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| Below lists a sequence of practice mapping fastq files to bam files, performing variant calling and variout quality checks. | | Below lists a sequence of practice mapping fastq files to bam files, performing variant calling and variout quality checks. |
| | | |
− | == Steps == | + | == Steps (Wednesday) == |
| + | |
| + | [[Media:Hmkang-20110309-practical.pdf | Low-level Processing - Practical Slides (PDF)]] |
| | | |
| 0. SETTING UP ENVIRONMENTAL VARIABLES | | 0. SETTING UP ENVIRONMENTAL VARIABLES |
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| 1. Understanding FASTQ format | | 1. Understanding FASTQ format |
| | | |
− | zcat ${IN}/NA12878.exon.sample.read1.fastq.gz | head | + | zcat ${IN}/NA12878.exon.sample.read1.fastq.gz | head |
− | zcat ${IN}/NA12878.exon.sample.read2.fastq.gz | head | + | zcat ${IN}/NA12878.exon.sample.read2.fastq.gz | head |
− | zcat ${IN}/NA12878.exon.sample.unpaired.fastq.gz | head | + | zcat ${IN}/NA12878.exon.sample.unpaired.fastq.gz | head |
| | | |
| press q to quit | | press q to quit |
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| ${BIN}/bwa samse ${REF}/human_g1k_v37_chr20.fa ${OUT}/NA12878.exon.sample.unpaired.fastq.gz.sai ${IN}/NA12878.exon.sample.unpaired.fastq.gz | ${BIN}/samtools-hybrid view -uhS - | ${BIN}/samtools-hybrid sort -m 10000000 - ${OUT}/NA12878.exon.sample.unpaired.bwa.sorted | | ${BIN}/bwa samse ${REF}/human_g1k_v37_chr20.fa ${OUT}/NA12878.exon.sample.unpaired.fastq.gz.sai ${IN}/NA12878.exon.sample.unpaired.fastq.gz | ${BIN}/samtools-hybrid view -uhS - | ${BIN}/samtools-hybrid sort -m 10000000 - ${OUT}/NA12878.exon.sample.unpaired.bwa.sorted |
| | | |
− | ${BIN}/bwa sampe ${REF}/human_g1k_v37_chr20.fa ${OUT}//NA12878.exon.sample.read1.fastq.gz.sai ${OUT}/NA12878.exon.sample.read2.fastq.gz.sai ${IN}/NA12878.exon.sample.read1.fastq.gz ${IN}/NA12878.exon.sample.read2.fastq.gz ${BIN}/samtools-hybrid view -uhS - ${BIN}/samtools-hybrid sort -m 10000000 - ${OUT}/NA12878.exon.sample.paired.bwa.sorted | + | ${BIN}/bwa sampe ${REF}/human_g1k_v37_chr20.fa ${OUT}//NA12878.exon.sample.read1.fastq.gz.sai ${OUT}/NA12878.exon.sample.read2.fastq.gz.sai ${IN}/NA12878.exon.sample.read1.fastq.gz ${IN}/NA12878.exon.sample.read2.fastq.gz | ${BIN}/samtools-hybrid view -uhS - | ${BIN}/samtools-hybrid sort -m 10000000 - ${OUT}/NA12878.exon.sample.paired.bwa.sorted |
| | | |
| 3. Merge multiple BAMs into one | | 3. Merge multiple BAMs into one |
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| | | |
| 4. View SAM/BAM format | | 4. View SAM/BAM format |
− | ${BIN}/samtools-hybrid view -h ${OUT}/NA12878.exon.sample.merged.bam | head -5 | + | ${BIN}/samtools-hybrid view -h ${OUT}/NA12878.exon.sample.merged.bam | less |
| + | |
| + | 5. Mark Duplicate Reads |
| + | ${BIN}/superDeDuper -i ${OUT}/NA12878.exon.sample.merged.bam -o ${OUT}/NA12878.exon.sample.deduped.bam -v |
| + | |
| + | 6. Visualize alignment to reference genome |
| + | ${BIN}/samtools-hybrid index ${OUT}/NA12878.exon.sample.deduped.bam |
| + | ${BIN}/samtools-hybrid tview ${OUT}/NA12878.exon.sample.deduped.bam ${REF}/human_g1k_v37_chr20.fa |
| + | |
| + | * Type 'g', and 20:19989392 |
| + | * TYPE 'g', and 20:20032998 |
| + | |
| + | 7. Use QPLOT to assess the quality |
| + | |
| + | ${BIN}/qplot --plot ${OUT}/NA12878.exon.sample.deduped.bam.qplot.pdf --stats ${OUT}/NA12878.exon.sample.deduped.bam.qplot.stats --reference ${REF}/human_g1k_v37_chr20.fa --dbsnp ${REF}/dbsnp.b130.ncbi37.chr20.tbl --gccontent ${REF}/ncbi37.chr20.gc ${OUT}/NA12878.exon.sample.deduped.bam |
| + | |
| + | == Steps (Thursday) == |
| + | |
| + | 0. SETTING UP ENVIRONMENTAL VARIABLES |
| + | |
