diff --git a/app/models/labware_creators/plate_split_to_tube_racks.rb b/app/models/labware_creators/plate_split_to_tube_racks.rb
index f10433435..60db9fd9a 100644
--- a/app/models/labware_creators/plate_split_to_tube_racks.rb
+++ b/app/models/labware_creators/plate_split_to_tube_racks.rb
@@ -60,8 +60,12 @@ class PlateSplitToTubeRacks < Base
     validates_nested :sequencing_csv_file, if: :sequencing_file
     validates_nested :contingency_csv_file, if: :contingency_file
 
+    # validations for duplications between the two tube rack files
+    validate :check_tube_rack_barcodes_differ_between_files
+    validate :check_tube_barcodes_differ_between_files
+
     # validate there are sufficient tubes in the racks for the number of parent wells
-    validate :must_have_sufficient_tubes_in_rack, if: :contingency_file
+    validate :must_have_sufficient_tubes_in_rack_files, if: :contingency_file
 
     # validate that the tube barcodes do not already exist in the system
     validate :tube_barcodes_are_unique?
@@ -137,6 +141,22 @@ def anchor
       'children_tab'
     end
 
+    # Returns a CsvFile object for the sequencing tube rack scan CSV file, or nil if the file doesn't exist.
+    #
+    # @return [CsvFile, nil] A CsvFile object for the sequencing tube rack scan CSV file, or nil if the file
+    # doesn't exist.
+    def sequencing_csv_file
+      @sequencing_csv_file ||= CsvFile.new(sequencing_file) if sequencing_file
+    end
+
+    # Returns a CsvFile object for the contingency tube rack scan CSV file, or nil if the file doesn't exist.
+    #
+    # @return [CsvFile, nil] A CsvFile object for the contingency tube rack scan CSV file, or nil if the file
+    # doesn't exist.
+    def contingency_csv_file
+      @contingency_csv_file ||= CsvFile.new(contingency_file) if contingency_file
+    end
+
     # Returns the number of unique sample UUIDs for the parent wells after applying the current well filter.
     #
     # @return [Integer] The number of unique sample UUIDs.
@@ -151,27 +171,93 @@ def num_parent_wells
       @num_parent_wells ||= well_filter.filtered.length
     end
 
-    # Validation method that checks whether there are enough tubes in the rack.
+    # Validation to compare the tube rack barcodes in the two files to check for duplication
     #
-    # @return [void]
-    def must_have_sufficient_tubes_in_rack
-      errors.add(:tube_rack_file, 'Must have sufficient tubes in rack') unless sufficient_tubes_in_racks?
+    # Sets errors if the tube rack barcodes are the same
+    def check_tube_rack_barcodes_differ_between_files
+      return unless contingency_file_correctly_parsed? && sequencing_file_correctly_parsed?
+
+      return unless same_tube_rack_barcode?
+
+      errors.add(
+        :contingency_csv_file,
+        'The tube rack barcodes within the contingency and sequencing files must be different'
+      )
+    end
+
+    def same_tube_rack_barcode?
+      seq_tube_rack = extract_tube_rack_barcode(sequencing_csv_file)
+      cont_tube_rack = extract_tube_rack_barcode(contingency_csv_file)
+
+      seq_tube_rack == cont_tube_rack
+    end
+
+    def extract_tube_rack_barcode(file)
+      file.position_details.values.first['tube_rack_barcode']
+    end
+
+    # Validation to compare the tube barcodes in the two files to check for duplication
+    #
+    # Sets errors if the tube rack barcodes are the same
+    def check_tube_barcodes_differ_between_files
+      return unless contingency_file_correctly_parsed? && sequencing_file_correctly_parsed?
+
+      seq_barcodes = extract_barcodes(sequencing_csv_file)
+      cont_barcodes = extract_barcodes(contingency_csv_file)
+
+      duplicate_barcodes = seq_barcodes & cont_barcodes
+      return if duplicate_barcodes.empty?
+
+      errors.add(
+        :contingency_csv_file,
+        "Tube barcodes are duplicated across contingency and sequencing files (#{duplicate_barcodes.join(', ')})"
+      )
+    end
+
+    def extract_barcodes(file)
+      file.position_details.values.pluck('tube_barcode')
     end
 
