@bgicli/bgicli 2.1.0 → 2.2.0

package/data/skills/bio-workflows-expression-to-pathways/SKILL.md

@@ -0,0 +1,329 @@
+<!--
+# COPYRIGHT NOTICE
+# This file is part of the "Universal Biomedical Skills" project.
+# Copyright (c) 2026 MD BABU MIA, PhD <md.babu.mia@mssm.edu>
+# All Rights Reserved.
+#
+# This code is proprietary and confidential.
+# Unauthorized copying of this file, via any medium is strictly prohibited.
+#
+# Provenance: Authenticated by MD BABU MIA
+
+-->
+
+---
+name: bio-workflows-expression-to-pathways
+description: Workflow from differential expression results to functional enrichment analysis. Covers GO, KEGG, Reactome enrichment with clusterProfiler and visualization. Use when taking DE results to pathway enrichment.
+tool_type: r
+primary_tool: clusterProfiler
+workflow: true
+depends_on:
+  - pathway-analysis/go-enrichment
+  - pathway-analysis/kegg-pathways
+  - pathway-analysis/reactome-pathways
+  - pathway-analysis/gsea
+  - pathway-analysis/enrichment-visualization
+qc_checkpoints:
+  - input_validation: "Valid gene IDs, sufficient DE genes"
+  - enrichment_qc: "Reasonable number of terms, p-values not all significant"
+measurable_outcome: Execute skill workflow successfully with valid output within 15 minutes.
+allowed-tools:
+  - read_file
+  - run_shell_command
+---
+
+# Expression to Pathways Workflow
+
+Convert differential expression results into biological insights through functional enrichment analysis.
+
+## Workflow Overview
+
+```
+DE Results (gene list or ranked list)
+    |
+    v
+[1. Gene ID Conversion] --> Convert to Entrez/Ensembl
+    |
+    v
+[2. Over-representation Analysis]
+    |
+    +---> GO Enrichment (BP, MF, CC)
+    |
+    +---> KEGG Pathways
+    |
+    +---> Reactome Pathways
+    |
+    v
+[3. GSEA (ranked genes)]
+    |
+    v
+[4. Visualization] -----> Dot plots, networks, bar plots
+    |
+    v
+Functional annotations and pathway insights
+```
+
+## Input Preparation
+
+### From DESeq2 Results
+
+```r
+library(DESeq2)
+library(clusterProfiler)
+library(org.Hs.eg.db)
+
+# Load DE results
+res <- read.csv('deseq2_results.csv', row.names = 1)
+
+# Significant genes for ORA
+sig_genes <- rownames(subset(res, padj < 0.05 & abs(log2FoldChange) > 1))
+
+# All genes for background
+all_genes <- rownames(res)
+
+# Ranked list for GSEA (by stat or log2FC)
+ranked_genes <- res$log2FoldChange
+names(ranked_genes) <- rownames(res)
+ranked_genes <- sort(ranked_genes, decreasing = TRUE)
+ranked_genes <- ranked_genes[!is.na(ranked_genes)]
+```
+
+### Gene ID Conversion
+
+```r
+# Convert gene symbols to Entrez IDs
+sig_entrez <- bitr(sig_genes, fromType = 'SYMBOL', toType = 'ENTREZID',
+                   OrgDb = org.Hs.eg.db)
+
+# For ranked list
+ranked_entrez <- bitr(names(ranked_genes), fromType = 'SYMBOL', toType = 'ENTREZID',
+                      OrgDb = org.Hs.eg.db)
+ranked_list <- ranked_genes[ranked_entrez$SYMBOL]
+names(ranked_list) <- ranked_entrez$ENTREZID
+```
+
