Repository: fulcrumgenomics/prymer
Issue category: Performance Issue

Speed up from O(n^2) iteration over all left/right primer hits in `OffTargetDetector._to_amplicons`
`OffTargetDetector._to_amplicons` uses `itertools.product` over the left and right primer hits and evaluates each of them in a loop to identify valid left/right hit combinations for the pair.

Suggestion:

1. Get hits by primer sequence from `mappings_of`
2. Split into left and right hits
3. Group hits by refname
4. For each refname, split into left +, right -, left -, right+ hits
5. Build amplicons from left + and right - (amplicon strand +), left - and right + (amplicon strand -)

Building amplicons - dealing with only positive & negative in correct orientation and known to be on the same reference, with known amplicon strand
1. `itertools.product` over positive & negative primer hits
2. Check product size against min/max amplicon size range
3. If in range, return the product hit as positive start to negative end with the known reference name and amplicon strand


Functions to edit:
prymer/offtarget/offtarget_detector.py:OffTargetDetector._build_off_target_result
prymer/offtarget/offtarget_detector.py:OffTargetDetector._to_amplicons

Patch:
diff --git a/prymer/offtarget/offtarget_detector.py b/prymer/offtarget/offtarget_detector.py
index d1c7868..71d3201 100644
--- a/prymer/offtarget/offtarget_detector.py
+++ b/prymer/offtarget/offtarget_detector.py
@@ -75,6 +75,7 @@
 """  # noqa: E501
 
 import itertools
+from collections import defaultdict
 from contextlib import AbstractContextManager
 from dataclasses import dataclass
 from dataclasses import field
@@ -83,6 +84,7 @@
 from types import TracebackType
 from typing import Optional
 from typing import Self
+from typing import TypeAlias
 from typing import TypeVar
 
 from ordered_set import OrderedSet
@@ -90,6 +92,7 @@
 from prymer.api.oligo import Oligo
 from prymer.api.primer_pair import PrimerPair
 from prymer.api.span import Span
+from prymer.api.span import Strand
 from prymer.offtarget.bwa import BWA_EXECUTABLE_NAME
 from prymer.offtarget.bwa import BwaAlnInteractive
 from prymer.offtarget.bwa import BwaHit
@@ -98,6 +101,9 @@
 
 PrimerType = TypeVar("PrimerType", bound=Oligo)
 
+ReferenceName: TypeAlias = str
+"""Alias for a reference sequence name."""
+
 
 @dataclass(init=True, frozen=True)
 class OffTargetResult:
@@ -334,27 +340,78 @@ def _build_off_target_result(
         result: OffTargetResult
 
         # Get the mappings for the left primer and right primer respectively
-        p1: BwaResult = hits_by_primer[primer_pair.left_primer.bases]
-        p2: BwaResult = hits_by_primer[primer_pair.right_primer.bases]
-
-        # Get all possible amplicons from the left_primer_mappings and right_primer_mappings
-        # primer hits, filtering if there are too many for either
-        if p1.hit_count > self._max_primer_hits or p2.hit_count > self._max_primer_hits:
+        left_bwa_result: BwaResult = hits_by_primer[primer_pair.left_primer.bases]
+        right_bwa_result: BwaResult = hits_by_primer[primer_pair.right_primer.bases]
+
+        # If there are too many hits, this primer pair will not pass. Exit early.
+        if (
+            left_bwa_result.hit_count > self._max_primer_hits
+            or right_bwa_result.hit_count > self._max_primer_hits
+        ):
             result = OffTargetResult(primer_pair=primer_pair, passes=False)
-        else:
-            amplicons = self._to_amplicons(p1.hits, p2.hits, self._max_amplicon_size)
-            result = OffTargetResult(
-                primer_pair=primer_pair,
-                passes=self._min_primer_pair_hits <= len(amplicons) <= self._max_primer_pair_hits,
-                spans=amplicons if self._keep_spans else [],
-                left_primer_spans=(
-                    [self._hit_to_span(h) for h in p1.hits] if self._keep_primer_spans else []
-                ),
-                right_primer_spans=(
-                    [self._hit_to_span(h) for h in p2.hits] if self._keep_primer_spans else []
-                ),
+            if self._cache_results:
+                self._primer_pair_cache[primer_pair] = replace(result, cached=True)
+            return result
+
+        # Map the hits by reference name
+        left_positive_hits: defaultdict[ReferenceName, list[BwaHit]] = defaultdict(list)
+        left_negative_hits: defaultdict[ReferenceName, list[BwaHit]] = defaultdict(list)
+        right_positive_hits: defaultdict[ReferenceName, list[BwaHit]] = defaultdict(list)
+        right_negative_hits: defaultdict[ReferenceName, list[BwaHit]] = defaultdict(list)
+
+        # Split the hits for left and right by reference name and strand
+        for hit in left_bwa_result.hits:
+            if hit.negative:
+                left_negative_hits[hit.refname].append(hit)
+            else:
+                left_positive_hits[hit.refname].append(hit)
+
+        for hit in right_bwa_result.hits:
+            if hit.negative:
+                right_negative_hits[hit.refname].append(hit)
+            else:
+                right_positive_hits[hit.refname].append(hit)
+
+        refnames: set[ReferenceName] = {
+            h.refname for h in itertools.chain(left_bwa_result.hits, right_bwa_result.hits)
+        }
+
+        # Build amplicons from hits on the same reference with valid relative orientation
+        amplicons: list[Span] = []
+        for refname in refnames:
+            amplicons.extend(
+                self._to_amplicons(
+                    positive_hits=left_positive_hits[refname],
+                    negative_hits=right_negative_hits[refname],
+                    max_len=self._max_amplicon_size,
+                    strand=Strand.POSITIVE,
+                )
+            )
+            amplicons.extend(
+                self._to_amplicons(
+                    positive_hits=right_positive_hits[refname],
+                    negative_hits=left_negative_hits[refname],
+                    max_len=self._max_amplicon_size,
+                    strand=Strand.NEGATIVE,
+                )
             )
 
