Analysis of Bead-level Data using beadarray

Mark Dunning

May 14, 2019

Introduction
beadarray is a package for the pre-processing and analysis of Illumina BeadArray.
The main advantage is being able to read raw data output by Illumina’s scanning
software. Data presented in this form are in the same format regardless of
the assay (i.e expression, genotyping, methylation) being performed. Thus,
beadarray is able to handle all these types of data. Many functions within
beadarray have been written to cope with this flexibility.
The BeadArray technology involves randomly arranged arrays of beads, with
beads having the same probe sequence attached colloquially known as a bead-
type. BeadArrays are combined in parallel on either a rectangular chip (Bead-
Chip) or a matrix of 8 by 12 hexagonal arrays (Sentrix Array Matrix or SAM).
The BeadChip is further divided into strips on the surface known as sections,
with each section giving rise to a different image when scanned by BeadScan.
These images, and associated text files, comprise the raw data for a beadarray
analysis. However, for BeadChips, the number of sections assigned to each bio-
logical sample may vary from 1 on HumanHT12 chips, 2 on HumanWG6 chips
or sometimes ten or more for SNP chips with large numbers of SNPs being
investigated.
This vignette demonstrates the processing of bead-level data using beadarray
using data from the beadarrayExampleData package. A more comprehensive
commentary on the analysis of Illumina BeadArray package is given in the
vignette of BeadArrayUseCases, including other analysis tools that are not part
of beadarray .
library(beadarrayExampleData)
library(beadarray)
data(exampleBLData)
Analysis of Bead-level Data using beadarray

Citing beadarray
If you use beadarray for the analysis or pre-processing of BeadArray data please
cite:
Dunning MJ, Smith ML, Ritchie ME, Tavaré S, beadarray: R classes and
methods for Illumina bead-based data, Bioinformatics, 23(16):2183-2184

1 Asking for help on beadarray

Wherever possible, questions about beadarray should be sent to the Biocon-
ductor mailing list1 . This way, all problems and solutions will be kept in a 1
http://www.bioconductor.o
searchable archive. When posting to this mailing list, please first consult the
posting guide. In particular, state the version of beadarray and R that you are
using2 , and try to provide a reproducible example of your problem. This will 2
This can be done
help us to diagnose the problem. by pasting the out-
put of running the
function session
Info().

2 Reading bead-level data into beadarray

2.1 File formats
The raw images and text files required to perform a bead-level analysis are
produced by Illumina’s BeadScan or iScan software. Usually, it will be necessary
for you to modify BeadScan’s default settings to obtain bead-level data, see
http://www.compbio.group.cam.ac.uk/Resources/illumina.

The command to read bead-level data from the current working directory is
as follows. However, raw data are not included with beadarrayExampleData or
beadarray . See the BeadArrayUseCases package for some example data to try
out this function.
BLData = readIllumina(useImages=FALSE, illuminaAnnotation = "Humanv3")

The useImages argument specifies whether beadarray will read foreground and
background intensities from the TIFF images present in the directory, allow-
ing users to experiment with strategies for image processing. Such strategies
are described in greater detail in the imageProcessing.pdf vignette. In this
example we set useImages=FALSE (often a convenient choice), and locally back-
ground corrected intensities will simply be extracted from the txt files. The

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Analysis of Bead-level Data using beadarray

optical background-correction that is referred to here is done by subtracting the

background pixel intensities surrounding each bead. It should not to be con-
fused with another background correction further along the analysis pipeline,
which may involve negative control beads to account for non-specific binding.
beadarray is able to use some of Illumina’s files during analysis. These include
.locs files, which contain the locations of all beads on an array (not just those
that were decoded), and .sdf files, which contain information about the physi-
cal layout of the chip. In combination, using these files can result in significant
time improvements to the detection of spatial artifacts and add additional infor-
mation to some QA plots. These files are not read automatically, but if present,
the path to these files is stored by beadarray for future use. If the metrics file
generated by BeadScan is present in the directory, it will be read unaltered and
stored.