| + | setenv BIN /home/hyun/wed/bin |
| + | setenv IN /home/hyun/wed/input |
| + | setenv REF /home/hyun/wed/ref |
| + | |
| + | setenv OUT ~/seq/wednesday/output |
| + | mkdir --p ${OUT} |
| + | |
| + | 1. EXON-TARGETTED DATA : COMPUTING GENOTYPE LIKELHOOD FROM BAM FILES |
| + | |
| + | ${BIN}/samtools-hybrid pileup -g -f ${REF}/human_g1k_v37_chr20.fa ${OUT}/NA12878.exon.sample.deduped.bam > ${OUT}/NA12878.exon.sample.glf |
| + | |
| + | 2. EXON-TARGETTED DATA : VIEW THE GENOTYPE LIKELIHOOD FORMAT |
| + | |
| + | ${BIN}/samtools-hybrid glfview ${OUT}/NA12878.exon.sample.glf | less |
| + | |
| + | TYPE 'q' to finish |
| + | |
| + | 3. EXON-TARGETTED DATA : SINGLE-SAMPLE GENOTYPE CALLING using GLFSINGLE |
| + | |
| + | ${BIN}/glfSingle --maxDepth 10000 --minMapQuality 20 -p 0.9 -g ${OUT}/NA12878.exon.sample.glf -b ${OUT}/NA12878.exon.sample.vcf |
| + | |
| + | 4. EXON-TARGETTED DATA : VIEW THE VCF FILES AND COUNT # OF SNPS |
| + | |
| + | less ${OUT}/NA12878.exon.sample.vcf |
| + | |
| + | grep -v ^# ${OUT}/NA12878.exon.sample.vcf | wc -l |
| + | |
| + | 5. DEEP-COVERAGE GENOME : COMPUTE THE GENOTYPE LIKELIHOOD |
| + | |
| + | ${BIN}/samtools-hybrid pileup -g -f ${REF}/human_g1k_v37_chr20.fa ${IN}/NA12878.highcov.sample.bam > ${OUT}/NA12878.highcov.sample.glf |
| + | |
| + | 6. DEEP-COVERAGE GENOME : SINGLE-SAMPLE VARIANT CALLING |
| + | |
| + | ${BIN}/glfSingle --maxDepth 10000 --minMapQuality 20 -p 0.9 -g ${OUT}/NA12878.highcov.sample.glf -b ${OUT}/NA12878.highcov.sample.vcf |
| + | |
| + | 7. DEEP-COVERAGE GENOME : VIEW THE VCF FILES AND COUNT # OF SNPS |
| + | |
| + | less ${OUT}/NA12878.highcov.sample.vcf |
| + | |
| + | 8. VIEW THE VCF FILES AND COUNT # OF SNPS |
| | | |
| + | grep -v ^# ${OUT}/NA12878.highcov.sample.vcf | wc -l |
| | | |
− | 5. Mark Deuplicate Reads
| + | 9. EVALUATE OVERLAP BETWEEN THE TWO SETS OF VARIANT CALLS |
− | ${BIN}/superDeDuper -i ${OUT}/NA12878.exon.sample.merged.bam \
| |
− | -o ${OUT}/NA12878.exon.sample.deduped.bam -v
| |
| | | |
− | 6. Visualize alignment to reference genome
| + | cat ${OUT}/NA12878.exon.sample.vcf ${OUT}/NA12878.highcov.sample.vcf | grep -v ^# | cut -f 1,2 | sort | uniq -d | wc -l |
− | ${BIN}/samtools-hybrid tview ${OUT}/NA12878.exon.sample.deduped.bam \ | + | |
− | ${REF}/human_g1k_v37_chr20.fa
| + | cat ${OUT}/NA12878.exon.sample.vcf ${OUT}/NA12878.highcov.sample.vcf | grep -v ^# | cut -f 1,2 | sort | uniq -d |
| + | |
| + | 10. VIEW ACTUAL ALIGNMENT AT SNP POSITIONS |
| + | |
| + | ${BIN}/samtools-hybrid tview ${OUT}/NA12878.exon.sample.deduped.bam ${REF}/human_g1k_v37_chr20.fa |
| + | |
| + | TYPE g, and 20:19989392 |
| + | TYPE g, and 20:20032998 |
| + | TYPE g, and 20:20139952 |
| + | |
| + | ${BIN}/samtools-hybrid tview ${IN}/NA12878.highcov.sample.bam ${REF}/human_g1k_v37_chr20.fa |
| + | |
| + | TYPE g, and 20:19989392 |
| + | TYPE g, and 20:20032998 |
| + | TYPE g, and 20:20139952 |
| + | |
| + | 11. SUMMARIZE VCF STATISTICS |
| + | |
| + | perl ${BIN}/vcfSummary.pl --vcf ${OUT}/NA12878.exon.sample.vcf --dbsnp ${REF}/dbsnp_129_b37.rod.chr20.map --bfile ${REF}/hapmap3_r3_b37_fwd.consensus.qc.poly.chr20 |
| + | |
| + | perl ${BIN}/vcfSummary.pl --vcf ${OUT}/NA12878.highcov.sample.vcf --dbsnp ${REF}/dbsnp_129_b37.rod.chr20.map --bfile ${REF}/hapmap3_r3_b37_fwd.consensus.qc.poly.chr20 |
| + | |
| + | == Filtering Examples in multisample calling == |
| + | * [[Media:Filtering example unfiltered.pdf | (BEFORE FILTERING)]] |
| + | * [[Media:Filtering example.pdf | (AFTER FILTERING)]] |
| + | |
| + | |
| + | == Where can you download this software? == |
| + | samtools-hybrid, glfSingle, superDeDuper, qplot can be downloaded at: |
| + | https://github.com/statgen/statgen |