Problem with reading raw Illumina Human HT-12 v4 data using beadarray
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jaro.slamecka ▴ 140
@jaroslamecka-7419
Last seen 2.2 years ago
Mitchell Cancer Institute, Mobile AL, U…

Hi,

I am new to microarray data analysis, trying to put the pieces of puzzle together. Trying to read raw Illumina Human HT-12 v4 data using beadarray. My raw data folder contains csv, txt, EIF, idat, locs, tif, xml file for each of the 12 samples. There is one sdf file, one metrics.txt file and 3 fiducial #N_Red_center.tif files.

Function readIllumina(useImages = FALSE, illuminaAnnotation = "Humanv4") gives the error below. Each txt file contains only two columns: Code Grn. Is the format of my raw data unsuitable for readIllumina? Or is there a way to instruct it so that it read the data properly? Thanks in advance!

Trying to read all Illumina files inD:\RData\HA1
Processing section 9992405085_A
Error in `[.data.frame`(lines, 1, 1:4) : undefined columns selected

> traceback()
6: stop("undefined columns selected")
5: `[.data.frame`(lines, 1, 1:4)
4: lines[1, 1:4]
3: numberOfColumns(file, sep = sep)
2: readBeadLevelTextFile(file.path(targets$directory[i], targets$textFile[i]),
       dec = dec)
1: readIllumina(useImages = FALSE, illuminaAnnotation = "Humanv4")

> sessionInfo()
R version 3.1.2 (2014-10-31)
Platform: x86_64-w64-mingw32/x64 (64-bit)

locale:
[1] LC_COLLATE=English_United States.1252
[2] LC_CTYPE=English_United States.1252   
[3] LC_MONETARY=English_United States.1252
[4] LC_NUMERIC=C                          
[5] LC_TIME=English_United States.1252    

attached base packages:
[1] parallel  stats     graphics  grDevices utils     datasets  methods  
[8] base     

other attached packages:
[1] beadarray_2.16.0    ggplot2_1.0.0       GEOquery_2.32.0    
[4] lumi_2.18.0         Biobase_2.26.0      BiocGenerics_0.12.1

loaded via a namespace (and not attached):
 [1] affy_1.44.0             affyio_1.34.0           annotate_1.44.0        
 [4] AnnotationDbi_1.28.1    base64_1.1              base64enc_0.1-2        
 [7] BatchJobs_1.5           BBmisc_1.8              BeadDataPackR_1.18.0   
[10] beanplot_1.2            BiocInstaller_1.16.1    BiocParallel_1.0.0     
[13] biomaRt_2.22.0          Biostrings_2.34.1       bitops_1.0-6           
[16] brew_1.0-6              bumphunter_1.6.0        checkmate_1.5.1        
[19] codetools_0.2-10        colorspace_1.2-4        DBI_0.3.1              
[22] digest_0.6.8            doRNG_1.6               fail_1.2               
[25] foreach_1.4.2           genefilter_1.48.1       GenomeInfoDb_1.2.4     
[28] GenomicAlignments_1.2.1 GenomicFeatures_1.18.3  GenomicRanges_1.18.4   
[31] grid_3.1.2              gtable_0.1.2            illuminaio_0.8.0       
[34] IRanges_2.0.1           iterators_1.0.7         KernSmooth_2.23-13     
[37] lattice_0.20-29         limma_3.22.4            locfit_1.5-9.1         
[40] MASS_7.3-37             Matrix_1.1-5            matrixStats_0.13.1     
[43] mclust_4.4              methylumi_2.12.0        mgcv_1.8-4             
[46] minfi_1.12.0            multtest_2.22.0         munsell_0.4.2          
[49] nleqslv_2.5             nlme_3.1-119            nor1mix_1.2-0          
[52] pkgmaker_0.22           plyr_1.8.1              preprocessCore_1.28.0  
[55] proto_0.3-10            quadprog_1.5-5          R.methodsS3_1.6.1      
[58] RColorBrewer_1.1-2      Rcpp_0.11.4             RCurl_1.95-4.5         
[61] registry_0.2            reshape_0.8.5           reshape2_1.4.1         
[64] rngtools_1.2.4          Rsamtools_1.18.2        RSQLite_1.0.0          
[67] rtracklayer_1.26.2      S4Vectors_0.4.0         scales_0.2.4           
[70] sendmailR_1.2-1         siggenes_1.40.0         splines_3.1.2          
[73] stats4_3.1.2            stringr_0.6.2           survival_2.37-7        
[76] tools_3.1.2             XML_3.98-1.1            xtable_1.7-4           
[79] XVector_0.6.0           zlibbioc_1.12.0

 

Jaro

Jaroslav Slamecka, PhD.
Mitchell Cancer Institute
University of South Alabama
1660 Springhill Ave.
Mobile, AL 36604

beadarray readIllumina • 2.7k views
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Entering edit mode
Mike Smith ★ 6.6k
@mike-smith
Last seen 2 hours ago
EMBL Heidelberg

Hi Jaro,

Thanks for bringing this to our attention.  Unfortunately, if your txt file only has two columns you can't use many of the features of beadarray (e.g. BASH) that rely on the coordinate information, and the software currently fails to read the data (admittedly with a not very helpful message).

That said there are still some advantages to working with bead-level data over Illumina's summarized version, such as being able to perform outlier removal on the log2 scale.  For the moment, if you wish to use the beadarray package you could fake the two coordinate columns by giving everything a value of 0.  beadarray should be able to read that, but you have to be careful not to make use of any of the spatial information as this would obviously be misleading.

If you have access to the iScan machine and want to ensure that the X,Y coordinates are included in the future, the instructions can be found on page 35 of the user guide here:

http://support.illumina.com/content/dam/illumina-support/documents/myillumina/f1805681-577a-4db3-9240-333ac009d333/iscan_system_user_guide_11313539_b.pdf

Let me know if you need anything more,

Mike

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@gordon-smyth
Last seen 2 hours ago
WEHI, Melbourne, Australia

You could try read.idat() in the limma package, which will read in the expression data from the idat files.

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Entering edit mode
jaro.slamecka ▴ 140
@jaroslamecka-7419
Last seen 2.2 years ago
Mitchell Cancer Institute, Mobile AL, U…

Thank you both so much for your help! Too bad the core lab didn't set the iScan to include the XY coordinate data, I'll make sure they do the next time. And I will look into read.idat in limma.

Jaro

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