Last updated: 2020-01-20

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Setup

library(ngsReports)
library(tidyverse)
library(magrittr)
library(edgeR)
library(limma)
library(AnnotationHub)
library(ensembldb)
library(scales)
library(pander)
library(cowplot)
library(corrplot)
if (interactive()) setwd(here::here())
theme_set(theme_bw())
panderOptions("big.mark", ",")
panderOptions("table.split.table", Inf)
panderOptions("table.style", "rmarkdown")
twoCols <- c(rgb(0.8, 0.1, 0.1), rgb(0.2, 0.2, 0.8))
ah <- AnnotationHub() %>%
    subset(species == "Danio rerio") %>%
    subset(rdataclass == "EnsDb")
ensDb <- ah[["AH74989"]]
grGenes <- genes(ensDb)
grTrans <- transcripts(ensDb)
trLengths <- exonsBy(ensDb, "tx") %>%
    width() %>%
    vapply(sum, integer(1))
mcols(grTrans)$length <- trLengths[names(grTrans)]
rRnaTrans <- grTrans %>%
    subset(grepl("rRNA", tx_biotype)) %>%
    mcols() %>%
    .[["tx_id_version"]]

In order to perform adequate QC, an EnsDb object was obtained for Ensembl release 98 using the AnnotationHub package. This provided the GC content and length for each of the 65,905 transcripts contained in that release.

samples <- read_csv("data/samples.csv") %>%
    mutate(reads = str_extract(fqName, "R[12]")) %>%
    dplyr::rename(Filename = fqName)
labels <- structure(
    samples$sampleID,
    names = samples$Filename
)

Metadata for each fastq file was also loaded. Reads were provided as paired-end reads, with \(n=4\) samples for each genotype.

Raw Data

Library Sizes

rawFqc <- list.files(
    path = "data/0_rawData/FastQC/",
    pattern = "zip",
    full.names = TRUE
    ) %>%
    FastqcDataList()

Library Sizes for the raw, unprocessed dataset ranged between 27,979,654 and 37,144,975 reads.

r1 <- grepl("R1", fqName(rawFqc))
plotReadTotals(rawFqc[r1], labels = labels[r1], barCols = twoCols)
*Library Sizes for each sample before any processing was undertaken.*

Library Sizes for each sample before any processing was undertaken.

GC Content

As this was a total RNA dataset, GC content will provide a clear marker of the success of rRNA depletion. This was plotted, with all R2 reads showing a far greater spike in GC content at 81%, which is likely due to incomplete rRNA depletion. In particular, the mutant samples appeared most significantly affected raising possibility that overall GC content may be affected in these samples.

gcPlots <- list(
    r1 = plotGcContent(
        x = rawFqc[r1], 
        labels = labels[r1],
        plotType = "line",
        gcType = "Transcriptome",
        species = "Drerio"
    ),
    r2 = plotGcContent(
        x = rawFqc[!r1], 
        labels = labels[!r1],
        plotType = "line",
        gcType = "Transcriptome",
        species = "Drerio"
    )
)
lg <- get_legend(gcPlots$r2) 
plot_grid(
    plotlist = list(
        r1 = gcPlots$r1 + 
            ggtitle("R1: GC Distribution", subtitle = c()) +
            theme(legend.position = "none"),
        r2 = gcPlots$r2 + 
            ggtitle("R2: GC Distribution", subtitle = c()) +
            theme(legend.position = "none"),
        lg = lg),
    nrow = 1,
    rel_widths = c(1,1,1)
)
*GC content for R1 and R2 reads. Notably, the R2 reads had clearer spikes in GC content above 70%*

GC content for R1 and R2 reads. Notably, the R2 reads had clearer spikes in GC content above 70%

rawGC <- getModule(rawFqc, "Per_sequence_GC") %>% 
    group_by(Filename) %>% 
    mutate(Freq = Count / sum(Count)) %>%
    dplyr::filter(GC_Content > 70) %>% 
    summarise(Freq = sum(Freq)) %>% 
    arrange(desc(Freq)) %>%
    left_join(samples) 
rawGC %>%
    ggplot(aes(sampleID, Freq, fill = reads)) +
    geom_bar(stat = "identity", position = "dodge") +
    facet_wrap(~genotype, scales = "free_x") +
    scale_y_continuous(labels = percent) +
    scale_fill_manual(values = twoCols) +
    labs(x = "Sample", y = "Percent of Total")
*Percentages of each library which contain >70% GC. Using the known theoretical distribution, this should be 0.19% of the total library.*

Percentages of each library which contain >70% GC. Using the known theoretical distribution, this should be 0.19% of the total library.

