Description Usage Arguments Details Value
Call or flag candidate alleles to construct a consensus genotype.
1 | callAllele(fb, tbase = NULL, clean = TRUE, verbose = FALSE)
|
fb |
A |
tbase |
A data.frame with thresholds which are locus specific. Thresholds are:
|
clean |
Logical. If |
verbose |
Logical. If |
Function works on an individual run (one PCR reaction). Apply this to sample * locus * run
combination.
The data should come from an NGS run as processed by de Barba et al. (2016).
For algorithm used to find stutters, see findStutter
.
De Barba, M., Miquel, C., Lobréaux, S., Quenette, P. Y., Swenson, J. E., & Taberlet, P. (2016). High-throughput microsatellite genotyping in ecology: improved accuracy, efficiency, standardization and success with low-quantity and degraded DNA. Molecular Ecology Resources, 1-16. https://doi.org/10.1111/1755-0998.12594
Key for abbreviations used in (pseudo)code:
A = allele
S = stutter
R = relative low threshold
L = low count threshold
D = disbalance
The result is appended three columns; one for called A, one for flagged alleles and if read is a stutter. Possible flags are:
L = low amplification threshold (if for some reason, number of total reads is very low, alleles get a flag)
N = no stutter (if there was enough reads but no stutter was present)
D = disbalance - alleles not in balance (expecting 1:1 for heterozygotes, those out of balance flagged)
M = multiple alleles (self explanatory)
Algorithm is as follows:
0. find max allele height
1. if allele has number of reads < L, flag it as "L"
2. see if allele has stutter
2a. if yes, mark as called
2aa. if A in disbalance (A < D), flag as "D"
2ab. mark stutter as such $stutter = TRUE
2b. if no, check AlleleWithNoStutterHeight
2ba. if x > AlleleWithNoStutterHeight, add flag "N"
2bb. if x < AlleleWithNoStutterHeight, ignore allele
3. if number of unflagged alleles is more than 2 (those marked with D are not counted), add flag "M" to all
Output should be all alleles and their stutters.
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