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.gitattributes

@@ -58,3 +58,17 @@ saved_model/**/* filter=lfs diff=lfs merge=lfs -text
 # Video files - compressed
 *.mp4 filter=lfs diff=lfs merge=lfs -text
 *.webm filter=lfs diff=lfs merge=lfs -text
+test/tumor_chr17.bai filter=lfs diff=lfs merge=lfs -text
+full/hcc1143_T_clean.bai filter=lfs diff=lfs merge=lfs -text
+test/normal_chr17.bai filter=lfs diff=lfs merge=lfs -text
+full/hcc1143_N_clean.bai filter=lfs diff=lfs merge=lfs -text
+hla/hla_candidates_R1.fastq filter=lfs diff=lfs merge=lfs -text
+hla/hla_unmapped.bam filter=lfs diff=lfs merge=lfs -text
+hla/hla_candidates_R2.fastq filter=lfs diff=lfs merge=lfs -text
+hla/hla_chr6_primary.bam filter=lfs diff=lfs merge=lfs -text
+test/normal_chr17.bam filter=lfs diff=lfs merge=lfs -text
+test/tumor_chr17.bam filter=lfs diff=lfs merge=lfs -text
+hla/hla_alt_contigs.bam filter=lfs diff=lfs merge=lfs -text
+hla/hla_candidates.bam filter=lfs diff=lfs merge=lfs -text
+full/hcc1143_T_clean.bam filter=lfs diff=lfs merge=lfs -text
+full/hcc1143_N_clean.bam filter=lfs diff=lfs merge=lfs -text

full/hcc1143_N_clean.bai

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full/hcc1143_N_clean.bam

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full/hcc1143_T_clean.bai

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full/hcc1143_T_clean.bam

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hla/hla_alt_contigs.bam

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hla/hla_candidates.bam

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hla/hla_candidates_R1.fastq

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hla/hla_candidates_R2.fastq

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hla/hla_chr6_primary.bam

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hla/hla_unmapped.bam

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hla/patient_hla.txt

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+# HCC1143 Clinical HLA Typing
+# Source: Boegel et al. (2014) PubMed 25960936
+#         Confirmed: Cellosaurus CVCL_1245
+# Method: seq2HLA from RNA-seq
+# Note:   HLA-A is homozygous A*31:01
+#         Computational tools (OptiType) may misresolved second A allele
+#         as A*23:01 due to homozygosity ambiguity — use this file as ground truth
+HLA-A*31:01
+HLA-A*31:01
+HLA-B*37:01
+HLA-B*35:08
+HLA-C*06:02
+HLA-C*04:01