Over-represented Sequences

The top 30 Overrepresented sequences were analysed using blastn and most were found to be mitochondrial rRNA sequences.

getModule(rawFqc, "Overrep") %>% 
    group_by(Sequence, Possible_Source) %>% 
    summarise(`Found In` = n(), `Highest Percentage` = max(Percentage)) %>% 
    arrange(desc(`Highest Percentage`), desc(`Found In`)) %>% 
    ungroup() %>% 
    dplyr::slice(1:30) %>%
    mutate(`Highest Percentage` = percent_format(0.01)(`Highest Percentage`/100)) %>%
    pander(
        justify = "llrr",
        caption = paste(
            "*Top", nrow(.),"Overrepresented sequences.",
            "The number of samples they were found in is shown,",
            "along with the percentage of the most 'contaminated' sample.*"
            )
    )
Top 30 Overrepresented sequences. The number of samples they were found in is shown, along with the percentage of the most ‘contaminated’ sample.
Sequence Possible_Source Found In Highest Percentage
GTGGGTTCAGGTAATTAATTTAAAGCTACTTTCGTGTTTGGGCCTCTAGC No Hit 12 1.72%
CTGGGGGAGCGGCCGCCCCGCGGCGCCCCCTCTCGTTCCCGTCTCCGGAG No Hit 10 1.69%
CCGCTGTATTACTCAGGCTGCACTGCAGTGTCTATTCACAGGCGCGATCC No Hit 12 1.32%
GGCCCGGCGCACGTCCAGAGTCGCCGCCGCACACCGCAGCGCATCCCCCC No Hit 9 1.31%
CTCCTGAAAAGGTTGTATCCTTTGTTAAAGGGGCTGTACCCTCTTTAACT No Hit 11 1.11%
GGTTCAGGTAATTAATTTAAAGCTACTTTCGTGTTTGGGCCTCTAGCATC No Hit 12 1.09%
GGGGTGTACGAAGCTGAACTTTTATTCATCTCCCAGACAACCAGCTATTG No Hit 12 1.07%
GGCCCGGCGCACGTCCAGAGTCGCCGCCGCGCACCGCAGCGCATCCCCCC No Hit 10 1.03%
CGAGAGGCTCTAGTTGATATACTACGGCGTAAAGGGTGGTTAAGGAACAA No Hit 12 1.01%
GGGGGAGCGGCCGCCCCGCGGCGCCCCCTCTCGTTCCCGTCTCCGGAGCG No Hit 9 0.87%
CCTCCTTCAAGTATTGTTTCATGTTACATTTTCGTATATTCTGGGGTAGA No Hit 12 0.82%
CCCGCTGTATTACTCAGGCTGCACTGCAGTGTCTATTCACAGGCGCGATC No Hit 12 0.79%
GTTCAGGTAATTAATTTAAAGCTACTTTCGTGTTTGGGCCTCTAGCATCT No Hit 12 0.79%
GGGTTCAGGTAATTAATTTAAAGCTACTTTCGTGTTTGGGCCTCTAGCAT No Hit 12 0.78%
CGGGTCGGGTGGGTGGCCGGCATCACCGCGGACCTCGGGCGCCCTTTTGG No Hit 12 0.73%
GGGCCTCTAGCATCTAAAAGCGTATAACAGTTAAAGGGCCGTTTGGCTTT No Hit 11 0.68%
CAGTGGCGTGCGCCTGTAATCCAAGCTACTGGGAGGCTGAGGCTGGCGGA No Hit 11 0.64%
CGGGTCGGGTGGGTAGCCGGCATCACCGCGGACCTCGGGCGCCCTTTTGG No Hit 12 0.57%
CTTAGACGACCTGGTAGTCCAAGGCTCCCCCAGGAGCACCATATCGATAC No Hit 11 0.54%
AGCTGGGGAGATCCGCGAGAAGGGCCCGGCGCACGTCCAGAGTCGCCGCC No Hit 11 0.53%
GGCCTCTAGCATCTAAAAGCGTATAACAGTTAAAGGGCCGTTTGGCTTTA No Hit 10 0.53%
CAGCCTATTTAACTTAGGGCCAACCCGTCTCTGTGGCAATAGAGTGGGAA No Hit 12 0.51%
GGGTGGGTGGCCGGCATCACCGCGGACCTCGGGCGCCCTTTTGGACGTGG No Hit 10 0.50%
GGGAGCGGCCGCCCCGCGGCGCCCCCTCTCGTTCCCGTCTCCGGAGCGCG No Hit 9 0.49%
CTGGGAGATGAATAAAAGTTCAGCTTCGTACACCCCAAATTAAAAAATTA No Hit 10 0.48%
GCCTATTTAACTTAGGGCCAACCCGTCTCTGTGGCAATAGAGTGGGAAGA No Hit 12 0.47%
GGTCGGGTGGGTGGCCGGCATCACCGCGGACCTCGGGCGCCCTTTTGGAC No Hit 11 0.45%
CCCCCGAACCCTTCCAAGCCGAACCGGAGCCGGTCGCGGCGCACCGCCGA No Hit 10 0.45%
GTCGGGTGGGTGGCCGGCATCACCGCGGACCTCGGGCGCCCTTTTGGACG No Hit 10 0.43%
GCCCACTACGACAACGTGTTTTGTAAATTATGATCTTTATTCTCCTGAAA No Hit 10 0.43%

