GA	SULT1A2	ENST00000335715	1	A/V	gCT/gTC	ENSG00000197165	ENST00000335715.9:c.491_492delinsTC	ENSP00000338742.4:p.Ala164Val	MELIQDISRPPLEYVKGVPLIKYFAEALGPLQSFQARPDDLLISTYPKSGTTWVSQILDMIYQGGDLEKCHRAPIFMRVPFLEFKVPGIPSGMETLKNTPAPRLLKTHLPLALLPQTLLDQKVKVVYVARNAKDVAVSYYHFYHMAKVYPHPGTWESFLEKFMAGEVSYGSWYQHVQEWWELSRTHPVLYLFYEDMKENPKREIQKILEFVGRSLPEETVDLMVEHTSFKEMKKNPMTNYTTVRREFMDHSISPFMRKGMAGDWKTTFTVAQNERFDADYAKKMAGCSLSFRSEL			missense	164	NA	0.0534405615378929	NA	NA	185	0.136	NA	NA	1.SULT1A2.ENST00000335715.missense.164A/V	
+chr16	28593449	28593450	AG	GA	SULT1A2	ENST00000395630	5	A/V	gCT/gTC	ENSG00000197165	ENST00000395630.5:c.491_492delinsTC	ENSP00000378992.1:p.Ala164Val	MELIQDISRPPLEYVKGVPLIKYFAEALGPLQSFQARPDDLLISTYPKSGTTWVSQILDMIYQGGDLEKCHRAPIFMRVPFLEFKVPGIPSGMETLKNTPAPRLLKTHLPLALLPQTLLDQKVKVVYVARNAKDVAVSYYHFYHMAKVYPHPGTWESFLEKFMAGEVSYGSWYQHVQEWWELSRTHPVLYLFYEDMKENPKREIQKILEFVGRSLPEETVDLMVEHTSFKEMKKNPMTNYTTVRREFMDHSISPFMRKGMAGDWKTTFTVAQNERFDADYAKKMAGCSLSFRSEL			missense	164	NA	0.0534405615378929	NA	NA	185	0.136	NA	NA	2.SULT1A2.ENST00000395630.missense.164A/V	
+chr16	28593449	28593450	AG	GA	SULT1A2	ENST00000526384	3	A/V	gCT/gTC	ENSG00000197165	ENST00000526384.1:c.491_492delinsTC	ENSP00000435358.1:p.Ala164Val	MELIQDISRPPLEYVKGVPLIKYFAEALGPLQSFQARPDDLLISTYPKSGTTWVSQILDMIYQGGDLEKCHRAPIFMRVPFLEFKVPGIPSGMETLKNTPAPRLLKTHLPLALLPQTLLDQKVKVVYVARNAKDVAVSYYHFYHMAKVYPHPGTWESFLEKFMAGEVSYGSWYQHVQEWWELSRTHPVLYLF			missense	164	NA	0.0534405615378929	NA	NA	185	0.136	NA	NA	3.SULT1A2.ENST00000526384.missense.164A/V	
+chr16	28593449	28593450	AG	GA	SULT1A2	ENST00000534108	2	GW/GR	ggCTgg/ggTCgg	ENSG00000197165	ENST00000534108.5:c.576_577delinsTC	ENSP00000433075.1:p.Trp193Arg	MELIQDISRPPLEYVKGVPLIKYFAEALGPLQSFQARPDDLLISTYPKSGTTWVSQILDMIYQGGDLEKCHRAPIFMRVPFLEFKVPGIPSGVCVLGARGVEEDRAGASAHQTFPDPLLRDGDSEKHTSPTTPEDTPAPGSAPPDSVGSEGQGGLCCPQRKGCGGFLLPLLPHGQSVPSPWDLGKLPGEVHGWRSVLWVLVPARARVVGAEPHPPCSLPLL			missense	192-193	NA	0.0534405615378929	NA	NA	185	0.136	NA	NA	4.SULT1A2.ENST00000534108.missense.192-193GW/GR	
+chr16	28593449	28593450	AG	GA	AC020765.6	ENST00000677940	NA	A/V	gCT/gTC	ENSG00000288656	ENST00000677940.1:c.257_258delinsTC	ENSP00000503077.1:p.Ala86Val	MLAKLLCDQVVGAPIAVSAFYAGMSILQGKDDIFLDLKQKFWNTYMVVYVARNAKDVAVSYYHFYHMAKVYPHPGTWESFLEKFMAGEVSYGSWYQHVQEWWELSRTHPVLYLFYEDMKENPKREIQKILEFVGRSLPEETVDLMVEHTSFKEMKKNPMTNYTTVRREFMDHSISPFMRKGMAGDWKTTFTVAQNERFDADYAKKMAGC			missense	86	NA	0.0	NA	NA	185	0.136	NA	NA	5.AC020765.6.ENST00000677940.missense.86A/V	
