--- title: "NF-GenePop Analysis" author: "Laura H Spencer" date: "1/22/2018" output: html_document --- ```{r setup, include=FALSE} knitr::opts_chunk$set(echo = TRUE) knitr::opts_knit$set(root.dir ="~/Documents/Roberts Lab/O.lurida_genetics") ``` #### In this notebook I will use the GenePop R program to analyze the 2016/2017 Fidalgo Bay (NF) Ostrea lurida microsatellite data; the results from each analysis are housed in a series of .txt files. Prior to importing the data, prepared the 2016/2017 NF data in Excel and exported into GenePop format; resulting file is available on [2018-01-22-Preparing-for-Genepop.md](https://github.com/laurahspencer/O.lurida_genetics/blob/master/Notebooks/2018-01-22-Preparing-for-Genepop.md) #### First, install GenePop program; ```{r} # install.packages("genepop") knitr::opts_chunk$set(library(genepop)) ``` #### Pull basic information on allele and genotype frequencies per locus and per sample ```{r} basic_info(inputFile="Data/Oly2016NFH+2017NFW_Merged.txt", outputFile = "Analyses/NF-Basic-Info.txt", verbose=T) ``` #### Resulting file: ["NF-Basic-Info.txt"](https://raw.githubusercontent.com/laurahspencer/O.lurida_genetics/master/Analyses/NF-Basic-Info.txt) #### Hetero- and homozygosity info pasted here: ### NF Wild | Loci | Olur10 | Olur11 | Olur12 | Olur13 | Olur15 | Olur19 | |------------------------|-------------|---------|---------|---------|---------|---------| | Expected Homozygotes | 4.99 | 24.59 | 11.83 | 6.68 | 6.66 | 6.64 | | Observed Homozygotes | 2 | 28 | 12 | 7 | 6 | 7 | | Expected Heterozygotes | 92.01 | 70.41 | 86.17 | 91.32 | 91.34 | 90.36 | | Observed Heterozygotes | 95 | 67 | 86 | 91 | 92 | 90 | ### NF Hatchery | Loci | Olur10 | Olur11 | Olur12 | Olur13 | Olur15 | Olur19 | |------------------------|-------------|---------|---------|---------|---------|---------| | Expected Homozygotes | 5.02 | 23.03 | 13.44 | 7.14 | 7.22 | 7.10 | | Observed Homozygotes | 7 | 27 | 5 | 6 | 6 | 7 | | Expected Heterozygotes | 92.98 | 70.97 | 86.56 | 88.86 | 90.78 | 88.90 | | Observed Heterozygotes | 91 | 67 | 95 | 90 | 92 | 89 | #### Assess whether loci are in Hardy-Weinberg Equilibrium ```{r} test_HW(inputFile = "Data/Oly2016NFH+2017NFW_Merged.txt", which="Proba", outputFile = "Analyses/NF-HWE.txt", enumeration = FALSE, dememorization = 10000, batches = 500, iterations = 2000, verbose = interactive()) ``` #### Resulting file: ["NF-HWE.txt"](https://raw.githubusercontent.com/laurahspencer/O.lurida_genetics/master/Analyses/NF-HWE.txt) #### All P-values across loci in each population are >>0.05, do not reject the null hypothesis that all loci are in HWE. Pop : NFW-2017 ----------------------------------------- Fis estimates --------------- locus P-val S.E. W&C R&H Steps ----------- ------- ------- ------- ------- ------ Olur10 0.2885 0.0155 -0.0327 -0.0097 22774 switches Olur11 0.7817 0.0096 0.0487 0.0823 32358 switches Olur12 0.9079 0.0055 0.0020 -0.0093 77677 switches Olur13 0.3355 0.0108 0.0035 0.0086 83321 switches Olur15 0.3031 0.0111 -0.0073 -0.0015 68376 switches Olur19 0.3528 0.0109 0.0040 0.0092 82550 switches All (Fisher's method): Chi2 : 9.8276 Df : 12.0000 Prob : 0.6311 Pop : NFH-2016 ----------------------------------------- Fis estimates --------------- locus P-val S.E. W&C R&H Steps ----------- ------- ------- ------- ------- ------ Olur10 0.6622 0.0165 0.0214 0.0184 19734 switches Olur11 0.7706 0.0105 0.0563 0.0169 29904 switches Olur12 0.8312 0.0070 -0.0981 -0.0520 86460 switches Olur13 0.1129 0.0057 -0.0129 -0.0057 112034 switches Olur15 0.0795 0.0057 -0.0135 -0.0123 93919 switches Olur19 0.1101 0.0065 -0.0012 0.0012 89921 switches All (Fisher's method): Chi2 : 15.5530 Df : 12.0000 Prob : 0.2126 ========================================== All locus, all populations ========================================== All (Fisher's method) : Chi2 : 