Trimming

trimFqc <- list.files(
    path = "data/1_trimmedData/FastQC/", 
    pattern = "zip", 
    full.names = TRUE
) %>%
    FastqcDataList()
trimStats <- readTotals(rawFqc) %>%
    dplyr::rename(Raw = Total_Sequences) %>%
    left_join(readTotals(trimFqc), by = "Filename") %>%
    dplyr::rename(Trimmed = Total_Sequences) %>%
    dplyr::filter(grepl("R1", Filename)) %>%
    mutate(
        Discarded = 1 - Trimmed/Raw,
        Retained = Trimmed / Raw
    )

After adapter trimming between 4.16% and 5.08% of reads were discarded. No other improvement was noted.

Aligned Data

Trimmed reads were:

  1. aligned to the reference genome using STAR 2.7.0d and summarised to each gene using featureCounts. These counts were to be used for all gene-level analysis
  2. aligned using kallisto to a modified transcriptome which contained the psen2S4Ter sequence as an ‘alternative’ transcript of psen2. This data was used for genotype checking, and assessing any GC bias in the data. Whilst transcript-level information is included for rRNA genes in the EnsDb object, these transcripts are excluded by default from the reference transcriptome provided by Ensembl.

Genotype Checking

cpm(kallisto$counts) %>%
    .[c("ENSDART00000006381.8", "psen2S4Ter"),] %>%
    as.data.frame() %>%
    rownames_to_column("psen2") %>%
    pivot_longer(cols = contains("kall"), names_to = "Sample", values_to = "CPM") %>%
    mutate(
        Sample = basename(Sample),
        psen2 = case_when(
            psen2 == "psen2S4Ter" ~ "S4Ter",
            psen2 != "psen2S4Ter" ~ "WT"
        )
    ) %>%
    left_join(samples, by = c("Sample" = "sampleName")) %>%
    ggplot(aes(sampleID, CPM, fill = psen2)) +
    geom_bar(stat = "identity") +
    scale_fill_manual(values = twoCols) +
    facet_wrap(~genotype, nrow = 1, scales = "free_x")
*CPM obtained for both WT and S4Ter mutant transcripts of psen2*

CPM obtained for both WT and S4Ter mutant transcripts of psen2

As all samples demonstrated the expected expression patterns, no mislabelling was detected in this dataset.

