diff --git a/htsinfer/get_library_type.py b/htsinfer/get_library_type.py
index 37dad4f..e5fd2da 100644
--- a/htsinfer/get_library_type.py
+++ b/htsinfer/get_library_type.py
@@ -127,6 +127,21 @@ def _evaluate_mate_relationship(
                 self.mapping.library_type.relationship = (
                     StatesTypeRelationship.split_mates
                 )
+            # Infer mate relationship, even when assumed to be single
+            elif (
+                self.results.file_1 == StatesType.single and
+                self.results.file_2 == StatesType.single
+            ) and (
+                self.library_source.file_1.short_name is not None or
+                self.library_source.file_2.short_name is not None
+            ):
+                LOGGER.debug("Determining mate relationship by alignment...")
+                self.mapping.library_type.relationship \
+                    = StatesTypeRelationship.not_available
+                self.mapping.library_source = self.library_source
+                self.mapping.paths = self.path_1, self.path_2
+                self.mapping.evaluate()
+                self._align_mates()
         elif (
             self.library_source.file_1.short_name is not None or
             self.library_source.file_2.short_name is not None