+## Step 1: GO Over-representation Analysis
+
+```r
+# Biological Process
+go_bp <- enrichGO(gene = sig_entrez$ENTREZID,
+                  OrgDb = org.Hs.eg.db,
+                  ont = 'BP',
+                  pAdjustMethod = 'BH',
+                  pvalueCutoff = 0.05,
+                  qvalueCutoff = 0.1,
+                  readable = TRUE)
+
+# Molecular Function
+go_mf <- enrichGO(gene = sig_entrez$ENTREZID,
+                  OrgDb = org.Hs.eg.db,
+                  ont = 'MF',
+                  pAdjustMethod = 'BH',
+                  pvalueCutoff = 0.05,
+                  readable = TRUE)
+
+# Cellular Component
+go_cc <- enrichGO(gene = sig_entrez$ENTREZID,
+                  OrgDb = org.Hs.eg.db,
+                  ont = 'CC',
+                  pAdjustMethod = 'BH',
+                  pvalueCutoff = 0.05,
+                  readable = TRUE)
+
+# Simplify redundant terms
+go_bp_simple <- simplify(go_bp, cutoff = 0.7, by = 'p.adjust')
+```
+
+## Step 2: KEGG Pathway Enrichment
+
+```r
+kegg <- enrichKEGG(gene = sig_entrez$ENTREZID,
+                   organism = 'hsa',
+                   pvalueCutoff = 0.05,
+                   qvalueCutoff = 0.1)
+
+# Convert KEGG IDs to readable names
+kegg <- setReadable(kegg, OrgDb = org.Hs.eg.db, keyType = 'ENTREZID')
+```
+
+## Step 3: Reactome Pathway Enrichment
+
+```r
+library(ReactomePA)
+
+reactome <- enrichPathway(gene = sig_entrez$ENTREZID,
+                          organism = 'human',
+                          pvalueCutoff = 0.05,
+                          readable = TRUE)
+```
+
+## Step 4: Gene Set Enrichment Analysis (GSEA)
+
+```r
+# GO GSEA
+gsea_go <- gseGO(geneList = ranked_list,
+                 OrgDb = org.Hs.eg.db,
+                 ont = 'BP',
+                 minGSSize = 10,
+                 maxGSSize = 500,
+                 pvalueCutoff = 0.05,
+                 verbose = FALSE)
+
+# KEGG GSEA
+gsea_kegg <- gseKEGG(geneList = ranked_list,
+                     organism = 'hsa',
+                     minGSSize = 10,
+                     maxGSSize = 500,
+                     pvalueCutoff = 0.05,
+                     verbose = FALSE)
+```
+
+## Step 5: Visualization
+
+```r
+library(enrichplot)
+library(ggplot2)
+
+# Dot plot
+dotplot(go_bp_simple, showCategory = 20) +
+    ggtitle('GO Biological Process Enrichment')
+ggsave('go_bp_dotplot.pdf', width = 10, height = 8)
+
+# Bar plot
+barplot(kegg, showCategory = 15) +
+    ggtitle('KEGG Pathway Enrichment')
+ggsave('kegg_barplot.pdf', width = 9, height = 6)
+
+# Enrichment map (network of related terms)
+go_bp_simple <- pairwise_termsim(go_bp_simple)
+emapplot(go_bp_simple, showCategory = 30) +
+    ggtitle('GO Term Similarity Network')
+ggsave('go_network.pdf', width = 10, height = 10)
+
+# Concept network (gene-term connections)
+cnetplot(go_bp, showCategory = 5, categorySize = 'pvalue') +
+    ggtitle('Gene-Concept Network')
+ggsave('cnet_plot.pdf', width = 12, height = 10)
+
+# GSEA plot for specific pathway