25.3806 Df : 24.0000 Prob : 0.3853 #### Assess whether any loci are linked ```{r} test_LD(inputFile = "Data/Oly2016NFH+2017NFW_Merged.txt", outputFile = "Analyses/NF-LD.txt", dememorization = 10000, batches = 100, iterations = 1000, verbose = TRUE) ``` #### Resulting file: ["NF-LD.txt"](https://raw.githubusercontent.com/laurahspencer/O.lurida_genetics/master/Analyses/NF-LD.txt) #### Interesting, results from this Linkage Disequilibrium test indicate that there are, in fact, linked loci: Pop Locus#1 Locus#2 P-Value S.E. Switches ---------- ------- ------- -------- -------- -------- NFW-2017 Olur10 Olur11 0.32418 0.045606 216 NFW-2017 Olur10 Olur12 0.372000 0.048411 65 NFW-2017 Olur11 Olur12 1.000000 0.000000 435 NFW-2017 Olur10 Olur13 1.000000 0.000000 42 NFW-2017 Olur11 Olur13 0.990730 0.006526 279 NFW-2017 Olur12 Olur13 0.144450 0.034977 90 NFW-2017 Olur10 Olur15 1.000000 0.000000 33 NFW-2017 Olur11 Olur15 0.975760 0.014058 232 NFW-2017 Olur12 Olur15 1.000000 0.000000 96 NFW-2017 Olur13 Olur15 0.065770 0.024413 35 NFW-2017 Olur10 Olur19 1.000000 0.000000 35 NFW-2017 Olur11 Olur19 1.000000 0.000000 229 NFW-2017 Olur12 Olur19 1.000000 0.000000 91 NFW-2017 Olur13 Olur19 0.060010 0.023868 16 NFW-2017 Olur15 Olur19 0.000000 0.000000 28 <------ Wild 15 & 19 linked NFH-2016 Olur10 Olur11 1.000000 0.000000 154 NFH-2016 Olur10 Olur12 0.208400 0.040527 72 NFH-2016 Olur11 Olur12 0.923220 0.024857 520 NFH-2016 Olur10 Olur13 1.000000 0.000000 45 NFH-2016 Olur11 Olur13 0.726700 0.043512 284 NFH-2016 Olur12 Olur13 0.000000 0.000000 151 <------ Hatchery 12 & 13 linked NFH-2016 Olur10 Olur15 1.000000 0.000000 36 NFH-2016 Olur11 Olur15 0.715690 0.043815 301 NFH-2016 Olur12 Olur15 0.049120 0.021532 165 NFH-2016 Olur13 Olur15 0.000000 0.000000 68 <------ Hatchery 13 & 15 linked NFH-2016 Olur10 Olur19 1.000000 0.000000 40 NFH-2016 Olur11 Olur19 0.716270 0.042213 282 NFH-2016 Olur12 Olur19 0.000000 0.000000 149 <------ Hatchery 12 & 19 linked NFH-2016 Olur13 Olur19 0.000000 0.000000 54 <------ Hatchery 13 & 19 linked NFH-2016 Olur15 Olur19 0.000000 0.000000 44 <------ Hatchery 15 & 19 linked P-value for each locus pair across all populations (Fisher's method) ----------------------------------------------------- Locus pair Chi2 df P-Value -------------------- -------- --- -------- Olur10 & Olur11 2.252913 4 0.689355 Olur10 & Olur12 5.114315 4 0.275768 Olur11 & Olur12 0.159775 4 0.996974 Olur10 & Olur13 0.000000 4 1.000000 Olur11 & Olur13 0.657110 4 0.956511 Olur12 & Olur13 >35.7544134 <0.000000 <------ 12 & 13 linked Olur10 & Olur15 0.000000 4 1.000000 Olur11 & Olur15 0.718094 4 0.949079 Olur12 & Olur15 6.026978 4 0.197143 Olur13 & Olur15 >37.3279524 <0.000000 <------ 13 & 15 linked Olur10 & Olur19 0.000000 4 1.000000 Olur11 & Olur19 0.667396 4 0.955288 Olur12 & Olur19 >31.8847694 <0.000002 <------ 12 & 19 linked Olur13 & Olur19 >37.5112584 <0.000000 <------ 13 & 19 linked Olur15 & Olur19 >63.7695394 <0.000000 <------ 15 & 19 linked #### Assess for null alleles ```{r} nulls(inputFile = "Data/Oly2016NFH+2017NFW_Merged.txt", outputFile = "Analyses/NF-null.txt", nullAlleleMethod = "B96", CIcoverage = 0.95, verbose = TRUE) ``` #### Resulting file: ["NF-null.txt"](https://raw.githubusercontent.com/laurahspencer/O.lurida_genetics/master/Analyses/NF-null.txt) #### Null allele frequences low; will compare with results from MicroChecker to confirm. (Locus by population) table of estimated null allele frequencies ================================================================ Locus: Populations (! names truncated to 6 characters): NFW-20 NFH-20 ----------------------------------------------------- Olur10 0.0000 0.0079 Olur11 0.0125 0.0159 Olur12 0.0000 0.0000 Olur13 0.0000 0.0000 Olur15 0.0000 0.0000 Olur19 0.0000 0.0000 ================================================================ Confidence intervals