evaluateCovAndBQ: Evaluate coverage and base quality

In sandmanns/appreci8R: appreci8R: an R/Bioconductor package for filtering SNVs and short indels with high sensitivity and high PPV

Description

appreci8R combines and filters the output of different variant calling tools according to the 'appreci8'-algorithm. In the 5th analysis step, all calls are evaluated with respect to coverage and basequality (using Rsamtools). Calls with insufficient coverage and/or basequality are filtered from further analysis. A GRanges object with all calls featuring sufficient coverage and basequality is returned.

Usage

evaluateCovAndBQ(output_folder, combined_calls_g, bam_folder,
                 dp = 50, nr_alt = 20, vaf = 0.01, bq = 15, bq_diff = 7)

Arguments

output_folder	The folder to write the output files into. If an empty string is provided, no files are written out.
combined_calls_g	A GRanges object; necessary input object. The GRanges object contains one call per line. CombineOutput()-output can directly be taken as input.
bam_folder	The folder containing the bam- and bai-files for every sample. The file names have to match the sample names, i.e. Sample1.bam and Sample1.bai if Sample1.vcf (without any defined suffixes) was analyzed for caller1.
dp	Minimum depth that is required for a call to pass the filter (default: 50).
nr_alt	Minimum number of reads carrying the alternate allele that is required for a call to pass the filter (default: 20).
vaf	Minimum VAF that is required for a call to pass the filter (default: 0.01).
bq	Minimum base quality that is required for a call to pass the filter (default: 15; values above 44 not recommended).
bq_diff	Maximum difference in basequality between reference and alternative that is allowed for a call to pass the filter (default: 7).

Details

The function evaluateCovAndBQ evaluates coverage and base quality of all calls and filteres them according to user-definable thresholds. Only those calls that feature sufficient coverage and base quality are reported. Filtration is performed in a 2-step procedure:

First, coverage is evaluated using Rsamtools pileup and a threshold of min_base_quality=0. If depth is below the defined threshold dp and/or the number of reads carrying the alternate allele is below the defined threshold nr_alt and/or VAF is below the defined threshold VAF, a call is excluded. However, if coverage is sufficient, base quality - which is more time-consuming - is evaluated.

To evaluate base quality, the threshold for min_base_quality is successively increased from 0 to 44 (thus, no values above 44 are recommended for bq). The average base quality for all reads carrying the reference and the alternate allele is calculated. For indels, which are always summed up as “+” by Rsamtools, no base quality can be determined. If the average base quality of the alternate allele is lower than bq, the call is filtered. If “average base quality reference allele” - “average base quality alternate allele” is higher than bq_diff, the call is filtered.

Coverage is reported with respect to the forward- and reverse reads separately. It is not yet evaluated. This is done in the 7th analysis step (finalFiltration).

Value

A GRanges object is returned containing all calls with sufficient coverage and base quality. Reported metadata columns are: SampleID, Ref, Alt, Location, c. (position of variant on cDNA level), p. (position of variant on protein level), AA_ref, AA_alt, Codon_ref, Codon_alt, Consequence, Gene, GeneID, TranscriptID, Caller1 to CallerX (dependent on the number of callers that is evaluated), Nr_Ref, Nr_Alt, DP, VAF, BQ_REF, BQ_ALT, Nr_Ref_fwd, Nr_Alt_fwd, DP_fwd, VAF_rev, Nr_Ref_rev, Nr_Alt_rev, DP_rev, VAF_rev.

If an output folder is provided, the output is saved as Results_Frequency.txt.

Author(s)

Sarah Sandmann <[e-mail]>

References

Rsamtools: http://bioconductor.org/packages/release/bioc/html/Rsamtools.html

See Also

appreci8R, appreci8Rshiny, filterTarget, normalize, annotate, combineOutput, determineCharacteristics, finalFiltration

Examples

library("GenomicRanges")
combined<-GRanges(seqnames = c("4","X"),
                  ranges = IRanges(start = c (106196951,15838366),
                                   end = c (106196951,15838366)),
                  SampleID = c("Sample2","Sample1"), Ref = c("A","C"),
                  Alt = c("G","A"), Location = c("coding,coding","coding"),
                  c. = c("5284,5347","864"), p. = c("1762,1783","288"),
                  AA_ref = c("I,I","N"), AA_alt = c("V,V","K"),
                  Codon_ref = c("ATA,ATA","AAC"),
                  Codon_alt = c("GTA,GTA","AAA"),
                  Consequence = c("nonsynonymous,nonsynonymous","nonsynonymous"),
                  Gene = c("TET2,TET2","ZRSR2"),
                  GeneID = c("54790,54790","8233"),
                  TranscriptID = c("18308,18309","75467"),
                  GATK = c(1,1), VarScan = c(NA,1))
bam_folder <- system.file("extdata", package = "appreci8R")
bam_folder <- paste(bam_folder, "/", sep="")

filtered<-evaluateCovAndBQ("", combined, bam_folder)

sandmanns/appreci8R documentation built on Dec. 7, 2020, 12:32 a.m.