+gseaplot2(gsea_kegg, geneSetID = 1:3, pvalue_table = TRUE)
+ggsave('gsea_plot.pdf', width = 10, height = 8)
+
+# Ridge plot for GSEA
+ridgeplot(gsea_go, showCategory = 15)
+ggsave('gsea_ridge.pdf', width = 8, height = 10)
+```
+
+## Step 6: Export Results
+
+```r
+# Export enrichment results
+write.csv(as.data.frame(go_bp), 'go_bp_enrichment.csv', row.names = FALSE)
+write.csv(as.data.frame(kegg), 'kegg_enrichment.csv', row.names = FALSE)
+write.csv(as.data.frame(reactome), 'reactome_enrichment.csv', row.names = FALSE)
+write.csv(as.data.frame(gsea_go), 'gsea_go_results.csv', row.names = FALSE)
+
+# Combine key results
+combined <- rbind(
+    data.frame(Database = 'GO_BP', as.data.frame(go_bp_simple)[1:10,]),
+    data.frame(Database = 'KEGG', as.data.frame(kegg)[1:10,]),
+    data.frame(Database = 'Reactome', as.data.frame(reactome)[1:10,])
+)
+write.csv(combined, 'top_enriched_pathways.csv', row.names = FALSE)
+```
+
+## Parameter Recommendations
+
+| Analysis | Parameter | Value |
+|----------|-----------|-------|
+| enrichGO | pvalueCutoff | 0.05 |
+| enrichGO | qvalueCutoff | 0.1 |
+| simplify | cutoff | 0.7 |
+| gseGO | minGSSize | 10 |
+| gseGO | maxGSSize | 500 |
+| GSEA | perm | 1000 (default) |
+
+## Troubleshooting
+
+| Issue | Likely Cause | Solution |
+|-------|--------------|----------|
+| No enriched terms | Too few genes, wrong IDs | Check gene IDs, relax thresholds |
+| All terms significant | Too many genes | Be more stringent with DE cutoffs |
+| Gene ID conversion fails | Wrong organism, format | Check OrgDb package, gene format |
+| GSEA no results | Poor ranking, small gene sets | Check ranked list, adjust minGSSize |
+
+## Complete Workflow Script
+
+```r
+library(clusterProfiler)
+library(org.Hs.eg.db)
+library(ReactomePA)
+library(enrichplot)
+library(ggplot2)
+
+# Configuration
+de_file <- 'deseq2_results.csv'
+output_dir <- 'pathway_analysis'
+dir.create(output_dir, showWarnings = FALSE)
+
+# Load and prepare data
+res <- read.csv(de_file, row.names = 1)
+sig_genes <- rownames(subset(res, padj < 0.05 & abs(log2FoldChange) > 1))
+cat('Significant genes:', length(sig_genes), '\n')
+
+# Convert IDs
+sig_entrez <- bitr(sig_genes, fromType = 'SYMBOL', toType = 'ENTREZID', OrgDb = org.Hs.eg.db)
+cat('Converted to Entrez:', nrow(sig_entrez), '\n')
+
+# Ranked list for GSEA
+ranked <- res$log2FoldChange
+names(ranked) <- rownames(res)
+ranked <- sort(ranked[!is.na(ranked)], decreasing = TRUE)
+ranked_entrez <- bitr(names(ranked), fromType = 'SYMBOL', toType = 'ENTREZID', OrgDb = org.Hs.eg.db)
+ranked_list <- ranked[ranked_entrez$SYMBOL]
+names(ranked_list) <- ranked_entrez$ENTREZID
+
+# GO enrichment
+go_bp <- enrichGO(sig_entrez$ENTREZID, OrgDb = org.Hs.eg.db, ont = 'BP', readable = TRUE)
+go_bp_simple <- simplify(go_bp, cutoff = 0.7)
+
+# KEGG
+kegg <- enrichKEGG(sig_entrez$ENTREZID, organism = 'hsa')
+kegg <- setReadable(kegg, OrgDb = org.Hs.eg.db, keyType = 'ENTREZID')
+
+# Reactome