GC Content

gcInfo <- kallisto$counts %>%
    as.data.frame() %>%
    rownames_to_column("tx_id") %>%
    dplyr::filter(
        tx_id != "psen2S4Ter"
    ) %>%
    as_tibble() %>%
    pivot_longer(
        cols = contains("kall"), 
        names_to = "sampleName", 
        values_to = "counts"
    ) %>%
    dplyr::filter(
        counts > 0
    ) %>%
    mutate(
        sampleName = basename(sampleName),
        tx_id_version = tx_id,
        tx_id = str_replace(tx_id, "(ENSDART[0-9]+).[0-9]+", "\\1")
    ) %>%
    left_join(
        mcols(grTrans) %>% as.data.frame()
    ) %>%
    dplyr::select(
        ends_with("id"), sampleName, counts, gc_content, length
    ) %>%
    split(f = .$sampleName) %>%
    lapply(function(x){
        DataFrame(
            gc = Rle(x$gc_content/100, x$counts),
            logLen = Rle(log10(x$length), x$counts)
        )
    }
    ) 
gcSummary <- gcInfo %>%
    vapply(function(x){
        c(mean(x$gc), sd(x$gc), mean(x$logLen), sd(x$logLen))
        }, numeric(4)
        ) %>%
    t() %>%
    set_colnames(
        c("mn_gc", "sd_gc", "mn_logLen", "sd_logLen")
    ) %>%
    as.data.frame() %>%
    rownames_to_column("sampleName") %>%
    as_tibble() %>%
    left_join(dplyr::filter(samples, reads == "R1")) %>%
    dplyr::select(starts_with("sample"), genotype, contains("_"))
gcCors <- rawGC %>%
    dplyr::filter(reads == "R1") %>%
    dplyr::select(starts_with("sample"), genotype, Freq, reads) %>%
    left_join(gcSummary) %>%
    dplyr::select(Freq, mn_gc, mn_logLen) %>%
    cor()

The initial QC steps identified an unusually large proportion of reads with >70% GC content. As such an exploration of any residual impacts this may have on the remainder of the library was performed.

A run length encoded (RLE) vector was formed for each sample taking the number of reads for each transcript as the run lengths, and both the GC content and length of each transcript as the values. Transcript lengths were transformed to the log10 scale due to the wide variety of lengths contained in the transcriptome.

From these RLEs, the mean GC and mean length was calculated for each sample, and these values were compared to the proportion of raw reads with > 70% GC, taking these values from the R1 libraries only.

  • A correlation of -30% was found between the mean GC content after summarisation to transcript-level counts and the proportion of the original sequence libraries with >70% GC. This makes intuitive sense and suggests that the amount of high-GC reads in the initial libraries, as representative of incomplete rRNA removal, negatively biases the GC composition of the non-ribosomal RNA component of the library.
  • A correlation of -80% was found was found between the mean transcript length of the libraries after summarisation to transcript-level counts and the proportion of the original sequence libraries with >70% GC. This is far less obvious mechanistically, but suggests that incomplete rRNA removal also biases the fragmentation or length selection process.

Given the more successful rRNA-removal in the wild-type samples, this dataset is likely to contain significant GC and Length bias which is non-biological in origin.

a <- gcSummary %>%
    left_join(rawGC) %>%
    dplyr::filter(reads == "R1") %>%
    ggplot(aes(Freq, mn_logLen)) +
    geom_point(aes(colour = genotype), size = 3) +
    geom_smooth(method = "lm") +
    scale_shape_manual(values = c(19, 1)) +
    labs(
        x = "Proportion of initial library with > 70% GC",
        y = "Mean log(length)",
        colour = "Genotype"
    ) 
b <- gcSummary %>%
    left_join(rawGC) %>%
        dplyr::filter(reads == "R1") %>%
    ggplot(aes(Freq, mn_gc)) +
    geom_point(aes(colour = genotype), size = 3) +
    geom_smooth(method = "lm") +
    scale_shape_manual(values = c(19, 1)) +
    scale_y_continuous(labels = percent) +
    labs(
        x = "Proportion of initial library with > 70% GC",
        y = "Mean GC Content",
        colour = "Genotype"
    ) 
plot_grid(
    a + theme(legend.position = "none"),
    b + theme(legend.position = "none"),
    get_legend(b),
    nrow = 1,
    rel_widths = c(4,4,1)
)
*Comparison of bias introduced by incomplete rRNA removal. Regression lines are shown along with standard error bands for each comparison.*

Comparison of bias introduced by incomplete rRNA removal. Regression lines are shown along with standard error bands for each comparison.

