Sequence Analysis Practice 2011/03/09 - Revision history

Mktrost at 17:29, 10 March 2011

2011-03-10T17:29:06Z

← Older revision		Revision as of 13:29, 10 March 2011
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	* [[Media:Filtering example unfiltered.pdf \| (BEFORE FILTERING)]]		* [[Media:Filtering example unfiltered.pdf \| (BEFORE FILTERING)]]
	* [[Media:Filtering example.pdf \| (AFTER 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

Hmkang at 16:04, 10 March 2011

2011-03-10T16:04:19Z

← Older revision		Revision as of 12:04, 10 March 2011
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	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		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 ==		== Filtering Examples in multisample calling ==
	* [[Media:Filtering example unfiltered.pdf \| (BEFORE FILTERING)]]		* [[Media:Filtering example unfiltered.pdf \| (BEFORE FILTERING)]]
	* [[Media:Filtering example.pdf \| (AFTER FILTERING)]]		* [[Media:Filtering example.pdf \| (AFTER FILTERING)]]

Hmkang at 16:03, 10 March 2011

2011-03-10T16:03:59Z

← Older revision		Revision as of 12:03, 10 March 2011
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	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		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)]]

Hmkang at 15:59, 10 March 2011

2011-03-10T15:59:31Z

← Older revision		Revision as of 11:59, 10 March 2011
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	${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		${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

			9. EVALUATE OVERLAP BETWEEN THE TWO SETS OF VARIANT CALLS

			cat ${OUT}/NA12878.exon.sample.vcf ${OUT}/NA12878.highcov.sample.vcf \| grep -v ^# \| cut -f 1,2 \| sort \| uniq -d \| wc -l

			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

Hmkang: /* Steps */

2011-03-10T15:55:54Z

Steps

← Older revision		Revision as of 11:55, 10 March 2011
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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)]]		[[Media:Hmkang-20110309-practical.pdf \| Low-level Processing - Practical Slides (PDF)]]

Hmkang at 21:56, 9 March 2011

2011-03-09T21:56:35Z

← Older revision		Revision as of 17:56, 9 March 2011
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	* Type 'g', and 20:19989392		* Type 'g', and 20:19989392
	* TYPE 'g', and 20:20032998		* 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

Hmkang at 21:32, 9 March 2011

2011-03-09T21:32:58Z

← Older revision		Revision as of 17:32, 9 March 2011
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	== Steps ==		== Steps ==

			[[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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	4. View SAM/BAM format		4. View SAM/BAM format
	${BIN}/samtools-hybrid view -h ${OUT}/NA12878.exon.sample.merged.bam \| less		${BIN}/samtools-hybrid view -h ${OUT}/NA12878.exon.sample.merged.bam \| less


	5. Mark Duplicate Reads		5. Mark Duplicate Reads

Hmkang at 21:22, 9 March 2011

2011-03-09T21:22:18Z

← Older revision		Revision as of 17:22, 9 March 2011
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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

Hmkang at 21:20, 9 March 2011

2011-03-09T21:20:50Z

← Older revision		Revision as of 17:20, 9 March 2011
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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

Hmkang at 21:19, 9 March 2011

2011-03-09T21:19:03Z

← Older revision		Revision as of 17:19, 9 March 2011
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	6. Visualize alignment to reference genome		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		${BIN}/samtools-hybrid tview ${OUT}/NA12878.exon.sample.deduped.bam ${REF}/human_g1k_v37_chr20.fa

	* Type 'g', and 20:19989392		* Type 'g', and 20:19989392
	* TYPE 'g', and 20:20032998		* TYPE 'g', and 20:20032998