+reactome <- enrichPathway(sig_entrez$ENTREZID, organism = 'human', readable = TRUE)
+
+# GSEA
+gsea_go <- gseGO(ranked_list, OrgDb = org.Hs.eg.db, ont = 'BP', verbose = FALSE)
+
+# Plots
+pdf(file.path(output_dir, 'enrichment_plots.pdf'), width = 10, height = 8)
+print(dotplot(go_bp_simple, showCategory = 20) + ggtitle('GO Biological Process'))
+print(barplot(kegg, showCategory = 15) + ggtitle('KEGG Pathways'))
+if (nrow(as.data.frame(reactome)) > 0) {
+    print(dotplot(reactome, showCategory = 15) + ggtitle('Reactome Pathways'))
+}
+dev.off()
+
+# Export
+write.csv(as.data.frame(go_bp_simple), file.path(output_dir, 'go_bp.csv'), row.names = FALSE)
+write.csv(as.data.frame(kegg), file.path(output_dir, 'kegg.csv'), row.names = FALSE)
+write.csv(as.data.frame(reactome), file.path(output_dir, 'reactome.csv'), row.names = FALSE)
+
+cat('\nResults saved to:', output_dir, '\n')
+cat('GO BP terms:', nrow(as.data.frame(go_bp_simple)), '\n')
+cat('KEGG pathways:', nrow(as.data.frame(kegg)), '\n')
+cat('Reactome pathways:', nrow(as.data.frame(reactome)), '\n')
+```
+
+## Related Skills
+
+- pathway-analysis/go-enrichment - GO enrichment details
+- pathway-analysis/kegg-pathways - KEGG analysis
+- pathway-analysis/reactome-pathways - Reactome analysis
+- pathway-analysis/gsea - GSEA methods
+- pathway-analysis/enrichment-visualization - Visualization options
+
+
+<!-- AUTHOR_SIGNATURE: 9a7f3c2e-MD-BABU-MIA-2026-MSSM-SECURE -->

package/data/skills/bio-workflows-fastq-to-variants/SKILL.md

@@ -0,0 +1,374 @@
+<!--
+# COPYRIGHT NOTICE
+# This file is part of the "Universal Biomedical Skills" project.
+# Copyright (c) 2026 MD BABU MIA, PhD <md.babu.mia@mssm.edu>
+# All Rights Reserved.
+#
+# This code is proprietary and confidential.
+# Unauthorized copying of this file, via any medium is strictly prohibited.
+#
+# Provenance: Authenticated by MD BABU MIA
+
+-->
+
+---
+name: bio-workflows-fastq-to-variants
+description: End-to-end DNA sequencing workflow from FASTQ files to variant calls. Covers QC, alignment with BWA, BAM processing, and variant calling with bcftools or GATK HaplotypeCaller. Use when calling variants from raw sequencing reads.
+tool_type: cli
+primary_tool: bcftools
+workflow: true
+depends_on:
+  - read-qc/fastp-workflow
+  - read-alignment/bwa-alignment
+  - alignment-files/alignment-sorting
+  - alignment-files/duplicate-handling
+  - variant-calling/variant-calling
+  - variant-calling/filtering-best-practices
+qc_checkpoints:
+  - after_qc: "Q30 >85%, adapter content <1%"
+  - after_alignment: "Mapping rate >95%, properly paired >90%"
+  - after_dedup: "Duplication rate <30% for WGS, <50% for exome"
+  - after_calling: "Ti/Tv ratio ~2.1 for WGS, dbSNP overlap >95%"
+measurable_outcome: Execute skill workflow successfully with valid output within 15 minutes.
+allowed-tools:
+  - read_file
+  - run_shell_command
+---
+
+# FASTQ to Variants Workflow
+
+Complete pipeline from raw DNA sequencing FASTQ files to filtered variant calls.