2.2 A note for those with iScan data

Data from Illumina’s newer iScan system come in a different format to the
previous BeadScan data. The scanner itself is capable of producing higher-
resolution images and there are two images of each array section (along with
two .locs files), which are labeled Swath1 and Swath2. These two images
are of the two halves of the array section, with an overlapping region in the
middle. However, there is only one bead-level text file (with the extension
perBeadFile.txt), with no indication as to which of the two images each entry
comes from. Given this, simply reading the bead-level text file will result in any
function that uses bead locations performing undesirably. However, the read
Illumina function is able to detect that iScan data is present and will advise
the user to run the processSwathData function, which will try and deconvolute
the bead-level data and create two files, one per swath, which can then be read
independently into beadarray.

2.3 Array annotation

The text files produced by the scanning software give a very limited annotation
for each bead that was scanned. All beads are associate with position on the
3
array and a numeric code (ArrayAddress that refers to the decoding oligonu- These decoding se-
quences are required
cleotide sequence attached to the bead3 . A collection of beads with the same
due to the random
ArrayAddress are known as a bead-type and have the same 50-base sequence nature of each ar-
attached. However, the sequence and the region of the genome that it targets ray. However, the
cannot be inferred from bead-level data alone. The mapping between ArrayAd- sequences them-
dress and hybridization sequence is provided on Illumina’s FTP site in a series selves have never
been disclosed

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Analysis of Bead-level Data using beadarray

of flat-files and we have built Bioconductor packages that can be accessed from
within beadarray. In order that the correct mappings are performed, users must
specify an annotation name for their data, which requires knowing the organism
being investigated and annotation revision number (e.g. Humanv4, Humanv3,
Humanv2, Humanv1, Mousev2, Mousev1p1, Mousev1 or Ratv1). The sugges
tAnnotation function may be used if you are unsure of which string to use.
This checks the overlap between the bead IDs found in the data with a col-
lection of IDs extracted from Illumina’s annotation files. For the example data
stored with the beadarrayExampleData, the suggested annotation is Humanv3.
Provided that the illuminaHumanv3.db package is present, beadarray will be
able to annotate the beadarrayExampleData object.
suggestAnnotation(exampleBLData,verbose=TRUE)

## Percentage of overlap with IDs on this array and known expression platforms
## HUMANREF8_V3_0_R1_11282963_A_WGDASL HumanHT12_V3_0_R3_11283641_A
## 48.78621 97.16521
## HumanHT12 V4 0 R1 15002873_B
_ _ _ _ HumanHT12 V4 0 R2 15002873_B
_ _ _ _
## 83.70782 84.05715
## HumanHT12 V4 0 R2 15002873 B_WGDASL
_ _ _ _ _ HumanRef8_V1
## 54.68237 37.33872
## HumanRef8_V2_0_R2_11223162_A HumanRef8_V2_0_R4_11223162_A
## 38.88553 38.88470
## HumanRef8_V3_0_R0_11282963_A HumanRef8_V3_0_R3_11282963_A
## 48.78621 48.78621
## HumanWG6_V1 HumanWG6 V2 0 R2 11223189_A
_ _ _ _
## 37.33872 81.32701
## HumanWG6 V2 0 R4 11223189_A
_ _ _ _ HumanWG6 V2 11223189_B
_ _
## 81.32619 81.32619
## HumanWG6_V3_0_R3_11282955_A MouseRef8_V1
## 97.16521 36.67984
## MouseRef8_V1_1_R4_11234312_A MouseRef8_V2_0_R3_11278551_A
## 38.10629 43.77615
## MouseWG6_V1 MouseWG6 V1 1 R4 11234304_A
_ _ _ _
## 36.67984 38.10629
## MouseWG6_V1_B MouseWG6 V2 0 R3 11278593_A
_ _ _ _
## 36.58768 78.45060
## RatRef12 V1 0 R5 11222119_A
_ _ _ _
## 36.06644
## [1] "Humanv3"

annotation(exampleBLData) <-"Humanv3"

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Analysis of Bead-level Data using beadarray

The verbose output of suggestAnnotation shows high overlap between the Ar-
rayAddress IDs in the exampleBLData object and the ArrayAddress IDs in the of-
ficial annotation files HumanHT12_V3_0_R3_11283641_A and HumanWG6_V3_0_R3_11282955_A.
However, both HT12 and WG6 arrays have the same probe sequences on them
and the difference is the number of sections on a chip. Hence, we can assign
the Humanv3 label to data from either platform.