+chr16	28593472	28593473	T	G	SULT1A2	ENST00000335715	1	E/D	gaA/gaC	ENSG00000197165	ENST00000335715.9:c.468A>C	ENSP00000338742.4:p.Glu156Asp	MELIQDISRPPLEYVKGVPLIKYFAEALGPLQSFQARPDDLLISTYPKSGTTWVSQILDMIYQGGDLEKCHRAPIFMRVPFLEFKVPGIPSGMETLKNTPAPRLLKTHLPLALLPQTLLDQKVKVVYVARNAKDVAVSYYHFYHMAKVYPHPGTWESFLEKFMAGEVSYGSWYQHVQEWWELSRTHPVLYLFYEDMKENPKREIQKILEFVGRSLPEETVDLMVEHTSFKEMKKNPMTNYTTVRREFMDHSISPFMRKGMAGDWKTTFTVAQNERFDADYAKKMAGCSLSFRSEL			missense	156	NA	0.0534405615378929	NA	NA	195	0.121	0	NA	6.SULT1A2.ENST00000335715.missense.156E/D	
+chr16	28593472	28593473	T	G	SULT1A2	ENST00000395630	5	E/D	gaA/gaC	ENSG00000197165	ENST00000395630.5:c.468A>C	ENSP00000378992.1:p.Glu156Asp	MELIQDISRPPLEYVKGVPLIKYFAEALGPLQSFQARPDDLLISTYPKSGTTWVSQILDMIYQGGDLEKCHRAPIFMRVPFLEFKVPGIPSGMETLKNTPAPRLLKTHLPLALLPQTLLDQKVKVVYVARNAKDVAVSYYHFYHMAKVYPHPGTWESFLEKFMAGEVSYGSWYQHVQEWWELSRTHPVLYLFYEDMKENPKREIQKILEFVGRSLPEETVDLMVEHTSFKEMKKNPMTNYTTVRREFMDHSISPFMRKGMAGDWKTTFTVAQNERFDADYAKKMAGCSLSFRSEL			missense	156	NA	0.0534405615378929	NA	NA	195	0.121	0	NA	7.SULT1A2.ENST00000395630.missense.156E/D	
+chr16	28593472	28593473	T	G	SULT1A2	ENST00000526384	3	E/D	gaA/gaC	ENSG00000197165	ENST00000526384.1:c.468A>C	ENSP00000435358.1:p.Glu156Asp	MELIQDISRPPLEYVKGVPLIKYFAEALGPLQSFQARPDDLLISTYPKSGTTWVSQILDMIYQGGDLEKCHRAPIFMRVPFLEFKVPGIPSGMETLKNTPAPRLLKTHLPLALLPQTLLDQKVKVVYVARNAKDVAVSYYHFYHMAKVYPHPGTWESFLEKFMAGEVSYGSWYQHVQEWWELSRTHPVLYLF			missense	156	NA	0.0534405615378929	NA	NA	195	0.121	0	NA	8.SULT1A2.ENST00000526384.missense.156E/D	
+chr16	28593472	28593473	T	G	SULT1A2	ENST00000534108	2	K/Q	Aag/Cag	ENSG00000197165	ENST00000534108.5:c.553A>C	ENSP00000433075.1:p.Lys185Gln	MELIQDISRPPLEYVKGVPLIKYFAEALGPLQSFQARPDDLLISTYPKSGTTWVSQILDMIYQGGDLEKCHRAPIFMRVPFLEFKVPGIPSGVCVLGARGVEEDRAGASAHQTFPDPLLRDGDSEKHTSPTTPEDTPAPGSAPPDSVGSEGQGGLCCPQRKGCGGFLLPLLPHGQSVPSPWDLGKLPGEVHGWRSVLWVLVPARARVVGAEPHPPCSLPLL			missense	185	NA	0.0534405615378929	NA	NA	195	0.121	0	NA	9.SULT1A2.ENST00000534108.missense.185K/Q	
+chr16	28593472	28593473	T	G	AC020765.6	ENST00000677940	NA	E/D	gaA/gaC	ENSG00000288656	ENST00000677940.1:c.234A>C	ENSP00000503077.1:p.Glu78Asp	MLAKLLCDQVVGAPIAVSAFYAGMSILQGKDDIFLDLKQKFWNTYMVVYVARNAKDVAVSYYHFYHMAKVYPHPGTWESFLEKFMAGEVSYGSWYQHVQEWWELSRTHPVLYLFYEDMKENPKREIQKILEFVGRSLPEETVDLMVEHTSFKEMKKNPMTNYTTVRREFMDHSISPFMRKGMAGDWKTTFTVAQNERFDADYAKKMAGC			missense	78	NA	0.0	NA	NA	195	0.121	0	NA	10.AC020765.6.ENST00000677940.missense.78E/D	