-    # Checks if there are sufficient tubes in the child tube racks for all the parent wells.
+    # Validation that checks if there are sufficient tubes in the child tube racks for all the parent wells.
     # This depends on the number of unique samples in the parent plate, and the number of parent wells,
     # as well as whether they are using both sequencing tubes and contingency tubes or just contingency.
     #
-    # @return [Boolean] `true` if there are sufficient tubes, `false` otherwise.
-    def sufficient_tubes_in_racks?
+    # Sets errors if there are insufficient tubes.
+    def must_have_sufficient_tubes_in_rack_files
+      return unless files_correctly_parsed?
+
       if require_contingency_tubes_only?
-        num_contingency_tubes >= num_parent_wells
+        add_error_if_insufficient_tubes(:contingency_csv_file, num_contingency_tubes, num_parent_wells)
       else
-        (num_sequencing_tubes >= num_parent_unique_samples) &&
-          (num_contingency_tubes >= (num_parent_wells - num_parent_unique_samples))
+        add_error_if_insufficient_tubes(
+          :contingency_csv_file,
+          num_contingency_tubes,
+          num_parent_wells - num_parent_unique_samples
+        )
+        add_error_if_insufficient_tubes(:sequencing_csv_file, num_sequencing_tubes, num_parent_unique_samples)
       end
     end
 
+    # Checks the files parsed correctly
+    def files_correctly_parsed?
+      return contingency_file_correctly_parsed? if require_contingency_tubes_only?
+
+      contingency_file_correctly_parsed? && sequencing_file_correctly_parsed?
+    end
+
+    def sequencing_file_correctly_parsed?
+      sequencing_file.present? && sequencing_csv_file.correctly_parsed?
+    end
+
+    def contingency_file_correctly_parsed?
+      contingency_file.present? && contingency_csv_file.correctly_parsed?
+    end
+
+    # Adds an error when there are insufficient tubes in the given file
+    def add_error_if_insufficient_tubes(file, num_tubes, required_tubes)
+      errors.add(file, 'contains insufficient tubes') unless num_tubes >= required_tubes
+    end
+
     # Validation that the tube barcodes are unique and do not already exist in the system.
     # NB. this checks all the tube barcodes in the uploaded tube rack scan files, not just the
     # ones that will be used.
@@ -261,22 +347,6 @@ def upload_tube_rack_files
       parent_v1.qc_files.create_from_file!(contingency_file, 'scrna_core_contingency_tube_rack_scan.csv')
     end
 
-    # Returns a CsvFile object for the sequencing tube rack scan CSV file, or nil if the file doesn't exist.
-    #
-    # @return [CsvFile, nil] A CsvFile object for the sequencing tube rack scan CSV file, or nil if the file
-    # doesn't exist.
-    def sequencing_csv_file
-      @sequencing_csv_file ||= CsvFile.new(sequencing_file) if sequencing_file
-    end
-
-    # Returns a CsvFile object for the contingency tube rack scan CSV file, or nil if the file doesn't exist.
-    #
-    # @return [CsvFile, nil] A CsvFile object for the contingency tube rack scan CSV file, or nil if the file
-    # doesn't exist.
-    def contingency_csv_file
-      @contingency_csv_file ||= CsvFile.new(contingency_file) if contingency_file
-    end
-
     # Returns true if only contingency tubes are required for the parent plate, false otherwise.
     #
     # @return [Boolean]
diff --git a/app/models/labware_creators/plate_split_to_tube_racks/csv_file.rb b/app/models/labware_creators/plate_split_to_tube_racks/csv_file.rb
index 048df81d7..048d8723e 100644
--- a/app/models/labware_creators/plate_split_to_tube_racks/csv_file.rb
+++ b/app/models/labware_creators/plate_split_to_tube_racks/csv_file.rb
@@ -13,8 +13,8 @@ module LabwareCreators
   # and the tube locations then used to create a driver file for the liquid handler.
   # The filename of the file should also contain the tube rack barcode.
   # Example of file content (NB. no header line):
-  # TR00012345, A1, FX05653780
-  # TR00012345, A2, NO READ
+  # TR00012345,A1,FX05653780
+  # TR00012345,A2,NO READ
   # etc.
   #
   class PlateSplitToTubeRacks::CsvFile
@@ -23,6 +23,9 @@ class PlateSplitToTubeRacks::CsvFile
 