3 The beadLevelData class

Once imported, the bead-level data are stored in an object of class beadLevel-
Data. This class can handle raw data from both single channel and two-colour
BeadArrays. Due to the random nature of the technology, each array generally
has a variable number of rows of intensity data, and we use an R environment
variable to store this information in a memory efficient way.
The beadLevelData class contains a number of slots useful for describing Illu-
mina data. The data that have been extracted from the text files are found in
the beadData slot. This can be thought of as a list, which can be indexed by
name or a numeric value representing a particular array-section. A data frame
holds the data for that array-section, with the number of rows being the number
of beads on the section. For convenience, the function getBeadData is used to
access data held in the beadData slot. The function insertBeadData can be
used to assign new data to this slot.
Data types with one value per array-section can be stored in the sectionData
slot. For instance, any metrics information present in the directory used by
readIllumina will be stored here. This is also a convenient place to store any
QC information derived during the pre-processing of the data, as we will see.
The numeric identifiers for the bead-types in the beadLevelData are known as
ArrayAddress IDs in Illumina’s annotation files. For downstream analysis it is
convenient to convert these into the form ILMN_... used in most annotation
packages. Mapping objects to convert these IDs are supplied with beadarray in
the extdata directory, but this conversion may be performed automatically if
the annotation of the beadLevelData object is known. For two-channel data,
the intensities from the Red channel and associated coordinates are also stored
in the object.
class(exampleBLData)

## [1] "beadLevelData"
## attr(,"package")
## [1] "beadarray"

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Analysis of Bead-level Data using beadarray

slotNames(exampleBLData)

## [1] "beadData" "sectionData" "experimentData" "history"

##Get the beadData for array-section 1

exampleBLData[[1]][1:10,]

## ProbeID GrnX GrnY Grn wts

## [1,] 10008 900.6661 10781.320 355 1
## [2,] 10008 1992.5400 11352.000 377 1
## [3,] 10008 1257.4790 7559.513 452 1
## [4,] 10008 1700.1600 6351.157 267 1
## [5,] 10008 1814.5210 3299.495 431 1
## [6,] 10008 2060.3440 8471.688 357 1
## [7,] 10008 609.0356 6028.458 408 1
## [8,] 10008 1487.7190 15933.790 431 1
## [9,] 10008 1517.5080 14928.520 351 1
## [10,] 10008 1619.3640 17650.690 235 1

##Alternative using accessor function

getBeadData(exampleBLData, array=1, what="Grn")[1:10]

## [1] 355 377 452 267 431 357 408 431 351 235

##Get unique ProbeIDs. These are the ArrayAddressIDs

uIDs = unique(getBeadData(exampleBLData, array=1, what="ProbeID"))
uIDs[1:10]

## [1] 10008 10010 10017 10019 10020 10021 10025 10035 10037 10039

4 Scan Metrics
The first view of array quality can be assessed using the metrics calculated by
the scanner. These include the 95th (P95) and 5th (P05) quantiles of all pixel
intensities on the image. A signal-to-noise ratio (SNR) can be calculated as the
ratio of these two quantities. These metrics can be viewed in real-time as the
arrays themselves are being scanned. By tracking these metrics over time, one
can potentially halt problematic experiments before they even reach the analysis
stage. The metrics information for the exampleBLData object can retrieved in
the following way. Illumina recommend that the SNR ratio should be above 10,
so these arrays are acceptable. However, the P95 and P05 values will fluctuate

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Analysis of Bead-level Data using beadarray

over time and are dependant upon the scanner setup. Including SNR values for
arrays other than those currently being analysed will give a better indication of
whether any outlier arrays exist.
metrics(exampleBLData)

## Date Matrix Section RegGrn FocusGrn SatGrn P95Grn P05Grn

## 1 3/13/2009 6:45:04 PM 4613710017 B 0.13 0.70 0 704 36
## 12 04/01/09 04:50 4616494005 A 0.13 0.59 0 678 38
## RegRed FocusRed SatRed P95Red P05Red
## 1 0 0 0 0 0
## 12 0 0 0 0 0

p95(exampleBLData, "Grn")

## [1] 704 678

snr(exampleBLData, "Grn")

## [1] 19.55556 17.84211

5 Transformation Functions
A more flexible way to obtain per-bead data from a beadLevelData object is
to define a transformation function that takes as arguments the beadLevelData
object and an array index. The function then manipulates the data in the desired
manner and returns a vector the same length as the number of beads on the
array. The logGreenChannelTransform is the default transformation in many
plotting / QA functions within beadarray. Users with two-channel data may
also wish to experiment with the similarly defined logRedChannelTransform or
logRatioTransform when plotting.