+        result = OffTargetResult(
+            primer_pair=primer_pair,
+            passes=self._min_primer_pair_hits <= len(amplicons) <= self._max_primer_pair_hits,
+            spans=amplicons if self._keep_spans else [],
+            left_primer_spans=(
+                [self._hit_to_span(h) for h in left_bwa_result.hits]
+                if self._keep_primer_spans
+                else []
+            ),
+            right_primer_spans=(
+                [self._hit_to_span(h) for h in right_bwa_result.hits]
+                if self._keep_primer_spans
+                else []
+            ),
+        )
+
         if self._cache_results:
             self._primer_pair_cache[primer_pair] = replace(result, cached=True)
 
@@ -410,19 +467,89 @@ def mappings_of(self, primers: list[PrimerType]) -> dict[str, BwaResult]:
         return hits_by_primer
 
     @staticmethod
-    def _to_amplicons(lefts: list[BwaHit], rights: list[BwaHit], max_len: int) -> list[Span]:
-        """Takes a set of hits for one or more left primers and right primers and constructs
-        amplicon mappings anywhere a left primer hit and a right primer hit align in F/R
-        orientation up to `maxLen` apart on the same reference.  Primers may not overlap.
+    def _to_amplicons(
+        positive_hits: list[BwaHit], negative_hits: list[BwaHit], max_len: int, strand: Strand
+    ) -> list[Span]:
+        """Takes lists of positive strand hits and negative strand hits and constructs amplicon
+        mappings anywhere a positive strand hit and a negative strand hit occur where the end of
+        the negative strand hit is no more than `max_len` from the start of the positive strand
+        hit.
+
+        Primers may not overlap.
+
+        Args:
+            positive_hits: List of hits on the positive strand for one of the primers in the pair.
+            negative_hits: List of hits on the negative strand for the other primer in the pair.
+            max_len: Maximum length of amplicons to consider.
+            strand: The strand of the amplicon to generate. Set to Strand.POSITIVE if
+                `positive_hits` are for the left primer and `negative_hits` are for the right
+                primer. Set to Strand.NEGATIVE if `positive_hits` are for the right primer and
+                `negative_hits` are for the left primer.
+
+        Raises:
+            ValueError: If any of the positive hits are not on the positive strand, or any of the
+                negative hits are not on the negative strand. If hits are present on more than one
+                reference.
         """
+        if any(h.negative for h in positive_hits):
+            raise ValueError("Positive hits must be on the positive strand.")
+        if any(not h.negative for h in negative_hits):
+            raise ValueError("Negative hits must be on the negative strand.")
+
+        refnames: set[ReferenceName] = {
+            h.refname for h in itertools.chain(positive_hits, negative_hits)
+        }
+        if len(refnames) > 1:
+            raise ValueError(f"Hits are present on more than one reference: {refnames}")
+
+        # Exit early if one of the hit lists is empty - this will save unnecessary sorting of the
+        # other list
+        if len(positive_hits) == 0 or len(negative_hits) == 0:
+            return []
+
+        # Sort the positive strand hits by start position and the negative strand hits by *end*
+        # position. The `max_len` cutoff is based on negative_hit.end - positive_hit.start + 1.
+        positive_hits_sorted = sorted(positive_hits, key=lambda h: h.start)
+        negative_hits_sorted = sorted(negative_hits, key=lambda h: h.end)
+
         amplicons: list[Span] = []
-        for h1, h2 in itertools.product(lefts, rights):
-            if h1.negative == h2.negative or h1.refname != h2.refname:  # not F/R orientation
-                continue
 