     validate :correctly_parsed?
     validates_nested :tube_rack_scan, if: :correctly_formatted?
+    validate :check_for_rack_barcodes_the_same, if: :correctly_formatted?
+    validate :check_no_duplicate_well_coordinates, if: :correctly_formatted?
+    validate :check_no_duplicate_tube_barcodes, if: :correctly_formatted?
 
     NO_TUBE_TEXTS = ['NO READ', 'NOSCAN'].freeze
 
@@ -97,6 +100,36 @@ def correctly_formatted?
       correctly_parsed?
     end
 
+    def check_for_rack_barcodes_the_same
+      tube_rack_barcodes = tube_rack_scan.group_by(&:tube_rack_barcode).keys
+
+      return unless tube_rack_barcodes.size > 1
+
+      barcodes_str = tube_rack_barcodes.join(',')
+      errors.add(:base, "Should not contain different rack barcodes (#{barcodes_str})")
+    end
+
+    def check_no_duplicate_well_coordinates
+      duplicated_well_coordinates =
+        tube_rack_scan.group_by(&:tube_position).select { |_position, tubes| tubes.size > 1 }.keys.join(',')
+
+      return if duplicated_well_coordinates.empty?
+
+      errors.add(:base, "Contains duplicate well coordinates (#{duplicated_well_coordinates})")
+    end
+
+    def check_no_duplicate_tube_barcodes
+      duplicates = tube_rack_scan.group_by(&:tube_barcode).select { |_tube_barcode, tubes| tubes.size > 1 }.keys
+
+      # remove any NO READ or NOSCAN values
+      ignore_list = ['NO READ', 'NOSCAN']
+      duplicates = duplicates.reject { |barcode| ignore_list.include?(barcode) }
+
+      return if duplicates.empty?
+
+      errors.add(:base, "Contains duplicate tube barcodes (#{duplicates.join(',')})")
+    end
+
     # Generates a hash of position details based on the tube rack scan data in the CSV file.
     #
     # @return [Hash] A hash of position details, where the keys are positions and the values
diff --git a/spec/fixtures/files/plate_split_to_tube_racks/tube_rack_scan_with_different_rack_barcodes.csv b/spec/fixtures/files/plate_split_to_tube_racks/tube_rack_scan_with_different_rack_barcodes.csv
new file mode 100644
index 000000000..ecc5ec4d4
--- /dev/null
+++ b/spec/fixtures/files/plate_split_to_tube_racks/tube_rack_scan_with_different_rack_barcodes.csv
@@ -0,0 +1,16 @@
+FX12345678, A1, AB10000001
+FX12345678, B1, AB10000002
+FX12345678, C1, AB10000003
+FX12345678, D1, AB10000004
+FX12345678, E1, AB10000005
+FX12345678, F1, AB10000006
+FX12345678, G1, AB10000007
+FX12345678, H1, AB10000008
+FX23838838, A2, AB10000009
+FX12345678, B2, AB10000010
+FX12345678, C2, AB10000011
+FX12345678, D2, AB10000012
+FX12345678, E2, AB10000013
+FX12345678, F2, AB10000014
+FX12345678, G2, AB10000015
+FX12345678, H2, AB10000016
\ No newline at end of file
diff --git a/spec/fixtures/files/plate_split_to_tube_racks/tube_rack_scan_with_duplicate_tubes.csv b/spec/fixtures/files/plate_split_to_tube_racks/tube_rack_scan_with_duplicate_tubes.csv
new file mode 100644
index 000000000..28fa282cd
--- /dev/null
+++ b/spec/fixtures/files/plate_split_to_tube_racks/tube_rack_scan_with_duplicate_tubes.csv
@@ -0,0 +1,16 @@
+FX12345678, A1, AB10000001
+FX12345678, B1, AB10000002
+FX12345678, C1, AB10000003
+FX12345678, D1, AB10000004
+FX12345678, E1, NO READ
+FX12345678, F1, AB10000006
+FX12345678, G1, AB10000007
+FX12345678, H1, AB10000008
+FX12345678, A2, AB10000009
+FX12345678, B2, AB10000009
+FX12345678, C2, AB10000011
+FX12345678, D2, AB10000011
+FX12345678, E2, AB10000013
+FX12345678, F2, AB10000014
+FX12345678, G2, NO READ
+FX12345678, H2, AB10000016
\ No newline at end of file