+
+## Workflow Overview
+
+```
+FASTQ files
+    |
+    v
+[1. QC & Trimming] -----> fastp
+    |
+    v
+[2. Alignment] ---------> bwa-mem2
+    |
+    v
+[3. BAM Processing] ----> sort, markdup, index
+    |
+    v
+[4. Variant Calling] ---> bcftools (primary) or GATK
+    |
+    v
+[5. Filtering] ---------> Quality filters
+    |
+    v
+Filtered VCF
+```
+
+## Primary Path: BWA + bcftools
+
+### Step 1: Quality Control with fastp
+
+```bash
+# Single sample
+fastp -i sample_R1.fastq.gz -I sample_R2.fastq.gz \
+    -o sample_R1.trimmed.fq.gz -O sample_R2.trimmed.fq.gz \
+    --detect_adapter_for_pe \
+    --qualified_quality_phred 20 \
+    --length_required 50 \
+    --html sample_fastp.html
+
+# Batch processing
+for sample in sample1 sample2 sample3; do
+    fastp -i ${sample}_R1.fastq.gz -I ${sample}_R2.fastq.gz \
+        -o trimmed/${sample}_R1.fq.gz -O trimmed/${sample}_R2.fq.gz \
+        --detect_adapter_for_pe \
+        --html qc/${sample}_fastp.html
+done
+```
+
+**QC Checkpoint 1:** Check fastp reports
+- Q30 bases >85% (DNA typically higher quality than RNA)
+- Adapter content <1%
+- No unusual GC distribution
+
+### Step 2: BWA-MEM2 Alignment
+
+```bash
+# Index reference (once)
+bwa-mem2 index reference.fa
+
+# Align with read group info
+for sample in sample1 sample2 sample3; do
+    bwa-mem2 mem -t 8 \
+        -R "@RG\tID:${sample}\tSM:${sample}\tPL:ILLUMINA\tLB:lib1" \
+        reference.fa \
+        trimmed/${sample}_R1.fq.gz \
+        trimmed/${sample}_R2.fq.gz \
+    | samtools view -bS - > aligned/${sample}.bam
+done
+```
+
+**QC Checkpoint 2:** Check alignment stats
+```bash
+samtools flagstat aligned/${sample}.bam
+```
+- Mapped reads >95%
+- Properly paired >90%
+
+### Step 3: BAM Processing
+
+```bash
+for sample in sample1 sample2 sample3; do
+    # Sort by coordinate
+    samtools sort -@ 8 -o aligned/${sample}.sorted.bam aligned/${sample}.bam
+
+    # Mark duplicates (samtools method)
+    samtools fixmate -m aligned/${sample}.sorted.bam - | \
+        samtools sort -@ 8 - | \
+        samtools markdup -@ 8 - aligned/${sample}.markdup.bam
+
+    # Index
+    samtools index aligned/${sample}.markdup.bam
+
+    # Cleanup intermediate
+    rm aligned/${sample}.bam aligned/${sample}.sorted.bam
+done
+```
+
+**QC Checkpoint 3:** Check duplication rate
+```bash
+samtools flagstat aligned/${sample}.markdup.bam | grep "duplicates"
+```
+- WGS: <30% duplicates
+- Exome/targeted: <50% duplicates
+
+### Step 4: Variant Calling with bcftools
+
+```bash
+# Single sample calling
+bcftools mpileup -Ou -f reference.fa aligned/sample1.markdup.bam | \
+    bcftools call -mv -Oz -o variants/sample1.vcf.gz
+
+# Multi-sample calling (joint calling)
+bcftools mpileup -Ou -f reference.fa \
+    aligned/sample1.markdup.bam \
+    aligned/sample2.markdup.bam \
+    aligned/sample3.markdup.bam | \
+    bcftools call -mv -Oz -o variants/cohort.vcf.gz
+
+bcftools index variants/cohort.vcf.gz
+```
+
+### Step 5: Variant Filtering
+
+```bash
+# Basic quality filter
+bcftools filter -Oz \
+    -e 'QUAL<20 || DP<10 || MQ<30' \
+    -o variants/cohort.filtered.vcf.gz \
+    variants/cohort.vcf.gz
+
+# More stringent filter
+bcftools filter -Oz \
+    -e 'QUAL<30 || DP<10 || DP>200 || MQ<40 || MQB<0.1' \
+    -s "LowQual" \
+    -o variants/cohort.filtered.vcf.gz \