-            plus, minus = (h2, h1) if h1.negative else (h1, h2)
-            if minus.start > plus.end and (minus.end - plus.start + 1) <= max_len:
-                amplicons.append(Span(refname=plus.refname, start=plus.start, end=minus.end))
+        # Track the position of the previously examined negative hit.
+        prev_negative_hit_index = 0
+        for positive_hit in positive_hits_sorted:
+            # Check only negative hits starting with the previously examined one.
+            for negative_hit_index, negative_hit in enumerate(
+                negative_hits_sorted[prev_negative_hit_index:],
+                start=prev_negative_hit_index,
+            ):
+                # TODO: Consider allowing overlapping positive and negative hits.
+                if (
+                    negative_hit.start > positive_hit.end
+                    and negative_hit.end - positive_hit.start + 1 <= max_len
+                ):
+                    # If the negative hit starts to the right of the positive hit, and the amplicon
+                    # length is <= max_len, add it to the list of amplicon hits to be returned.
+                    amplicons.append(
+                        Span(
+                            refname=positive_hit.refname,
+                            start=positive_hit.start,
+                            end=negative_hit.end,
+                            strand=strand,
+                        )
+                    )
+
+                if negative_hit.end - positive_hit.start + 1 > max_len:
+                    # Stop searching for negative hits to pair with this positive hit.
+                    # All subsequence negative hits will have amplicon length > max_len
+                    break
+
+                if negative_hit.end < positive_hit.start:
+                    # This positive hit is genomically right of the current negative hit.
+                    # All subsequent positive hits will also be genomically right of this negative
+                    # hit, so we should start at the one after this. If this index is past the end
+                    # of the list, the slice `negative_hits_sorted[prev_negative_hit_index:]` will
+                    # be empty.
+                    prev_negative_hit_index = negative_hit_index + 1
 
         return amplicons
 

Test patch: diff --git a/tests/offtarget/test_offtarget.py b/tests/offtarget/test_offtarget.py
index f8f5f8c..a2f3a46 100644
--- a/tests/offtarget/test_offtarget.py
+++ b/tests/offtarget/test_offtarget.py
@@ -11,6 +11,7 @@
 from prymer.offtarget.bwa import BWA_EXECUTABLE_NAME
 from prymer.offtarget.bwa import BwaHit
 from prymer.offtarget.bwa import BwaResult
+from prymer.offtarget.bwa import Query
 from prymer.offtarget.offtarget_detector import OffTargetDetector
 from prymer.offtarget.offtarget_detector import OffTargetResult
 
@@ -171,68 +172,159 @@ def test_mappings_of(ref_fasta: Path, cache_results: bool) -> None:
             assert results_dict[p2.bases].hits[0] == expected_hit2
 