diff --git a/spec/fixtures/files/plate_split_to_tube_racks/tube_rack_scan_with_duplicate_well_positions.csv b/spec/fixtures/files/plate_split_to_tube_racks/tube_rack_scan_with_duplicate_well_positions.csv
new file mode 100644
index 000000000..dd1360f97
--- /dev/null
+++ b/spec/fixtures/files/plate_split_to_tube_racks/tube_rack_scan_with_duplicate_well_positions.csv
@@ -0,0 +1,16 @@
+FX12345678, A1, AB10000001
+FX12345678, B1, AB10000002
+FX12345678, C1, AB10000003
+FX12345678, D1, AB10000004
+FX12345678, E1, AB10000005
+FX12345678, F1, AB10000006
+FX12345678, G1, AB10000007
+FX12345678, H1, AB10000008
+FX12345678, A2, AB10000009
+FX12345678, A2, AB10000010
+FX12345678, C2, AB10000011
+FX12345678, D2, AB10000012
+FX12345678, E2, AB10000013
+FX12345678, E2, AB10000014
+FX12345678, G2, AB10000015
+FX12345678, H2, AB10000016
\ No newline at end of file
diff --git a/spec/models/labware_creators/plate_split_to_tube_racks/csv_file_spec.rb b/spec/models/labware_creators/plate_split_to_tube_racks/csv_file_spec.rb
index bdfdbb3f1..d8b2c09b6 100644
--- a/spec/models/labware_creators/plate_split_to_tube_racks/csv_file_spec.rb
+++ b/spec/models/labware_creators/plate_split_to_tube_racks/csv_file_spec.rb
@@ -300,4 +300,69 @@
       end
     end
   end
+
+  # there should be only one rack barcode in the file and it should be the same for all rows
+  context 'A file with inconsistant rack barcodes' do
+    let(:file) do
+      fixture_file_upload(
+        'spec/fixtures/files/plate_split_to_tube_racks/tube_rack_scan_with_different_rack_barcodes.csv',
+        'sequencescape/qc_file'
+      )
+    end
+
+    describe '#valid?' do
+      it 'should be invalid' do
+        expect(subject.valid?).to be false
+      end
+
+      it 'reports the errors' do
+        subject.valid?
+        expect(subject.errors.full_messages).to include(
+          'Should not contain different rack barcodes (FX12345678,FX23838838)'
+        )
+      end
+    end
+  end
+
+  # the same well position should not appear more than once in the file
+  context 'A file with duplicated well positions' do
+    let(:file) do
+      fixture_file_upload(
+        'spec/fixtures/files/plate_split_to_tube_racks/tube_rack_scan_with_duplicate_well_positions.csv',
+        'sequencescape/qc_file'
+      )
+    end
+
+    describe '#valid?' do
+      it 'should be invalid' do
+        expect(subject.valid?).to be false
+      end
+
+      it 'reports the errors' do
+        subject.valid?
+        expect(subject.errors.full_messages).to include('Contains duplicate well coordinates (A2,E2)')
+      end
+    end
+  end
+
+  # the same tube barcode should not appear more than once in the file
+  context 'A file with duplicated tube barcodes' do
+    let(:file) do
+      fixture_file_upload(
+        'spec/fixtures/files/plate_split_to_tube_racks/tube_rack_scan_with_duplicate_tubes.csv',
+        'sequencescape/qc_file'
+      )
+    end
+
+    describe '#valid?' do
+      it 'should not be valid' do
+        expect(subject.valid?).to be false
+      end
+
+      it 'reports the errors' do
+        subject.valid?
+        expect(subject.errors.full_messages).to include('Contains duplicate tube barcodes (AB10000009,AB10000011)')
+      end
+    end
+  end
 end
diff --git a/spec/models/labware_creators/plate_split_to_tube_racks_spec.rb b/spec/models/labware_creators/plate_split_to_tube_racks_spec.rb
index 553832d2e..d6f1dd32b 100644
--- a/spec/models/labware_creators/plate_split_to_tube_racks_spec.rb
+++ b/spec/models/labware_creators/plate_split_to_tube_racks_spec.rb
@@ -186,6 +186,14 @@
     )
   end
 