for null allele frequencies ================================================= Frequency 0.0250 0.9750 Locus Population estimate bound bound ------------------------------------------------- Olur10 NFW-2017 0.0000 (No info for CI) NFH-2016 0.0079 0.0000 0.0422 Olur11 NFW-2017 0.0125 0.0000 0.0694 NFH-2016 0.0159 0.0000 0.0724 Olur12 NFW-2017 0.0000 (No info for CI) NFH-2016 0.0000 (No info for CI) Olur13 NFW-2017 0.0000 (No info for CI) NFH-2016 0.0000 (No info for CI) Olur15 NFW-2017 0.0000 (No info for CI) NFH-2016 0.0000 (No info for CI) Olur19 NFW-2017 0.0000 (No info for CI) NFH-2016 0.0000 (No info for CI) ================================================= #### Exact conditional contingency-table test or genotypic differentiation. Assesses the distribution of diploid genotypes in the various populations. The null hypothesis tested is Ho: "genotypes are drawn from the same distribution in all populations" ```{r} test_diff(inputFile = "Data/Oly2016NFH+2017NFW_Merged.txt", outputFile = "Analyses/NF-Diff.txt", genic=FALSE, pairs=TRUE, dememorization = 10000, batches = 100, iterations = 1000, verbose = TRUE) ``` #### Resulting file: ["NF-Diff.txt"](https://raw.githubusercontent.com/laurahspencer/O.lurida_genetics/master/Analyses/NF-Diff.txt) #### Results indicate that genotypes are drawn from the same distribution, as P>>.01 for all loci. Locus Population pair P-Value S.E. Switches ----------- --------------------- ------- ------- -------- Olur10 NFH-2016 & NFW-2017 0.32784 0.01552 27018 Olur11 NFH-2016 & NFW-2017 0.54191 0.01155 36148 Olur12 NFH-2016 & NFW-2017 0.67662 0.01140 38491 Olur13 NFH-2016 & NFW-2017 0.08560 0.00745 32174 Olur15 NFH-2016 & NFW-2017 0.06505 0.00713 32414 Olur19 NFH-2016 & NFW-2017 0.15165 0.01146 32502 P-value for each population pair across all loci (Fisher's method) ----------------------------------------------------- Population pair Chi2 df P-Value -------------------- -------- --- -------- NFW-2017 & NFH-2016 18.39076 12 0.104331 #### Calculate Fst for each population, which is a measure of genetic structure (developed by Sewall Wright, 1969, 1978), and is related to statistical analysis of variance (ANOVA). FST is the proportion of the total genetic variance contained in a subpopulation (the S subscript) relative to the total genetic variance (the T subscript). Values can range from 0 to 1. High FST implies a considerable degree of differentiation among populations. ```{r} Fst(inputFile = "Data/Oly2016NFH+2017NFW_Merged.txt", outputFile = "Analyses/NF-Fst.txt", sizes=F, pairs=TRUE, dataType="Diploid", verbose = TRUE) ``` #### Resulting file: ["NF-Fst.txt"](https://raw.githubusercontent.com/laurahspencer/O.lurida_genetics/master/Analyses/NF-Fst.txt) #### Values are very close to zero, which indicates that there is little genetic differentiation among wild and hatchery populations. Indices for populations: ---- ------------- 1 NFW-2017 2 NFH-2016 ---------------------- Estimates for each locus: ======================== Locus: Olur10 --------------------------------- pop 1 2 -0.0007 Locus: Olur11 --------------------------------- pop 1 2 -0.0027 Locus: Olur12 --------------------------------- pop 1 2 -0.0020 Locus: Olur13 --------------------------------- pop 1 2 0.0030 Locus: Olur15 --------------------------------- pop 1 2 0.0036 Locus: Olur19 --------------------------------- pop 1 2 0.0027 Estimates for all loci (diploid): ========================= pop 1 2 0.0008 #### Generate stats on allelic diversity. Interpretation TBD. ```{r} genedivFis(inputFile="Data/Oly2016NFH+2017NFW_Merged.txt", sizes=FALSE, outputFile = "Analyses/NF-DivFis.txt", dataType = "Diploid", verbose=interactive()) ``` #### Resulting file: ["NF-DivFis.txt"](https://raw.githubusercontent.com/laurahspencer/O.lurida_genetics/master/Analyses/NF-DivFis.txt) ```{r} sessionInfo() ```