Molecular studies using trace DNA, such as from museum specimens, ancient or forensic samples and samples obtained noninvasively, often have a common problem of low quality of DNA templates. Amplification errors, such as allelic dropout and false allele, may arise during polymerase chain reaction (PCR) using such samples. A mathematical model which treats homozygotes and heterozygotes discriminately has been developed to measure sample quality and compute the confidence level of using multiple-tube approaches. We use plucked hair samples collected from 26 individual Sichuan snub-nosed monkeys (Rhinopithecus roxellana) to test the model. In this case, a confidence level of 99% can be achieved by three positive PCRs. If the sample quality is very poor and requires many PCR replicates, an alternative multiple-step genotyping method is recommended. This model enables researchers to optimize experimental protocols through pilot studies and obtain reliable genetic information using noninvasive sampling method.
Consensus Genotype
Genotype file of 26 individuals at 5 SSR loci.
Calculation_Program_of_Fig.3 (C Program Source File)
Source file of the program calculating the estimated number of replicates and the reduction in the number of replicates of heterozygotes and homozygotes using multiple-steps method at the 99% confidence level.
Calculation_Program_of_Fig.3.c
Data of Fig.3
The estimated number of replicates and the reduction in the number of replicates of heterozygotes and homozygotes using multiple-steps method at the 99% confidence level.
Sheet1 are the estimated numbers of replicates of heterozygotes.
Sheet1 are the estimated numbers of replicates of homozygotes.
Sheet3 and Sheet4 are selected genotyping approaches.
Fig.3 data.xls
Calculation Program of Fig.3 (win32 application)
Calculation Program of Fig.3
(win32 application compiled with Visual Studio 2008)
Calculation Program of Fig.3 win32.exe
Reliability Calculator (win32 application)
Reliability Calculator
(win32 application complied with Visual Studio 2008)
Reliability Calculator for Microsatellite Genotyping Using low quality DNA templates
Functions:
1. Generate a table of minimum thresholds of R1 and R2 at a given confidence level;
2. Find approaches reach a given confidence level for given R1 and R2;
3. Generate a table of minimum thresholds of R1 and R2 at a given confidence level (P(E1) is calculated by equation (4));
4. Find approaches reach a given confidence level for given R1 and R2 (P(E1) iscalculated by equation (4));
5. Calculate the lower limit of one-sided confidence interval;
6. Simulate the expected number of replicates for given R1, R2, ms and ns;
Reliability Calculator win32.exe
Reliability Calculator (C Program Source File)
Reliability Calculator (C Program Source File)
Reliability Calculator.c