 
+# Test building an OffTargetResult for a primer pair with left/right hits on different references
+# and in different orientations
+def test_build_off_target_result(ref_fasta: Path) -> None:
+    hits_by_primer: dict[str, BwaResult] = {
+        "A" * 100: BwaResult(
+            query=Query(
+                id="left",
+                bases="A" * 100,
+            ),
+            hit_count=3,
+            hits=[
+                BwaHit.build("chr1", 100, False, "100M", 0),
+                BwaHit.build("chr1", 400, True, "100M", 0),
+                BwaHit.build("chr2", 100, False, "100M", 0),
+                BwaHit.build("chr3", 700, True, "100M", 0),
+            ],
+        ),
+        "C" * 100: BwaResult(
+            query=Query(
+                id="right",
+                bases="C" * 100,
+            ),
+            hit_count=2,
+            hits=[
+                BwaHit.build("chr1", 800, False, "100M", 0),
+                BwaHit.build("chr1", 200, True, "100M", 0),
+                BwaHit.build("chr3", 600, False, "100M", 0),
+            ],
+        ),
+    }
+
+    primer_pair = PrimerPair(
+        left_primer=Oligo(
+            tm=50,
+            penalty=0,
+            span=Span(refname="chr10", start=100, end=199, strand=Strand.POSITIVE),
+            bases="A" * 100,
+        ),
+        right_primer=Oligo(
+            tm=50,
+            penalty=0,
+            span=Span(refname="chr10", start=300, end=399, strand=Strand.NEGATIVE),
+            bases="C" * 100,
+        ),
+        amplicon_tm=100,
+        penalty=0,
+    )
+
+    with _build_detector(
+        ref_fasta=ref_fasta, max_primer_hits=10, max_primer_pair_hits=10
+    ) as detector:
+        off_target_result: OffTargetResult = detector._build_off_target_result(
+            primer_pair=primer_pair,
+            hits_by_primer=hits_by_primer,
+        )
+
+    assert set(off_target_result.spans) == {
+        Span(refname="chr1", start=100, end=299, strand=Strand.POSITIVE),
+        Span(refname="chr3", start=600, end=799, strand=Strand.NEGATIVE),
+    }
+
+
 # Test that using the cache (or not) does not affect the results
 @pytest.mark.parametrize("cache_results", [True, False])
 @pytest.mark.parametrize(
-    "test_id, left, right, expected",
+    "test_id, positive, negative, strand, expected",
     [
-        (
-            "No mappings - different refnames",
-            BwaHit.build("chr1", 100, False, "100M", 0),
-            BwaHit.build("chr2", 100, True, "100M", 0),
-            [],
-        ),
-        (
-            "No mappings - FF pair",
-            BwaHit.build("chr1", 100, True, "100M", 0),
-            BwaHit.build("chr1", 100, True, "100M", 0),
-            [],
-        ),
-        (
-            "No mappings - RR pair",
-            BwaHit.build("chr1", 100, False, "100M", 0),
-            BwaHit.build("chr1", 100, False, "100M", 0),
-            [],
-        ),
         (
             "No mappings - overlapping primers (1bp overlap)",
             BwaHit.build("chr1", 100, False, "100M", 0),
             BwaHit.build("chr1", 199, True, "100M", 0),
+            Strand.POSITIVE,
             [],
         ),
         (
             "No mappings - amplicon size too big (1bp too big)",
             BwaHit.build("chr1", 100, False, "100M", 0),
             BwaHit.build("chr1", 151, True, "100M", 0),
+            Strand.POSITIVE,
             [],
         ),
         (
             "Mappings - FR pair (R1 F)",
             BwaHit.build("chr1", 100, False, "100M", 0),
             BwaHit.build("chr1", 200, True, "100M", 0),
-            [Span(refname="chr1", start=100, end=299)],
+            Strand.POSITIVE,
+            [Span(refname="chr1", start=100, end=299, strand=Strand.POSITIVE)],
         ),
         (
             "Mappings - FR pair (R1 R)",
-            BwaHit.build("chr1", 200, True, "100M", 0),
             BwaHit.build("chr1", 100, False, "100M", 0),
-            [Span(refname="chr1", start=100, end=299)],
+            BwaHit.build("chr1", 200, True, "100M", 0),
+            Strand.NEGATIVE,
+            [Span(refname="chr1", start=100, end=299, strand=Strand.NEGATIVE)],
         ),
     ],
 )
 def test_to_amplicons(
     ref_fasta: Path,
     test_id: str,
-    left: BwaHit,
-    right: BwaHit,
+    positive: BwaHit,
+    negative: BwaHit,
+    strand: Strand,
     expected: list[Span],
     cache_results: bool,
 ) -> None:
     with _build_detector(ref_fasta=ref_fasta, cache_results=cache_results) as detector:
-        actual = detector._to_amplicons(lefts=[left], rights=[right], max_len=250)
+        actual = detector._to_amplicons(
+            positive_hits=[positive], negative_hits=[negative], max_len=250, strand=strand
+        )
         assert actual == expected, test_id
 
 
+@pytest.mark.parametrize("cache_results", [True, False])
+@pytest.mark.parametrize(
+    "positive, negative, expected_error",
+    [
+        (
+            # No mappings - different refnames
+            BwaHit.build("chr1", 100, False, "100M", 0),
+            BwaHit.build("chr2", 100, True, "100M", 0),
+            "Hits are present on more than one reference",
+        ),
+        (
+            # No mappings - FF pair
+            BwaHit.build("chr1", 100, True, "100M", 0),
+            BwaHit.build("chr1", 100, True, "100M", 0),
+            "Positive hits must be on the positive strand",
+        ),
+        (
+            # No mappings - RR pair
+            BwaHit.build("chr1", 100, False, "100M", 0),
+            BwaHit.build("chr1", 100, False, "100M", 0),
+            "Negative hits must be on the negative strand",
+        ),
+    ],
+)
+def test_to_amplicons_value_error(
+    ref_fasta: Path,
+    positive: BwaHit,
+    negative: BwaHit,
+    expected_error: str,
+    cache_results: bool,
+) -> None:
+    with (
+        _build_detector(ref_fasta=ref_fasta, cache_results=cache_results) as detector,
+        pytest.raises(ValueError, match=expected_error),
+    ):
+        detector._to_amplicons(
+            positive_hits=[positive], negative_hits=[negative], max_len=250, strand=Strand.POSITIVE
+        )
+
+
 def test_generic_filter(ref_fasta: Path) -> None:
     """
     This test isn't intended to validate any runtime assertions, but is a minimal example for the