tcga/TCGA-SKCM.maf.gz

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test/create_test_data.sh

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+# ── Subset full HCC1143 BAMs to chr17 for pipeline development ───────────────
+# Input:  full WES BAMs (wherever you downloaded them)
+# Output: hcc1143_T_clean.bam / hcc1143_N_clean.bam (chr17 only)
+#
+# Why chr17: TP53 is on chr17 — the known HCC1143 driver mutation
+# we use as our validated anchor epitope. Subsetting to chr17 keeps
+# the test data small (~50MB vs ~23GB) while retaining the mutation
+# we actually care about.
+# ─────────────────────────────────────────────────────────────────────────────
+
+TUMOR_FULL=~/melanoma-pipeline/data/full/hcc1143_T_clean.bam
+NORMAL_FULL=~/melanoma-pipeline/data/full/hcc1143_N_clean.bam
+OUT=~/melanoma-pipeline/data/test
+
+mkdir -p $OUT
+
+# Subset tumor to chr17
+# -b: output BAM format, -h: include header, -@ 8: 8 threads
+samtools view \
+    -b \
+    -h \
+    -@ 8 \
+    $TUMOR_FULL \
+    chr17 \
+    -o $OUT/tumor_chr17.bam
+
+# Index tumor subset
+samtools index \
+    -@ 8 \
+    $OUT/tumor_chr17.bam \
+    $OUT/tumor_chr17.bai
+
+# Subset normal to chr17
+samtools view \
+    -b \
+    -h \
+    -@ 8 \
+    $NORMAL_FULL \
+    chr17 \
+    -o $OUT/normal_chr17.bam
+
+# Index normal subset
+samtools index \
+    -@ 8 \
+    $OUT/normal_chr17.bam \
+    $OUT/normal_chr17.bai
+
+# Verify read counts
+echo "Tumor chr17 reads:"
+samtools flagstat $OUT/tumor_chr17.bam
+
+echo "Normal chr17 reads:"
+samtools flagstat $OUT/normal_chr17.bam
+
+
+# ── Extract HLA reads from normal BAM for OptiType ───────────────────────────
+# Always use normal BAM for HLA typing — tumor DNA can have LOH
+# (Loss of Heterozygosity) at HLA loci which gives wrong alleles.
+#
+# Three-bucket strategy to avoid missing HLA reads due to ALT contigs:
+#   Bucket 1 — primary MHC region on chr6
+#   Bucket 2 — ALT contigs (reads that overflowed from primary chr6 assembly)
+#   Bucket 3 — unmapped reads (too divergent to map anywhere in hg38)
+# All three are merged and remapped against OptiType's HLA-specific reference.
+# ─────────────────────────────────────────────────────────────────────────────
+
+OUT_HLA=~/melanoma-pipeline/data/hla
+mkdir -p $OUT_HLA
+
+# Bucket 1 — primary MHC region on chr6
+# 28510120-33480577 = full MHC locus including HLA-A, B, C and flanking genes
+samtools view \
+    -b \
+    -h \
+    -@ 8 \
+    $NORMAL_FULL \
+    chr6:28510120-33480577 \
+    -o $OUT_HLA/hla_chr6_primary.bam
+
+# Bucket 2 — ALT contigs
+# Reads from patients with HLA alleles too divergent for the primary reference
+# — most common in non-European populations. Dynamically finds all ALT/HLA
+# contigs in the BAM header so this works regardless of hg38 build variant.
+ALT_CONTIGS=$(samtools view -H $NORMAL_FULL \
+    | grep "^@SQ" \
+    | grep -iE "_alt|HLA" \
+    | awk '{print $2}' \
+    | sed 's/SN://')
+
+if [ -n "$ALT_CONTIGS" ]; then
+    ALT_TMP=$(mktemp -d)
+    while IFS= read -r contig; do
+        safe_name="${contig//\//_}"
+        samtools view -b -h -@ 8 $NORMAL_FULL "$contig" -o "$ALT_TMP/${safe_name}.bam"
+    done <<< "$ALT_CONTIGS"
+    samtools merge -f -@ 8 $OUT_HLA/hla_alt_contigs.bam "$ALT_TMP"/*.bam
+    rm -rf "$ALT_TMP"
+else
+    # No ALT contigs — create an empty BAM so downstream merge still works
+    samtools view -b -h $NORMAL_FULL -o $OUT_HLA/hla_alt_contigs.bam /dev/null 2>/dev/null || \
+        samtools view -H $NORMAL_FULL -b -o $OUT_HLA/hla_alt_contigs.bam
+fi
+
+# Bucket 3 — unmapped reads
+# Small fraction of BAM but catches rare/unusual alleles that couldn't
+# map anywhere in hg38 due to extreme divergence from the reference
+# -f 4: flag 4 = read unmapped
+samtools view \
+    -b \
+    -f 4 \
+    -@ 8 \
+    $NORMAL_FULL \
+    -o $OUT_HLA/hla_unmapped.bam
+
+# Merge all three buckets into one candidate pool
+samtools merge \
+    -f \
+    -@ 8 \
+    $OUT_HLA/hla_candidates.bam \
+    $OUT_HLA/hla_chr6_primary.bam \
+    $OUT_HLA/hla_alt_contigs.bam \
+    $OUT_HLA/hla_unmapped.bam
+
+# Convert merged BAM to paired FASTQ for remapping
+# Sort by name first (-n) so read pairs are adjacent
+samtools sort -n -@ 8 $OUT_HLA/hla_candidates.bam \
+    | samtools fastq \
+    -1 $OUT_HLA/hla_candidates_R1.fastq \
+    -2 $OUT_HLA/hla_candidates_R2.fastq \
+    -0 /dev/null \
+    -s /dev/null
+
+# Remap against OptiType's HLA-specific reference
+# This is the critical step — reads that were on ALT contigs or unmapped
+# now find their correct HLA allele sequence. The HLA reference contains
+# sequences for thousands of known alleles across all populations.
+# hla_reference_dna.fasta is bundled inside the fred2/optitype Docker image
+# at /usr/local/bin/data/hla_reference_dna.fasta
+# Extract it once and reuse:
+#   docker run --rm fred2/optitype cat /usr/local/bin/data/hla_reference_dna.fasta \
+#       > ~/melanoma-pipeline/reference/hla_reference_dna.fasta
+HLA_REF=~/melanoma-pipeline/reference/hla_reference_dna.fasta
+
+# Index HLA reference if not already done
+if [ ! -f "${HLA_REF}.bwt" ]; then
+    echo "Indexing HLA reference..."
+    bwa index $HLA_REF
+fi
+
+bwa mem \
+    -t 8 \
+    $HLA_REF \
+    $OUT_HLA/hla_candidates_R1.fastq \
+    $OUT_HLA/hla_candidates_R2.fastq \
+    | samtools sort -n -@ 8 \
+    | samtools fastq \
+    -F 4 \
+    -1 $OUT_HLA/hla_fished_R1.fastq \
+    -2 $OUT_HLA/hla_fished_R2.fastq \
+    -0 /dev/null \
+    -s /dev/null
+
+# hla_fished_R1/R2.fastq are now ready to pass to OptiType (Step 3 of the pipeline)
+echo "HLA read extraction complete."
+echo "Fished read pairs: $(( $(wc -l < $OUT_HLA/hla_fished_R1.fastq) / 4 ))"
+echo "Ready for OptiType: $OUT_HLA/hla_fished_R1.fastq + hla_fished_R2.fastq"

test/normal_chr17.bai

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test/normal_chr17.bam

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test/tumor_chr17.bai

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test/tumor_chr17.bam

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