tests/test_data/fasta_generator/proximal_dnp/output.cut_off.fasta

@@ -0,0 +1,16 @@
+>1
+SFLEKFMAGEVSYGS
+>2
+SFLEKFMVGEVSYGS
+>3
+KLPGEVHGWRSVLWVL
+>4
+QLPGEVHGRRSVLWVL
+>5
+YPHPGTWESFLEKFM
+>6
+YPHPGTWDSFLEKFM
+>7
+PSPWDLGKLPGEVHG
+>8
+PSPWDLGQLPGEVHG

tests/test_data/fasta_generator/proximal_dnp/output.cut_off.key

@@ -0,0 +1,28 @@
+1:
+- WT.1.SULT1A2.ENST00000335715.missense.164A/V
+- WT.2.SULT1A2.ENST00000395630.missense.164A/V
+- WT.3.SULT1A2.ENST00000526384.missense.164A/V
+- WT.5.AC020765.6.ENST00000677940.missense.86A/V
+2:
+- MT.1.SULT1A2.ENST00000335715.missense.164A/V
+- MT.2.SULT1A2.ENST00000395630.missense.164A/V
+- MT.3.SULT1A2.ENST00000526384.missense.164A/V
+- MT.5.AC020765.6.ENST00000677940.missense.86A/V
+3:
+- WT.4.SULT1A2.ENST00000534108.missense.192-193GW/GR
+4:
+- MT.4.SULT1A2.ENST00000534108.missense.192-193GW/GR
+5:
+- WT.6.SULT1A2.ENST00000335715.missense.156E/D
+- WT.7.SULT1A2.ENST00000395630.missense.156E/D
+- WT.8.SULT1A2.ENST00000526384.missense.156E/D
+- WT.10.AC020765.6.ENST00000677940.missense.78E/D
+6:
+- MT.6.SULT1A2.ENST00000335715.missense.156E/D
+- MT.7.SULT1A2.ENST00000395630.missense.156E/D
+- MT.8.SULT1A2.ENST00000526384.missense.156E/D
+- MT.10.AC020765.6.ENST00000677940.missense.78E/D
+7:
+- WT.9.SULT1A2.ENST00000534108.missense.185K/Q
+8:
+- MT.9.SULT1A2.ENST00000534108.missense.185K/Q