log2(exampleBLData[[1]][1:10,2])

## [1] 9.814849 10.960393 10.296319 10.731455 10.825373 11.008670 9.250383

## [8] 10.538886 10.567488 10.661212

logGreenChannelTransform

## function (BLData, array)

## {
## x = getBeadData(BLData, array = array, what = "Grn")
## return(log2.na(x))
## }
## <bytecode: 0x56069f2a6f40>

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Analysis of Bead-level Data using beadarray

## <environment: namespace:beadarray>

logGreenChannelTransform(exampleBLData, array=1)[1:10]

## [1] 8.471675 8.558421 8.820179 8.060696 8.751544 8.479780 8.672425 8.751544

## [9] 8.455327 7.876517

logRedChannelTransform

## function (BLData, array)

## {
## x = getBeadData(BLData, array = array, what = "Red")
## return(log2.na(x))
## }
## <bytecode: 0x5606a1100368>
## <environment: namespace:beadarray>

6 Boxplots and imageplots

Two standard quality assessment plots supported by beadarray are the imageplot
and boxplot. Boxplots can be used to compare foreground and background
intensities between arrays. Image plots can be used to identify spatial artifacts
on the array surface that can occur from mis-handling or scanning problems.
With the raw bead-level data, we can plot false images of each array. This kind
of visualization is not possible when using the summarized BeadStudio output,
as the summary values are averaged over spatial positions. Image plots in R are
also more convenient than scrutinizing the original tiffs, as multiple arrays can
be visualized on the one page. By default, the array surface is plotted with the
longest edge going horizontally. Both the boxplot and imageplot functions
take a transformation function as an argument, with the default to do a log2
transformation on the green channel.
The imageplot can be configured in many ways (see manual page for more
details). Sections from a BeadChip often have one edge that is much longer
than the other, and it is important to recognise this when producing the plots.
By default, beadarray makes imageplots with the longest edge on the x-axis
(suitable for widescreen monitors). However, with horizontal = FALSE, the
imageplot will be displayed in the same orientation as the original TIFF image
from the directory. With the squareSize we can control how many pixels from
the original image make up the pixels in the resulting imageplot. The following

8
Analysis of Bead-level Data using beadarray

code produces imageplots for all array-sections in the example dataset. Note
that we also change the colour scheme to represent low and high intensities by
light and dark green respectively.
If .locs information is available to beadarray , it will be able to determine the
optimal squareSize parameter. If not (as with our example dataset), the user
may have to experiment with different values for squareSize.
imageplot(exampleBLData, array=1, low="lightgreen", high="darkgreen")

imageplot(exampleBLData, array=2, low="lightgreen", high="darkgreen")

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Analysis of Bead-level Data using beadarray

7 BASH
BASH is a method for managing the spatial artefacts that may be found on an
array as described in Cairns et al (2008). BASH uses the methodology developed
for the Harshlight package, but altered to exploit the availability of replicated
observations on the same array. The algorithm first identifies Extended defects,
where an array has gradual but significant shifts across the surface. BASH also
seeks to find more localized artifacts on arrays by classifying features that have
unusual intensities as outliers and then finding outliers close to each other on
the array. Two separate algorithms then search for areas with a larger num-
bers of outliers than would be expected by chance (Diffuse Defects) and large
connected clusters of outliers (Compact defects). The random nature (both in
position and numbers of each feature type) of Illumina arrays mean that the
Harshlight algorithm must proceed in a different way to the original Harshlight
implementation. Whereas Affymetrix probes have replicates on other arrays,
Illumina beads are replicated on the same array. We can therefore generate
an error image based on how much each bead differs from the median of its
replicates’ intensities, instead of replicates on other arrays. Having performed
manipulations to the error image, we can then find outliers on this image by bead
type, determining which beads are more than 3 Median Absolute Devations, or
MADs, from the median.
Finally, since Illumina arrays are randomly arranged and use a hexagonal grid
rather than rectangular, BASH has it’s own method for creating networks of
beads on the array. However, if .locs files are available to beadarray the time
taken for this step will be improved considerably.
The following command can be used to run BASH with the default settings
bsh = BASH(exampleBLData, array=1:2)

The weights and QC can be stored using setWeights and insertSectionData.

for(i in 1:2){

BLData <- setWeights(exampleBLData, wts=bsh$wts[[i]], array=i)

BLData <- insertSectionData(exampleBLData, what="BASHQC", data = bsh$QC)

We have already saved the weights into the exampleBLData object and they can
be retrieved in the following way. A weight of zero meaning that the bead will
be excluded from an outlier calculations or summarisation procedures.

