fix: infer library type relationship

htsinfer/get_library_type.py

@@ -113,7 +113,7 @@ def _evaluate_mate_relationship(
             ids_2: As `ids_1` but for the putative second mate file.
         """
         self.results.relationship = StatesTypeRelationship.not_mates
-        if ids_1 == ids_2 and len(ids_1) > 0 and len(ids_2) > 0:
+        if ids_1 and ids_2 and ids_1 == ids_2:
             if (
                 self.results.file_1 == StatesType.first_mate and
                 self.results.file_2 == StatesType.second_mate
@@ -140,8 +140,7 @@ def _evaluate_mate_relationship(
         else:
             self.results.relationship = StatesTypeRelationship.not_available
             LOGGER.debug(
-                "Library source is not determined, "
-                "mate relationship cannot be inferred by alignment."
+                "Mate relationship cannot be determined."
             )
 
     def _align_mates(self):
@@ -159,57 +158,66 @@ def _align_mates(self):
         previous_seq_id2 = None
 
         reads1 = []  # List to store alignments for one read from file1
-        mate1 = []  # List to store alignments for each read
+        mate1 = []  # List to store alignments for each read from file1
         reads2 = []  # List to store alignments for one read from file2
+        mate2 = []  # List to store alignments for each read from file2
 
         concordant = 0
 
         for read1 in samfile1:
-            if not read1.flag & (1 << 2) and isinstance(read1.query_name, str):
-                seq_id1 = read1.query_name
-                if (
-                    seq_id1 != previous_seq_id1 and
-                    previous_seq_id1 is not None
-                ):
-                    mate1.append(reads1.copy())
-                    reads1.clear()
-                if read1.reference_end:
-                    reads1.append(read1)
+            seq_id1 = read1.query_name
+            if (
+                seq_id1 != previous_seq_id1 and
+                previous_seq_id1 is not None
+            ):
+                mate1.append(reads1.copy())
+                reads1.clear()
+            if read1.reference_end:
+                reads1.append(read1)
             previous_seq_id1 = seq_id1
         mate1.append(reads1.copy())
 
         read_counter = 0
         for read2 in samfile2:
-            if not read2.flag & (1 << 2) and isinstance(read2.query_name, str):
-                seq_id2 = read2.query_name
-                if seq_id2 != previous_seq_id2 \
-                        and previous_seq_id2 is not None:
-                    if read_counter < len(mate1) and self._compare_alignments(
-                        mate1[read_counter], reads2
-                    ):
-                        concordant += 1
-                    reads2.clear()
-                    read_counter += 1
-                if read2.reference_end:
-                    reads2.append(read2)
+            seq_id2 = read2.query_name
+            if (
+                seq_id2 != previous_seq_id2 and
+                previous_seq_id2 is not None
+            ):
+                mate2.append(reads2.copy())
+                if self._compare_alignments(
+                    mate1[read_counter], reads2
+                ):
+                    concordant += 1
+                reads2.clear()
+                read_counter += 1
+            if read2.reference_end:
+                reads2.append(read2)
             previous_seq_id2 = seq_id2
-
-        if read_counter < len(mate1) and self._compare_alignments(
+        mate2.append(reads2.copy())
+        if self._compare_alignments(
             mate1[read_counter], reads2
         ):
             concordant += 1
-        LOGGER.debug(f"Number of aligned reads file 1: {len(mate1)}")
-        LOGGER.debug(f"Number of aligned reads file 2: {read_counter}")
+
+        aligned_mate1 = len(list(filter(None, mate1)))
+        aligned_mate2 = len(list(filter(None, mate2)))
+
+        LOGGER.debug(f"Number of aligned reads file 1: {aligned_mate1}")
+        LOGGER.debug(f"Number of aligned reads file 2: {aligned_mate2}")
         LOGGER.debug(f"Number of concordant reads: {concordant}")
-        self._update_relationship(concordant, read_counter)
+
+        self._update_relationship(
+            concordant, min(aligned_mate1, aligned_mate2)
+        )
 
         samfile1.close()
         samfile2.close()
 
-    def _update_relationship(self, concordant, read_counter):
+    def _update_relationship(self, concordant, aligned_reads):
         """Helper function to update relationship based on alignment."""
         try:
-            ratio = concordant / read_counter
+            ratio = concordant / aligned_reads
         except ZeroDivisionError:
             self.results.relationship = (
                 StatesTypeRelationship.not_available
@@ -219,8 +227,9 @@ def _update_relationship(self, concordant, read_counter):
                 self.results.relationship = (
                     StatesTypeRelationship.split_mates
                 )
-                self.mapping.library_type.relationship \
-                    = StatesTypeRelationship.split_mates
+                self.mapping.library_type.relationship = (
+                    StatesTypeRelationship.split_mates
+                )
                 self.mapping.mapped = False
                 self.mapping.star_dirs = []
             else:

htsinfer/get_read_orientation.py

@@ -82,8 +82,7 @@ def evaluate(self) -> ResultsOrientation:
             self.mapping.evaluate()
         else:
             LOGGER.debug(
-                "Library source is not determined, "
-                "read orientation cannot be inferred by alignment."
+                "Read orientation cannot be determined."
             )
 
         return self.process_alignments(star_dirs=self.mapping.star_dirs)

htsinfer/mapping.py

@@ -299,6 +299,7 @@ def build_star_command(
                 "--runThreadN", f"{str(self.threads_star)}",
                 "--genomeDir", f"{str(index_dir)}",
                 "--outFilterMultimapNmax", "50",
+                "--outSAMorder", "PairedKeepInputOrder",
                 "--outSAMunmapped", "Within", "KeepPairs",
             ]
             cmd: List[str] = cmd_base[:]

tests/test_mapping.py

@@ -187,7 +187,8 @@ def test_prepare_star_alignment_commands(self, tmpdir):
         file1_alignment_path = tmpdir / 'alignments/file_1'
         cmd = "STAR --alignIntronMax 1 --alignEndsType Local --runThreadN 1" \
             + " --genomeDir " + str(index_dir) + " --outFilterMultimapNmax " \
-            + "50 --outSAMunmapped Within KeepPairs --readFilesIn " \
+            + "50 --outSAMorder PairedKeepInputOrder " \
+            + "--outSAMunmapped Within KeepPairs --readFilesIn " \
             + str(FILE_2000_RECORDS) + " --outFileNamePrefix " \
             + str(file1_alignment_path) + "/"
         results = test_instance.prepare_star_alignment_commands(