PCA

transDGE <- kallisto$counts %>%
    set_colnames(basename(colnames(.))) %>%
    divide_by(kallisto$annotation$Overdispersion) %>%
    .[rowSums(.) > 4,] %>%
    DGEList(
        samples = tibble(
            sampleName = colnames(.)
        ) %>%
            left_join(samples) %>%
            dplyr::select(sampleName, sample = sampleID, genotype) %>%
            distinct(sampleName, .keep_all = TRUE)
    ) 
pca <- cpm(transDGE, log = TRUE) %>%
    t() %>%
    prcomp()
pca$x %>%
    as.data.frame() %>%
    rownames_to_column("sampleName") %>%
    left_join(gcSummary) %>%
    as_tibble() %>% 
    left_join(
        dplyr::filter(rawGC, reads == "R1")
        ) %>% 
    dplyr::select(
        PC1, PC2, PC3, 
        Mean_GC = mn_gc, 
        Mean_Length = mn_logLen, 
        Initial_GC70 = Freq,
        genotype
    ) %>% 
    mutate(genotype = as.numeric(as.factor(genotype))) %>%
    cor() %>% 
    corrplot(
        type = "lower", 
        diag = FALSE, 
        addCoef.col = 1, addCoefasPercent = TRUE
    )
*Correlations between the first three principal components and measured variables. Genotypes were converted to an ordered categorical variable for the purposes of visualisation*

Correlations between the first three principal components and measured variables. Genotypes were converted to an ordered categorical variable for the purposes of visualisation

a <- pca$x %>%
    as.data.frame() %>%
    rownames_to_column("sampleName") %>%
    left_join(
        dplyr::filter(rawGC, reads == "R1")
    ) %>%
    as_tibble() %>%
    ggplot(aes(PC1, PC2, colour = genotype, size = Freq)) +
    geom_point() +
    labs(
        x = paste0("PC1 (", percent(summary(pca)$importance["Proportion of Variance","PC1"]),")"),
        y = paste0("PC2 (", percent(summary(pca)$importance["Proportion of Variance","PC2"]),")"),
        size = "Raw GC > 70%",
        colour = "Genotype"
    )
b <- pca$x %>%
    as.data.frame() %>%
    rownames_to_column("sampleName") %>%
    left_join(gcSummary) %>%
    left_join(
        dplyr::filter(rawGC, reads == "R1")
    ) %>%
    as_tibble() %>%
    ggplot(aes(PC1, mn_gc)) +
    geom_point(aes(colour = genotype,  size = Freq)) +
    geom_smooth(method = "lm") +
    scale_y_continuous(labels = percent) +
    labs(
        x = paste0("PC1 (", percent(summary(pca)$importance["Proportion of Variance","PC1"]),")"),
        y = "Mean GC",
        size = "Raw GC > 70%",
        colour = "Genotype"
    )
plot_grid(
    plot_grid(
        a + theme(legend.position = "none"), 
        b + theme(legend.position = "none")
    ),
    get_legend(b + theme(legend.position = "bottom")),
    nrow = 2, 
    rel_heights = c(4,1)
)
*PCA plot showing genotype, initial GC content > 70% and mean GC content after summarisation to transcript-level.*

PCA plot showing genotype, initial GC content > 70% and mean GC content after summarisation to transcript-level.


devtools::session_info()
─ Session info ───────────────────────────────────────────────────────────────
 setting  value                       
 version  R version 3.6.2 (2019-12-12)
 os       Ubuntu 18.04.3 LTS          
 system   x86_64, linux-gnu           
 ui       X11                         
 language en_AU:en                    
 collate  en_AU.UTF-8                 
 ctype    en_AU.UTF-8                 
 tz       Australia/Adelaide          
 date     2020-01-20                  