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Analysis of Bead-level Data using beadarray

table(getBeadData(exampleBLData, array=1, what="wts"))

##
## 0 1
## 13380 1074989

table(getBeadData(exampleBLData, array=2, what="wts"))

##
## 0 1
## 143923 956850

Before combining the observations for each bead-type on an array, Illumina

remove any observations with outlying intensity (more than 3 median absolute
deviations from the median). This step can be repeated in beadarray and can
be adjusted so that other outlier removal schemes can be run. It is useful to see
where these outliers are located on the array surface. Often, they will coincide
with beads masked by BASH or with any spatial artefacts that may be seen.
The locations of beads that have been masked by BASH can be visualised using
the showArrayMask function.
showArrayMask(exampleBLData, array=2)

8 Using control information

Illumina have designed a number of control probes for each expression plat-
form. Two particular controls on expression arrays are housekeeping and biotin
controls. With the poscontPlot function, we can plot the intensities of any

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Analysis of Bead-level Data using beadarray

ArrayAddressIDs that are annotated as belonging to the Housekeeping or Biotin

group in the ExpressionControlData object. The mapping of controls to Ar-
rayAddressID is possible by having the illuminaHumanv3.db package installed
and setting the annotation of the object accordingly.
p <- combinedControlPlot(exampleBLData)

9 Summarization
The summarization procedure takes the BLData object, where each bead-type
is represented by differing numbers of observations on each array, and produces
a summarized object to make comparisons between arrays. For each array
section represented in the BLData object, all observations are extracted, trans-
formed, and then grouped together according to their ArrayAddressID. Outliers
are removed and the mean and standard deviation of the remaining beads are
calculated.
The illuminaChannel class is used to define how summarization proceeds with
specification of a transformation function, a function to remove outliers and
function to calculate the means and standard deviation. The default options
to summarize apply a log2 transformation, remove outliers using the Illumina 3
M.A.D cut-off, and report the mean and standard deviation for each bead type.
BSData <- summarize(exampleBLData)

## No sample factor specified. Summarizing each section separately

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Analysis of Bead-level Data using beadarray

## Finding list of unique probes in beadLevelData

## 49895 unique probeIDs found
## Number of unmapped probes removed: 319
## Summarizing G channel
## Processing Array 1
## Removing outliers
## Using exprFun
## Using varFun
## Summarizing G channel
## Processing Array 2
## Removing outliers
## Using exprFun
## Using varFun
## Making summary object

The code below creates a different summarized object; one which reports median
and standard errors and does not log transform the data.
myMedian <- function(x) median(x, na.rm=TRUE)
myMad <- function(x) mad(x, na.rm=TRUE)

greenChannel2 <- new("illuminaChannel", greenChannelTransform, illuminaOutlierMethod,

myMedian, myMad,"G")

BSData2 <- summarize(exampleBLData, list(greenChannel2))

The BSData object is very similar to the ExpressionSet class in Biobase. How-
ever, to accommodate the unique features of Illumina data we have added an
nObservations slot, which gives the number of beads that we used to create
the summary values for each bead-type on each array after outlier removal.
BSData

## ExpressionSetIllumina (storageMode: list)

## assayData: 49576 features, 2 samples
## element names: exprs, se.exprs, nObservations
## protocolData: none
## phenoData
## rowNames: 4613710017_B 4616494005_A
## varLabels: sampleID SampleFac
## varMetadata: labelDescription
## featureData
## featureNames: ILMN_1802380 ILMN_1893287 ... ILMN_1846115 (49576

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Analysis of Bead-level Data using beadarray

## total)
## fvarLabels: ArrayAddressID IlluminaID Status
## fvarMetadata: labelDescription
## experimentData: use 'experimentData(object)'
## Annotation: Humanv3
## QC Information
## Available Slots:
## QC Items: Date, Matrix, ..., SampleGroup, numBeads
## sampleNames: 4613710017_B, 4616494005_A