+    variants/cohort.vcf.gz
+
+# Stats
+bcftools stats variants/cohort.filtered.vcf.gz > variants/vcf_stats.txt
+```
+
+**QC Checkpoint 4:** Check variant stats
+- Ti/Tv ratio ~2.1 for whole genome
+- Ti/Tv ratio ~2.8-3.0 for exome
+- >95% overlap with dbSNP for known sites
+
+## Alternative Path: BWA + GATK HaplotypeCaller
+
+### Step 4 Alternative: GATK Variant Calling
+
+```bash
+# Create sequence dictionary (once)
+gatk CreateSequenceDictionary -R reference.fa
+
+# Index reference (once)
+samtools faidx reference.fa
+
+# Base Quality Score Recalibration (BQSR)
+gatk BaseRecalibrator \
+    -R reference.fa \
+    -I aligned/sample1.markdup.bam \
+    --known-sites dbsnp.vcf.gz \
+    -O recal_data.table
+
+gatk ApplyBQSR \
+    -R reference.fa \
+    -I aligned/sample1.markdup.bam \
+    --bqsr-recal-file recal_data.table \
+    -O aligned/sample1.recal.bam
+
+# HaplotypeCaller (per-sample GVCF mode)
+gatk HaplotypeCaller \
+    -R reference.fa \
+    -I aligned/sample1.recal.bam \
+    -O variants/sample1.g.vcf.gz \
+    -ERC GVCF
+
+# Joint genotyping (for multiple samples)
+gatk GenomicsDBImport \
+    -V variants/sample1.g.vcf.gz \
+    -V variants/sample2.g.vcf.gz \
+    -V variants/sample3.g.vcf.gz \
+    --genomicsdb-workspace-path genomicsdb \
+    -L intervals.bed
+
+gatk GenotypeGVCFs \
+    -R reference.fa \
+    -V gendb://genomicsdb \
+    -O variants/cohort.vcf.gz
+```
+
+### Step 5 Alternative: GATK Variant Filtering
+
+```bash
+# Hard filtering (for small cohorts)
+gatk VariantFiltration \
+    -R reference.fa \
+    -V variants/cohort.vcf.gz \
+    --filter-expression "QD < 2.0" --filter-name "LowQD" \
+    --filter-expression "FS > 60.0" --filter-name "HighFS" \
+    --filter-expression "MQ < 40.0" --filter-name "LowMQ" \
+    --filter-expression "MQRankSum < -12.5" --filter-name "LowMQRS" \
+    --filter-expression "ReadPosRankSum < -8.0" --filter-name "LowRPRS" \
+    -O variants/cohort.filtered.vcf.gz
+
+# VQSR (for large cohorts >30 samples)
+gatk VariantRecalibrator \
+    -R reference.fa \
+    -V variants/cohort.vcf.gz \
+    --resource:hapmap,known=false,training=true,truth=true,prior=15.0 hapmap.vcf.gz \
+    --resource:omni,known=false,training=true,truth=false,prior=12.0 omni.vcf.gz \
+    --resource:1000G,known=false,training=true,truth=false,prior=10.0 1000G.vcf.gz \
+    --resource:dbsnp,known=true,training=false,truth=false,prior=2.0 dbsnp.vcf.gz \
+    -an QD -an MQ -an MQRankSum -an ReadPosRankSum -an FS -an SOR \
+    -mode SNP \
+    -O cohort.snp.recal \
+    --tranches-file cohort.snp.tranches
+
+gatk ApplyVQSR \
+    -R reference.fa \
+    -V variants/cohort.vcf.gz \
+    -O variants/cohort.vqsr.vcf.gz \
+    --recal-file cohort.snp.recal \
+    --tranches-file cohort.snp.tranches \
+    -mode SNP \
+    --truth-sensitivity-filter-level 99.5
+```
+
+## Parameter Recommendations
+
+| Step | Parameter | WGS | Exome/Targeted |
+|------|-----------|-----|----------------|
+| bwa-mem2 | -t | 8-16 | 8 |
+| samtools markdup | - | Required | Required |
+| bcftools mpileup | -d | 250 (default) | 1000 |
+| bcftools mpileup | -q | 20 | 20 |
+| bcftools filter | QUAL | >20 | >30 |
+| bcftools filter | DP | >10, <2x mean | >20 |
+| GATK | intervals | - | Target BED |
+
+## Choosing Between bcftools and GATK
+
+| Criterion | bcftools | GATK |