+  let(:sequencing_file) do
+    fixture_file_upload('spec/fixtures/files/scrna_core_sequencing_tube_rack_scan.csv', 'sequencescape/qc_file')
+  end
+
+  let(:contingency_file) do
+    fixture_file_upload('spec/fixtures/files/scrna_core_contingency_tube_rack_scan.csv', 'sequencescape/qc_file')
+  end
+
   before do
     # need both child tubes to have a purpose config here
     create(
@@ -220,7 +228,7 @@
     end
   end
 
-  context '#sufficient_tubes_in_racks?' do
+  context '#must_have_sufficient_tubes_in_rack_files' do
     let(:num_parent_wells) { 96 }
     let(:num_parent_unique_samples) { 48 }
     let(:num_sequencing_tubes) { 48 }
@@ -242,13 +250,13 @@
 
     context 'when a contingency file is not present' do
       it 'does not call the validation' do
-        expect(subject).not_to receive(:sufficient_tubes_in_racks?)
+        subject.validate
+        expect(subject).not_to be_valid
+        expect(subject).not_to receive(:must_have_sufficient_tubes_in_rack_files)
       end
     end
 
     context 'when require_contingency_tubes_only? is true' do
-      let(:contingency_file) { 'somefile' }
-
       let(:form_attributes) do
         {
           user_uuid: user_uuid,
@@ -258,29 +266,29 @@
         }
       end
 
-      before { allow(subject).to receive(:require_contingency_tubes_only?).and_return(true) }
+      before do
+        allow(subject).to receive(:require_contingency_tubes_only?).and_return(true)
+        subject.must_have_sufficient_tubes_in_rack_files
+      end
 
       context 'when there are enough contingency tubes' do
         let(:num_contingency_tubes) { 96 }
 
-        it 'returns true' do
-          expect(subject.sufficient_tubes_in_racks?).to be true
+        it 'is valid' do
+          expect(subject.errors[:contingency_csv_file]).to be_empty
         end
       end
 
       context 'when there are not enough contingency tubes' do
         let(:num_contingency_tubes) { 47 }
 
-        it 'returns false' do
-          expect(subject.sufficient_tubes_in_racks?).to be false
+        it 'is not valid' do
+          expect(subject.errors.full_messages).to include('Contingency csv file contains insufficient tubes')
         end
       end
     end
 
     context 'when require_contingency_tubes_only? is false' do
-      let(:sequencing_file) { 'somefile' }
-      let(:contingency_file) { 'somefile' }
-
       let(:form_attributes) do
         {
           user_uuid: user_uuid,
@@ -291,32 +299,191 @@
         }
       end
 