It is possible to have multiple channels, each of which is summarized in a dif-

ferent manner, in the same ExpressionSetIllumina object. This is achieved
by passing a list of illuminaChannel objects to summarize. This would be es-
pecially useful for two-channel data, where the Red and Green channels, and
some combination of the two would be of interest in the analysis.
The detection score is a standard measure for Illumina expression experiments,
and can be viewed as an empirical estimate of the p-value for the null hy-
pothesis that there is no expression. These can be calculated for summarized
data provided that the identity of the negative controls on the array is known.
For further analysis of the summarized object, see the separate beadsummary
vignette beadsummary.pdf.
det = calculateDetection(BSData)

##
|
| | 0%
|
|======================================================================| 100%

head(det)

## 4613710017_B 4616494005_A
## ILMN_1802380 0.00000000 0.00000000
## ILMN_1893287 0.27309237 0.43658211
## ILMN_1736104 0.55555556 0.73564753
## ILMN_1792389 0.00000000 0.00000000
## ILMN_1854015 0.05756359 0.01869159
## ILMN_1904757 0.21686747 0.40987984

Detection(BSData) <- det

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sessionInfo()

## R version 3.6.0 (2019-04-26)

## Platform: x86_64-pc-linux-gnu (64-bit)
## Running under: Ubuntu 18.04.2 LTS
##
## Matrix products: default
## BLAS: /home/biocbuild/bbs-3.10-bioc/R/lib/libRblas.so
## LAPACK: /home/biocbuild/bbs-3.10-bioc/R/lib/libRlapack.so
##
## locale:
## [1] LC_CTYPE=en_US.UTF-8 LC_NUMERIC=C
## [3] LC_TIME=en_US.UTF-8 LC_COLLATE=C
## [5] LC_MONETARY=en_US.UTF-8 LC_MESSAGES=en_US.UTF-8
## [7] LC_PAPER=en_US.UTF-8 LC_NAME=C
## [9] LC_ADDRESS=C LC_TELEPHONE=C
## [11] LC_MEASUREMENT=en_US.UTF-8 LC_IDENTIFICATION=C
##
## attached base packages:
## [1] stats4 parallel stats graphics grDevices utils datasets
## [8] methods base
##
## other attached packages:
## [1] illuminaHumanv3.db_1.26.0 org.Hs.eg.db_3.8.2
## [3] AnnotationDbi_1.47.0 IRanges_2.19.3
## [5] S4Vectors_0.23.3 beadarrayExampleData_1.23.0
## [7] beadarray_2.35.1 hexbin_1.27.3
## [9] Biobase_2.45.0 BiocGenerics_0.31.2
## [11] knitr_1.22
##
## loaded via a namespace (and not attached):
## [1] tidyselect_0.2.5 xfun_0.7 reshape2_1.4.3
## [4] purrr_0.3.2 lattice_0.20-38 colorspace_1.4-1
## [7] htmltools_0.3.6 yaml_2.2.0 blob_1.1.1
## [10] BeadDataPackR_1.37.0 rlang_0.3.4 pillar_1.4.0
## [13] glue_1.3.1 DBI_1.0.0 bit64_0.9-7
## [16] GenomeInfoDbData 1.2.1 plyr_1.8.4
_ stringr_1.4.0
## [19] zlibbioc_1.31.0 munsell_0.5.0 gtable_0.3.0
## [22] memoise_1.1.0 evaluate_0.13 labeling_0.3
## [25] GenomeInfoDb_1.21.0 highr_0.8 illuminaio_0.27.0
## [28] Rcpp_1.0.1 openssl_1.3 scales_1.0.0
## [31] BiocManager_1.30.4 limma_3.41.0 base64_2.0

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## [34] XVector_0.25.0 bit_1.1-14 askpass_1.1

## [37] BiocStyle_2.13.0 ggplot2_3.1.1 digest_0.6.18
## [40] stringi_1.4.3 dplyr_0.8.1 GenomicRanges_1.37.4
## [43] grid_3.6.0 tools_3.6.0 bitops_1.0-6
## [46] magrittr_1.5 RCurl_1.95-4.12 lazyeval_0.2.2
## [49] RSQLite_2.1.1 tibble_2.1.1 crayon_1.3.4
## [52] pkgconfig_2.0.2 assertthat_0.2.1 rmarkdown_1.12
## [55] R6_2.4.0 compiler_3.6.0

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