Generic Exome Analysis Plan - Revision history

Goncalo: /* Read Mapping */

2010-05-21T17:07:56Z

Read Mapping

← Older revision		Revision as of 13:07, 21 May 2010
Line 21:		Line 21:

	; Map Reads with Appropriate Read Mapper		; Map Reads with Appropriate Read Mapper
	: Currently, [bio-bwa.sourceforge.net/bwa.shtm BWA] is a convenient, widely used read mapper.		: Currently, [http://bio-bwa.sourceforge.net/bwa.shtm BWA] is a convenient, widely used read mapper.

	; Mark Duplicate Reads		; Mark Duplicate Reads

Goncalo: /* Variant Filters */

2010-05-05T18:29:04Z

Variant Filters

← Older revision		Revision as of 14:29, 5 May 2010
Line 105:		Line 105:
	; Read Depth		; Read Depth
	: Consider filtering out variants at sites where total read depth is unusually low. Unfortunately, capture protocols introduce very large amounts of variation in sequencing depth and it is usually not possible to accurately filter out sites sequenced at very high depth.		: Consider filtering out variants at sites where total read depth is unusually low. Unfortunately, capture protocols introduce very large amounts of variation in sequencing depth and it is usually not possible to accurately filter out sites sequenced at very high depth.

			; Strand Bias
			: Check whether each variant allele is supported by reads that map to both strands. Variants that are supported by reads on only one strand are almost always false positives.

	= Special Considerations for Admixed Samples =		= Special Considerations for Admixed Samples =

Goncalo: /* Read Mapping */

2010-04-21T15:51:03Z

Read Mapping

← Older revision		Revision as of 11:51, 21 April 2010
Line 34:		Line 34:

	; Recalibrate Base Quality Scores		; Recalibrate Base Quality Scores
	: Base quality scores can be updated by comparing sites that are unlikely to vary (such as those not currently reported as variants in dbSNP or in the most recent [http://www.1000genomes.org 1000 Genomes Project]] analyses.		: Base quality scores can be updated by comparing sites that are unlikely to vary (such as those not currently reported as variants in dbSNP or in the most recent [http://www.1000genomes.org 1000 Genomes Project] analyses.

	; Update Base Quality Score Metrics		; Update Base Quality Score Metrics

Goncalo: /* Read Mapping */

2010-04-21T15:50:37Z

Read Mapping

← Older revision		Revision as of 11:50, 21 April 2010
Line 34:		Line 34:

	; Recalibrate Base Quality Scores		; Recalibrate Base Quality Scores
	: Base quality scores can be updated by comparing sites that are unlikely to vary (such as those not currently reported as variants in dbSNP or in the most recent [[1000 ~~Genome~~ Project]] analyses.		: Base quality scores can be updated by comparing sites that are unlikely to vary (such as those not currently reported as variants in dbSNP or in the most recent [http://www.1000genomes.org 1000 Genomes Project]] analyses.

	; Update Base Quality Score Metrics		; Update Base Quality Score Metrics

Goncalo: /* Variant Filters */

2010-04-21T12:02:57Z

Variant Filters

← Older revision		Revision as of 08:02, 21 April 2010
Line 101:		Line 101:

	; Local Realignment Filter		; Local Realignment Filter
	: Consider removing single nucleotide variants at sites where local realignment of all covering sequence reads suggests that ~~a length~~ polymorphism is present in the population.		: Consider removing single nucleotide variants at sites where local realignment of all covering sequence reads suggests that an [[indel]] polymorphism is present in the population.

	; Read Depth		; Read Depth
	: Consider filtering out variants at sites where total read depth is unusually low. Unfortunately, capture protocols introduce very large amounts of variation in sequencing depth and it is usually not possible to accurately filter out sites sequenced at very high depth.		: Consider filtering out variants at sites where total read depth is unusually low. Unfortunately, capture protocols introduce very large amounts of variation in sequencing depth and it is usually not possible to accurately filter out sites sequenced at very high depth.

	= Special Considerations for Admixed Samples =		= Special Considerations for Admixed Samples =

Tblackw at 12:01, 21 April 2010

2010-04-21T12:01:52Z

← Older revision		Revision as of 08:01, 21 April 2010
Line 136:		Line 136:
	== Burden Tests ==		== Burden Tests ==

	The same analyses that were originally carried for single variants should be carried out for groups of rare variants. In principle, one could ~~simple~~ use the presence of a rare variant (or a particular class of rare variant, such as a non-synonymous variant or a newly discovered variant) as a predictor and repeat the logistic regression, linear regression or genotype regression described above. For an initial pass, I think the precise form of this analysis is not critical, because the next step is to...		The same analyses that were originally carried for single variants should be carried out for groups of rare variants. In principle, one could simply use the presence of a rare variant (or a particular class of rare variant, such as a non-synonymous variant or a newly discovered variant) as a predictor and repeat the logistic regression, linear regression or genotype regression described above. For an initial pass, I think the precise form of this analysis is not critical, because the next step is to...

	== More Q-Q Plots ==		== More Q-Q Plots ==

Tblackw at 11:54, 21 April 2010

2010-04-21T11:54:41Z

← Older revision		Revision as of 07:54, 21 April 2010
Line 101:		Line 101:

	; Local Realignment Filter		; Local Realignment Filter
	: Consider removing single nucleotide variants at sites where local realignment of all covering sequence reads suggests that a ~~short indel~~ polymorphism is present in the population.		: Consider removing single nucleotide variants at sites where local realignment of all covering sequence reads suggests that a length polymorphism is present in the population.