-      before { allow(subject).to receive(:require_contingency_tubes_only?).and_return(false) }
+      before do
+        allow(subject).to receive(:require_contingency_tubes_only?).and_return(false)
+        subject.must_have_sufficient_tubes_in_rack_files
+      end
 
       context 'when there are enough tubes' do
-        it 'returns true' do
-          expect(subject.sufficient_tubes_in_racks?).to be true
+        it 'is valid' do
+          expect(subject.errors.full_messages).to be_empty
         end
       end
 
       context 'when there are not enough sequencing tubes' do
         let(:num_sequencing_tubes) { 47 }
 
-        it 'returns false' do
-          expect(subject.sufficient_tubes_in_racks?).to be false
+        it 'is not valid' do
+          expect(subject.errors.full_messages).to include('Sequencing csv file contains insufficient tubes')
         end
       end
 
       context 'when there are not enough contingency tubes' do
         let(:num_contingency_tubes) { 47 }
 
-        it 'returns false' do
-          expect(subject.sufficient_tubes_in_racks?).to be false
+        it 'is not valid' do
+          expect(subject.errors.full_messages).to include('Contingency csv file contains insufficient tubes')
         end
       end
     end
   end
 
+  context '#check_tube_rack_barcodes_differ_between_files' do
+    before do
+      stub_v2_plate(
+        parent_plate,
+        stub_search: false,
+        custom_includes:
+          'wells.aliquots,wells.aliquots.sample,wells.downstream_tubes,' \
+            'wells.downstream_tubes.custom_metadatum_collection'
+      )
+      allow(Sequencescape::Api::V2::Tube).to receive(:find_by).with(barcode: 'FX00000001').and_return(nil)
+      allow(Sequencescape::Api::V2::Tube).to receive(:find_by).with(barcode: 'FX00000002').and_return(nil)
+      allow(Sequencescape::Api::V2::Tube).to receive(:find_by).with(barcode: 'FX00000011').and_return(nil)
+      allow(Sequencescape::Api::V2::Tube).to receive(:find_by).with(barcode: 'FX00000012').and_return(nil)
+      allow(Sequencescape::Api::V2::Tube).to receive(:find_by).with(barcode: 'FX00000013').and_return(nil)
+      allow(Sequencescape::Api::V2::Tube).to receive(:find_by).with(barcode: 'FX00000014').and_return(nil)
+      allow(Sequencescape::Api::V2::Tube).to receive(:find_by).with(barcode: 'FX00000015').and_return(nil)
+    end
+
+    context 'when files are not present' do
+      before { subject.valid? }
+
+      it 'does not call the validation' do
+        expect(subject).not_to receive(:check_tube_rack_barcodes_differ_between_files)
+      end
+    end
+
+    context 'when a file is not correctly parsed' do
+      let(:form_attributes) do
+        {
+          user_uuid: user_uuid,
+          purpose_uuid: child_contingency_tube_purpose_uuid,
+          parent_uuid: parent_uuid,
+          sequencing_file: sequencing_file,
+          contingency_file: contingency_file
+        }
+      end
+
+      before do
+        allow(subject.sequencing_csv_file).to receive(:correctly_parsed?).and_return(false)
+        subject.valid?
+      end
+
+      it 'does not call the validation' do
+        expect(subject).not_to receive(:check_tube_rack_barcodes_differ_between_files)
+      end
+    end
+
+    context 'when the tube rack barcodes are the same' do
+      let(:form_attributes) do
+        {
+          user_uuid: user_uuid,
+          purpose_uuid: child_contingency_tube_purpose_uuid,
+          parent_uuid: parent_uuid,
+          sequencing_file: contingency_file,
+          contingency_file: contingency_file
+        }
+      end
+
+      it 'is not valid' do
+        expect(subject.valid?).to be false
+        expect(subject.errors[:contingency_csv_file]).to include(
+          'The tube rack barcodes within the contingency and sequencing files must be different'
+        )
+      end
+    end
+  end
+
+  context '#check_tube_barcodes_differ_between_files' do