+|-----------|----------|------|
+| Speed | Faster | Slower |
+| Memory | Lower | Higher |
+| Best for | Germline SNPs/indels | Germline, somatic |
+| Cohort size | Any | Scales well |
+| BQSR | Not supported | Recommended |
+| VQSR | Not supported | For large cohorts |
+
+## Troubleshooting
+
+| Issue | Likely Cause | Solution |
+|-------|--------------|----------|
+| Low mapping rate | Wrong reference, contamination | Verify reference genome version |
+| High duplication | PCR over-amplification, low input | Check library prep, may need more input DNA |
+| Low Ti/Tv | False positives | Increase quality filters |
+| Missing variants | Too stringent filters, low depth | Relax filters, check coverage |
+| Many indels at homopolymers | Sequencing errors | Filter homopolymer regions |
+
+## Complete Pipeline Script
+
+```bash
+#!/bin/bash
+set -e
+
+# Configuration
+THREADS=8
+REF="reference.fa"
+SAMPLES="sample1 sample2 sample3"
+OUTDIR="results"
+
+mkdir -p ${OUTDIR}/{trimmed,aligned,variants,qc}
+
+echo "=== Step 1: QC with fastp ==="
+for sample in $SAMPLES; do
+    fastp -i ${sample}_R1.fastq.gz -I ${sample}_R2.fastq.gz \
+        -o ${OUTDIR}/trimmed/${sample}_R1.fq.gz \
+        -O ${OUTDIR}/trimmed/${sample}_R2.fq.gz \
+        --detect_adapter_for_pe \
+        --html ${OUTDIR}/qc/${sample}_fastp.html \
+        -w ${THREADS}
+done
+
+echo "=== Step 2: Alignment with bwa-mem2 ==="
+for sample in $SAMPLES; do
+    bwa-mem2 mem -t ${THREADS} \
+        -R "@RG\tID:${sample}\tSM:${sample}\tPL:ILLUMINA" \
+        ${REF} \
+        ${OUTDIR}/trimmed/${sample}_R1.fq.gz \
+        ${OUTDIR}/trimmed/${sample}_R2.fq.gz | \
+    samtools view -@ ${THREADS} -bS - > ${OUTDIR}/aligned/${sample}.bam
+done
+
+echo "=== Step 3: BAM Processing ==="
+for sample in $SAMPLES; do
+    samtools fixmate -@ ${THREADS} -m ${OUTDIR}/aligned/${sample}.bam - | \
+    samtools sort -@ ${THREADS} - | \
+    samtools markdup -@ ${THREADS} - ${OUTDIR}/aligned/${sample}.markdup.bam
+    samtools index ${OUTDIR}/aligned/${sample}.markdup.bam
+    rm ${OUTDIR}/aligned/${sample}.bam
+done
+
+echo "=== Step 4: Joint Variant Calling ==="
+bcftools mpileup -Ou -f ${REF} ${OUTDIR}/aligned/*.markdup.bam | \
+    bcftools call -mv -Oz -o ${OUTDIR}/variants/cohort.vcf.gz
+bcftools index ${OUTDIR}/variants/cohort.vcf.gz
+
+echo "=== Step 5: Filtering ==="
+bcftools filter -Oz \
+    -e 'QUAL<20 || DP<10 || MQ<30' \
+    -o ${OUTDIR}/variants/cohort.filtered.vcf.gz \
+    ${OUTDIR}/variants/cohort.vcf.gz
+bcftools index ${OUTDIR}/variants/cohort.filtered.vcf.gz
+
+echo "=== Stats ==="
+bcftools stats ${OUTDIR}/variants/cohort.filtered.vcf.gz > ${OUTDIR}/variants/stats.txt
+
+echo "=== Pipeline Complete ==="
+echo "Filtered VCF: ${OUTDIR}/variants/cohort.filtered.vcf.gz"
+```
+
+## Related Skills
+
+- read-qc/fastp-workflow - Detailed QC options
+- read-alignment/bwa-alignment - BWA-MEM2 parameters
+- alignment-files/duplicate-handling - Duplicate marking details
+- variant-calling/variant-calling - bcftools calling options
+- variant-calling/gatk-variant-calling - GATK HaplotypeCaller details
+- variant-calling/filtering-best-practices - Advanced filtering strategies
+- variant-calling/variant-annotation - Annotate variants with VEP
+
+
+<!-- AUTHOR_SIGNATURE: 9a7f3c2e-MD-BABU-MIA-2026-MSSM-SECURE -->