	; Read Depth		; Read Depth

Tblackw at 11:51, 21 April 2010

2010-04-21T11:51:20Z

← Older revision		Revision as of 07:51, 21 April 2010
Line 88:		Line 88:

	; If Nuclear Families are Available, Assess Mendelian Consistency		; If Nuclear Families are Available, Assess Mendelian Consistency
	: When reporting rates of Mendelian inconsistencies, report these not on a ~~per site basis~~ but~~, instead, based on the number~~ of sites with at least one non-reference call in the trio.		: When reporting rates of Mendelian inconsistencies, report these not as a fraction of all sites, but as a fraction of sites with at least one non-reference call in the trio.

	== Variant Filters ==		== Variant Filters ==

	Initial sets of SNP calls ~~invariable~~ will include many false positives. ~~As a proportion~~ of ~~total~~ variants called~~, the number of false positives~~ is likely to increase as more and more samples are sequenced. To keep the ~~number~~ of false positives under control, it is important to both apply an increasingly strict set of quality control filters but also to experimentally validate some newly discovered variants. Many of these filters are currently implemented in [[GATK]].		Initial sets of SNP calls invariably will include many false positives. The fraction of false positives among all variants called is likely to increase as more and more samples are sequenced. To keep the fraction of false positives under control, it is important to both apply an increasingly strict set of quality control filters but also to experimentally validate some newly discovered variants. Many of these filters are currently implemented in [[GATK]].

	; Mapping Quality Filter		; Mapping Quality Filter
	: Consider removing variants at ~~sides~~ with low mapping quality scores. Even if the average mapping quality score for a site is high, consider removing variants at sites where a ~~large~~ fraction of reads have low mapping quality scores.		: Consider removing variants at sites with low mapping quality scores. Even if the average mapping quality score for a site is high, consider removing variants at sites where a noticeable fraction of reads have low mapping quality scores.

	; Allele Balance Filter		; Allele Balance Filter
	: Among individuals who are assigned an heterozygous genotype, check the proportion of reads supporting each allele. Consider filtering out variants where one of the alleles accounts for <30% of reads.		: Among individuals who are assigned an heterozygous genotype, check the proportion of reads supporting each allele. Consider filtering out variants where one of the alleles accounts for <30% of reads.

			; Local Realignment Filter
			: Consider removing single nucleotide variants at sites where local realignment of all covering sequence reads suggests that a short indel polymorphism is present in the population.

	; Read Depth		; Read Depth
	: Consider filtering out variants at sites where total read depth is unusually low. Unfortunately, capture protocols introduce very large amounts of variation in sequencing depth and it is usually not possible to ~~also~~ accurately filter out sites sequenced at very high depth.		: Consider filtering out variants at sites where total read depth is unusually low. Unfortunately, capture protocols introduce very large amounts of variation in sequencing depth and it is usually not possible to accurately filter out sites sequenced at very high depth.

	= Special Considerations for Admixed Samples =		= Special Considerations for Admixed Samples =
Line 112:		Line 115:
	= Initial Association Analyses =		= Initial Association Analyses =

	We anticipate that, at least early on, the initial association analysis of whole ~~genome~~ datasets in the context of complex trait association studies will focus on identifying and resolving quality control issues that might result in unexpected artifacts.		We anticipate that, at least early on, the initial association analysis of whole exome datasets in the context of complex trait association studies will focus on identifying and resolving quality control issues that might result in unexpected artifacts.

	== Initial Single SNP Tests ==		== Initial Single SNP Tests ==

Goncalo: /* Read Mapping */

2010-04-21T11:14:24Z

Read Mapping

← Older revision		Revision as of 07:14, 21 April 2010
Line 22:		Line 22:
	; Map Reads with Appropriate Read Mapper		; Map Reads with Appropriate Read Mapper
	: Currently, [bio-bwa.sourceforge.net/bwa.shtm BWA] is a convenient, widely used read mapper.		: Currently, [bio-bwa.sourceforge.net/bwa.shtm BWA] is a convenient, widely used read mapper.

			; Mark Duplicate Reads
			: Duplicate reads, whether generated as PCR artifacts during library preparation or optical duplicates during image analysis and base-calling, can mislead variant calling algorithms. To avoid problems, one typically removes from consideration all reads that appear to map at exactly the same location (for paired ends, only reads for which both ends map to the same locations are excluded.)

	; Basic Mapping Statistics		; Basic Mapping Statistics

Goncalo: /* Read Mapping */

2010-04-21T04:30:13Z

Read Mapping

← Older revision		Revision as of 00:30, 21 April 2010
Line 36:		Line 36:
	: Generate new curves with base quality scores per position.		: Generate new curves with base quality scores per position.
	: Calculate the number of mapped bases that reach at least Q20. Potentially, calculate Q20 ''equivalent'' bases by summing the quality scores for bases with base quality >Q20 and dividing the total by 20.		: Calculate the number of mapped bases that reach at least Q20. Potentially, calculate Q20 ''equivalent'' bases by summing the quality scores for bases with base quality >Q20 and dividing the total by 20.

			; Evaluate Coverage as Function of GC Content
			: For each target region, calculate read depth and also the proportion of GC bases in the reference genome. Flag samples where coverage varies strongly as a function of GC content.

	== Verify Sample Identities ==		== Verify Sample Identities ==