tests/test_data/fasta_generator/proximal_dnp/output.fasta

@@ -0,0 +1,16 @@
+>1
+WESFLEKFMAGEVSYGSWY
+>2
+WDSFLEKFMVGEVSYGSWY
+>3
+LGKLPGEVHGWRSVLWVLVP
+>4
+LGQLPGEVHGRRSVLWVLVP
+>5
+KVYPHPGTWESFLEKFMAG
+>6
+KVYPHPGTWDSFLEKFMVG
+>7
+SVPSPWDLGKLPGEVHGWR
+>8
+SVPSPWDLGQLPGEVHGRR

tests/test_data/fasta_generator/proximal_dnp/output.key

@@ -0,0 +1,28 @@
+1:
+- WT.1.SULT1A2.ENST00000335715.missense.164A/V
+- WT.2.SULT1A2.ENST00000395630.missense.164A/V
+- WT.3.SULT1A2.ENST00000526384.missense.164A/V
+- WT.5.AC020765.6.ENST00000677940.missense.86A/V
+2:
+- MT.1.SULT1A2.ENST00000335715.missense.164A/V
+- MT.2.SULT1A2.ENST00000395630.missense.164A/V
+- MT.3.SULT1A2.ENST00000526384.missense.164A/V
+- MT.5.AC020765.6.ENST00000677940.missense.86A/V
+3:
+- WT.4.SULT1A2.ENST00000534108.missense.192-193GW/GR
+4:
+- MT.4.SULT1A2.ENST00000534108.missense.192-193GW/GR
+5:
+- WT.6.SULT1A2.ENST00000335715.missense.156E/D
+- WT.7.SULT1A2.ENST00000395630.missense.156E/D
+- WT.8.SULT1A2.ENST00000526384.missense.156E/D
+- WT.10.AC020765.6.ENST00000677940.missense.78E/D
+6:
+- MT.6.SULT1A2.ENST00000335715.missense.156E/D
+- MT.7.SULT1A2.ENST00000395630.missense.156E/D
+- MT.8.SULT1A2.ENST00000526384.missense.156E/D
+- MT.10.AC020765.6.ENST00000677940.missense.78E/D
+7:
+- WT.9.SULT1A2.ENST00000534108.missense.185K/Q
+8:
+- MT.9.SULT1A2.ENST00000534108.missense.185K/Q

tests/test_data/fasta_generator/proximal_dnp/proximal_variants.tsv

@@ -0,0 +1,11 @@
+chromosome_name	start	stop	reference	variant	amino_acid_change	codon_change	protein_position	type	main_somatic_variant
+chr16	28593472	28593473	T	G	E/D	gaA/gaC	156	somatic	1.SULT1A2.ENST00000335715.missense.164A/V
+chr16	28593472	28593473	T	G	E/D	gaA/gaC	156	somatic	2.SULT1A2.ENST00000395630.missense.164A/V
+chr16	28593472	28593473	T	G	E/D	gaA/gaC	156	somatic	3.SULT1A2.ENST00000526384.missense.164A/V
+chr16	28593472	28593473	T	G	K/Q	Aag/Cag	185	somatic	4.SULT1A2.ENST00000534108.missense.192-193GW/GR
+chr16	28593472	28593473	T	G	E/D	gaA/gaC	78	somatic	5.AC020765.6.ENST00000677940.missense.86A/V
+chr16	28593449	28593450	AG	GA	A/V	gCT/gTC	164	somatic	6.SULT1A2.ENST00000335715.missense.156E/D
+chr16	28593449	28593450	AG	GA	A/V	gCT/gTC	164	somatic	7.SULT1A2.ENST00000395630.missense.156E/D
+chr16	28593449	28593450	AG	GA	A/V	gCT/gTC	164	somatic	8.SULT1A2.ENST00000526384.missense.156E/D
+chr16	28593449	28593450	AG	GA	GW/GR	ggCTgg/ggTCgg	192-193	somatic	9.SULT1A2.ENST00000534108.missense.185K/Q
+chr16	28593449	28593450	AG	GA	A/V	gCT/gTC	86	somatic	10.AC020765.6.ENST00000677940.missense.78E/D

