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Validate that reference index files exist (`.0123`, `.amb`, `.ann`, `.bwt.2bit.64`, `.pac`, `.fai`, `.dict`).\n2. Construct a read group string from sample metadata if one is not provided (fields: ID, SM, PL, LB, PU).\n3. Run `bwa-mem2 mem` with the read group, piping output to `samtools sort` to produce a coordinate-sorted BAM.\n4. Index the sorted BAM with `samtools index`.\n5. Mark duplicates with `picard MarkDuplicates` (or `samtools markdup` if selected).\n6. Index the deduplicated BAM.\n7. Report alignment summary: total reads, mapped reads, properly paired reads, duplicate reads, mapping rate.\n\n## Output Contract\n\n- Coordinate-sorted deduplicated BAM file (`.bam`).\n- BAM index file (`.bai`).\n- Duplicate metrics file (picard MarkDuplicates output).\n- Alignment summary statistics (total, mapped, properly paired, duplicates, mapping rate).\n\n## Limits\n\n- BWA-MEM2, samtools, and picard must be installed and available on `$PATH`.\n- The BWA-MEM2 index must be pre-built (`bwa-mem2 index \u003Cref.fa>`).\n- Memory scales with reference genome size (~8 GB for human GRCh38).\n- Picard requires a Java runtime (Java 8+).\n- This skill handles single-sample alignment only; multi-sample merging is out of scope.\n- Common failure cases:\n  - Reference index files missing or incomplete (e.g., `.bwt.2bit.64` absent).\n  - Read group string malformed or missing required fields (ID, SM).\n  - Insufficient memory for BWA-MEM2 index loading (~8 GB for human genome).\n","# Skill: genome-read-alignment\n\n## Use When\n\n- User needs to align FASTQ reads to a reference genome.\n- User wants to produce a coordinate-sorted, indexed BAM file.\n- User needs to mark PCR or optical duplicates in aligned reads.\n- User wants to add read group information (@RG) to alignments.\n\n## Inputs\n\n- Required:\n  - Trimmed FASTQ file(s) (single-end or paired-end).\n  - Reference FASTA, pre-indexed for BWA-MEM2.\n- Optional:\n  - Read group string (`@RG\\tID:...\\tSM:...\\tPL:...\\tLB:...\\tPU:...`).\n  - Number of threads (default: 4).\n  - Output directory (default: current directory).\n  - Temporary directory for sorting\u002Fdedup intermediates.\n  - Optical duplicate pixel distance (default: 2500 for patterned flowcells).\n  - Mark-duplicate tool choice: `picard` (default) or `samtools markdup`.\n\n## Workflow\n\n1. Validate that reference index files exist (`.0123`, `.amb`, `.ann`, `.bwt.2bit.64`, `.pac`, `.fai`, `.dict`).\n2. Construct a read group string from sample metadata if one is not provided (fields: ID, SM, PL, LB, PU).\n3. Run `bwa-mem2 mem` with the read group, piping output to `samtools sort` to produce a coordinate-sorted BAM.\n4. Index the sorted BAM with `samtools index`.\n5. Mark duplicates with `picard MarkDuplicates` (or `samtools markdup` if selected).\n6. Index the deduplicated BAM.\n7. Report alignment summary: total reads, mapped reads, properly paired reads, duplicate reads, mapping rate.\n\n## Output Contract\n\n- Coordinate-sorted deduplicated BAM file (`.bam`).\n- BAM index file (`.bai`).\n- Duplicate metrics file (picard MarkDuplicates output).\n- Alignment summary statistics (total, mapped, properly paired, duplicates, mapping rate).\n\n## Limits\n\n- BWA-MEM2, samtools, and picard must be installed and available on `$PATH`.\n- The BWA-MEM2 index must be pre-built (`bwa-mem2 index \u003Cref.fa>`).\n- Memory scales with reference genome size (~8 GB for human GRCh38).\n- Picard requires a Java runtime (Java 8+).\n- This skill handles single-sample alignment only; multi-sample merging is out of scope.\n- Common failure cases:\n  - Reference index files missing or incomplete (e.g., `.bwt.2bit.64` absent).\n  - Read group string malformed or missing required fields (ID, SM).\n  - Insufficient memory for BWA-MEM2 index loading (~8 GB for human 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