─ Packages ───────────────────────────────────────────────────────────────────
 package                * version   date       lib source        
 AnnotationDbi          * 1.48.0    2019-10-29 [2] Bioconductor  
 AnnotationFilter       * 1.10.0    2019-10-29 [2] Bioconductor  
 AnnotationHub          * 2.18.0    2019-10-29 [2] Bioconductor  
 askpass                  1.1       2019-01-13 [2] CRAN (R 3.6.0)
 assertthat               0.2.1     2019-03-21 [2] CRAN (R 3.6.0)
 backports                1.1.5     2019-10-02 [2] CRAN (R 3.6.1)
 Biobase                * 2.46.0    2019-10-29 [2] Bioconductor  
 BiocFileCache          * 1.10.2    2019-11-08 [2] Bioconductor  
 BiocGenerics           * 0.32.0    2019-10-29 [2] Bioconductor  
 BiocManager              1.30.10   2019-11-16 [2] CRAN (R 3.6.1)
 BiocParallel             1.20.1    2019-12-21 [2] Bioconductor  
 BiocVersion              3.10.1    2019-06-06 [2] Bioconductor  
 biomaRt                  2.42.0    2019-10-29 [2] Bioconductor  
 Biostrings               2.54.0    2019-10-29 [2] Bioconductor  
 bit                      1.1-15.1  2020-01-14 [2] CRAN (R 3.6.2)
 bit64                    0.9-7     2017-05-08 [2] CRAN (R 3.6.0)
 bitops                   1.0-6     2013-08-17 [2] CRAN (R 3.6.0)
 blob                     1.2.0     2019-07-09 [2] CRAN (R 3.6.1)
 broom                    0.5.3     2019-12-14 [2] CRAN (R 3.6.2)
 callr                    3.4.0     2019-12-09 [2] CRAN (R 3.6.2)
 cellranger               1.1.0     2016-07-27 [2] CRAN (R 3.6.0)
 cli                      2.0.1     2020-01-08 [2] CRAN (R 3.6.2)
 cluster                  2.1.0     2019-06-19 [2] CRAN (R 3.6.1)
 colorspace               1.4-1     2019-03-18 [2] CRAN (R 3.6.0)
 corrplot               * 0.84      2017-10-16 [2] CRAN (R 3.6.0)
 cowplot                * 1.0.0     2019-07-11 [2] CRAN (R 3.6.1)
 crayon                   1.3.4     2017-09-16 [2] CRAN (R 3.6.0)
 curl                     4.3       2019-12-02 [2] CRAN (R 3.6.2)
 data.table               1.12.8    2019-12-09 [2] CRAN (R 3.6.2)
 DBI                      1.1.0     2019-12-15 [2] CRAN (R 3.6.2)
 dbplyr                 * 1.4.2     2019-06-17 [2] CRAN (R 3.6.0)
 DelayedArray             0.12.2    2020-01-06 [2] Bioconductor  
 desc                     1.2.0     2018-05-01 [2] CRAN (R 3.6.0)
 devtools                 2.2.1     2019-09-24 [2] CRAN (R 3.6.1)
 digest                   0.6.23    2019-11-23 [2] CRAN (R 3.6.1)
 dplyr                  * 0.8.3     2019-07-04 [2] CRAN (R 3.6.1)
 edgeR                  * 3.28.0    2019-10-29 [2] Bioconductor  
 ellipsis                 0.3.0     2019-09-20 [2] CRAN (R 3.6.1)
 ensembldb              * 2.10.2    2019-11-20 [2] Bioconductor  
 evaluate                 0.14      2019-05-28 [2] CRAN (R 3.6.0)
 FactoMineR               2.1       2020-01-17 [2] CRAN (R 3.6.2)
 fansi                    0.4.1     2020-01-08 [2] CRAN (R 3.6.2)
 farver                   2.0.3     2020-01-16 [2] CRAN (R 3.6.2)
 fastmap                  1.0.1     2019-10-08 [2] CRAN (R 3.6.1)
 flashClust               1.01-2    2012-08-21 [2] CRAN (R 3.6.1)
 forcats                * 0.4.0     2019-02-17 [2] CRAN (R 3.6.0)
 fs                       1.3.1     2019-05-06 [2] CRAN (R 3.6.0)
 generics                 0.0.2     2018-11-29 [2] CRAN (R 3.6.0)
 GenomeInfoDb           * 1.22.0    2019-10-29 [2] Bioconductor  
 GenomeInfoDbData         1.2.2     2019-11-21 [2] Bioconductor  
 GenomicAlignments        1.22.1    2019-11-12 [2] Bioconductor  
 GenomicFeatures        * 1.38.0    2019-10-29 [2] Bioconductor  
 GenomicRanges          * 1.38.0    2019-10-29 [2] Bioconductor  
 ggdendro                 0.1-20    2016-04-27 [2] CRAN (R 3.6.0)
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 haven                    2.2.0     2019-11-08 [2] CRAN (R 3.6.1)
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[1] /home/steveped/R/x86_64-pc-linux-gnu-library/3.6
[2] /usr/local/lib/R/site-library
[3] /usr/lib/R/site-library
[4] /usr/lib/R/library