tests/test_fasta_generator.py

@@ -747,6 +747,52 @@ def test_multiple_proximal_variants_on_same_codon_results_in_stop_codon(self):
         self.assertEqual(os.path.getsize(generate_fasta_output_file.name), 0)
         self.assertEqual(os.path.getsize(generate_fasta_key_output_file.name), 0)
 
+    def test_proximal_dnp(self):
+        test_data_dir                  = os.path.join(self.test_data_dir, 'proximal_dnp')
+        generate_fasta_input_file      = os.path.join(test_data_dir, 'input.tsv')
+        generate_fasta_proximal_variants_file = os.path.join(test_data_dir, 'proximal_variants.tsv')
+        generate_fasta_output_file     = tempfile.NamedTemporaryFile()
+        generate_fasta_key_output_file = tempfile.NamedTemporaryFile()
+
+        generate_fasta_params = {
+            'input_file'                : generate_fasta_input_file,
+            'epitope_length'            : 10,
+            'flanking_sequence_length'  : 9,
+            'output_file'               : generate_fasta_output_file.name,
+            'output_key_file'           : generate_fasta_key_output_file.name,
+            'proximal_variants_file'    : generate_fasta_proximal_variants_file,
+        }
+        generator = FastaGenerator(**generate_fasta_params)
+
+        self.assertFalse(generator.execute())
+        expected_output_file = os.path.join(test_data_dir, 'output.fasta')
+        self.assertTrue(cmp(generate_fasta_output_file.name, expected_output_file))
+        expected_key_output_file = os.path.join(test_data_dir, 'output.key')
+        self.assertTrue(cmp(generate_fasta_key_output_file.name, expected_key_output_file))
+
+    def test_proximal_dnp_cut_off(self):
+        test_data_dir                  = os.path.join(self.test_data_dir, 'proximal_dnp')
+        generate_fasta_input_file      = os.path.join(test_data_dir, 'input.tsv')
+        generate_fasta_proximal_variants_file = os.path.join(test_data_dir, 'proximal_variants.tsv')
+        generate_fasta_output_file     = tempfile.NamedTemporaryFile()
+        generate_fasta_key_output_file = tempfile.NamedTemporaryFile()
+
+        generate_fasta_params = {
+            'input_file'                : generate_fasta_input_file,
+            'epitope_length'            : 8,
+            'flanking_sequence_length'  : 7,
+            'output_file'               : generate_fasta_output_file.name,
+            'output_key_file'           : generate_fasta_key_output_file.name,
+            'proximal_variants_file'    : generate_fasta_proximal_variants_file,
+        }
+        generator = FastaGenerator(**generate_fasta_params)
+
+        self.assertFalse(generator.execute())
+        expected_output_file = os.path.join(test_data_dir, 'output.cut_off.fasta')
+        self.assertTrue(cmp(generate_fasta_output_file.name, expected_output_file))
+        expected_key_output_file = os.path.join(test_data_dir, 'output.cut_off.key')
+        self.assertTrue(cmp(generate_fasta_key_output_file.name, expected_key_output_file))
+
     def test_distance_from_start_works_as_expected(self):
         generate_fasta_input_file      = tempfile.NamedTemporaryFile()
         generate_fasta_output_file     = tempfile.NamedTemporaryFile()