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To test the limits of SNP detection, discovery by setting a minimum variant threshold of 10% was also performed.
We also optimised mapping parameters for SNP detections and confirmed that the 20% minimum variant threshold for detecting SNPs in pooled samples of four individuals was appropriate.
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The higher specificity value achieved by Dindel is likely due to the minimum variant call threshold used in analysis.
Furthermore, the minimum variant allele frequency threshold for VarScan was set to, likewise for CRISP the number of haplotypes in each pool was set to 300.
Additionally, non-specific matches and broken pairs were ignored, and the program enforced a 20-fold minimum coverage threshold and 90% minimum variant frequency.
False-positive SNVs were excluded using the following thresholds: Fisher's exact test P < 0.05, coverage ≥10×, Phred base quality score ≥20, minimum variant allele frequency ≥20% and high-quality reads supporting variants allele ≥4.
An optional minimum variant support, v, used in extending ambiguity.
SNP calling parameters were set to minimum coverage = 5, and minimum variant frequency = 0.2.
The threshold TH denotes the mean of maximum and minimum thresholds; the threshold region TR denotes the value between maximum and minimum thresholds.
In order to assure sufficient power to detect variants, the minimum coverage threshold of a coding base was set to 30.
SAMtools varFilter script was applied to allow a maximum variant coverage of 10,000 to a minimum quality threshold (Phred-score) of 20 for the final SNP calls.
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