+    before do
+      stub_v2_plate(
+        parent_plate,
+        stub_search: false,
+        custom_includes:
+          'wells.aliquots,wells.aliquots.sample,wells.downstream_tubes,' \
+            'wells.downstream_tubes.custom_metadatum_collection'
+      )
+      allow(Sequencescape::Api::V2::Tube).to receive(:find_by).with(barcode: 'FX00000001').and_return(nil)
+      allow(Sequencescape::Api::V2::Tube).to receive(:find_by).with(barcode: 'FX00000002').and_return(nil)
+      allow(Sequencescape::Api::V2::Tube).to receive(:find_by).with(barcode: 'FX00000011').and_return(nil)
+      allow(Sequencescape::Api::V2::Tube).to receive(:find_by).with(barcode: 'FX00000012').and_return(nil)
+      allow(Sequencescape::Api::V2::Tube).to receive(:find_by).with(barcode: 'FX00000013').and_return(nil)
+      allow(Sequencescape::Api::V2::Tube).to receive(:find_by).with(barcode: 'FX00000014').and_return(nil)
+      allow(Sequencescape::Api::V2::Tube).to receive(:find_by).with(barcode: 'FX00000015').and_return(nil)
+    end
+
+    context 'when files are not present' do
+      before { subject.valid? }
+
+      it 'does not call the validation' do
+        expect(subject).not_to receive(:check_tube_barcodes_differ_between_files)
+      end
+    end
+
+    context 'when a file is not correctly parsed' do
+      let(:form_attributes) do
+        {
+          user_uuid: user_uuid,
+          purpose_uuid: child_contingency_tube_purpose_uuid,
+          parent_uuid: parent_uuid,
+          sequencing_file: sequencing_file,
+          contingency_file: contingency_file
+        }
+      end
+
+      before do
+        allow(subject.sequencing_csv_file).to receive(:correctly_parsed?).and_return(false)
+        subject.valid?
+      end
+
+      it 'does not call the validation' do
+        expect(subject).not_to receive(:check_tube_barcodes_differ_between_files)
+      end
+    end
+
+    context 'when there are duplicate tube barcodes between files' do
+      let(:form_attributes) do
+        {
+          user_uuid: user_uuid,
+          purpose_uuid: child_contingency_tube_purpose_uuid,
+          parent_uuid: parent_uuid,
+          sequencing_file: sequencing_file,
+          contingency_file: contingency_file
+        }
+      end
+      let(:seq_tube_details) do
+        {
+          'A1' => {
+            'tube_rack_barcode' => 'TR00000001',
+            'tube_barcode' => 'FX00000001'
+          },
+          'B1' => {
+            'tube_rack_barcode' => 'TR00000001',
+            'tube_barcode' => 'FX00000002'
+          },
+          'C1' => {
+            'tube_rack_barcode' => 'TR00000001',
+            'tube_barcode' => 'FX00000011'
+          },
+          'D1' => {
+            'tube_rack_barcode' => 'TR00000001',
+            'tube_barcode' => 'FX00000012'
+          }
+        }
+      end
+
+      before { allow(subject.sequencing_csv_file).to receive(:position_details).and_return(seq_tube_details) }
+
+      it 'is not valid' do
+        expect(subject.valid?).to be false
+        expect(subject.errors[:contingency_csv_file]).to include(
+          'Tube barcodes are duplicated across contingency and sequencing files (FX00000011, FX00000012)'
+        )
+      end
+    end
+  end
+
   context '#check_tube_rack_scan_file' do
     let(:tube_rack_file) { double('tube_rack_file') } # don't need an actual file for this test
     let(:tube_posn) { 'A1' }
@@ -444,14 +611,6 @@
     end
 
     context 'with both sequencing and contingency files' do
-      let(:sequencing_file) do
-        fixture_file_upload('spec/fixtures/files/scrna_core_sequencing_tube_rack_scan.csv', 'sequencescape/qc_file')
-      end
-
-      let(:contingency_file) do
-        fixture_file_upload('spec/fixtures/files/scrna_core_contingency_tube_rack_scan.csv', 'sequencescape/qc_file')
-      end
-
       let(:form_attributes) do
         {
           user_uuid: user_uuid,