RNbeads Sample pairing issue

Cameron Lassiter

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Feb 18, 2016, 1:10:35 PM2/18/16

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Hello,

I have been attempting to run RNbeads on a set of Illum 450 arrays. I have age and gender as covar and sample pairing for a few test.

So my options are set:

rnb.options(

filtering.sex.chromosomes.removal=TRUE,

identifiers.column="Sample_ID",

"covariate.adjustment.columns"=c("Age","Sex"),

"differential.comparison.columns"=c(

"MRIT0_noVsyesAllodynia",

"MRIT1_noVsyesAllodynia",

"NoAllodynia_MRIT0vsT1",

"YesAllodynia_MRIT0vsT1",

"T0_noVsyesAllodynia",

"T1__noVsyesAllodynia",

"NoAllodynia_T0vsT1",

"YesAllodynia_T0vsT1",

"MRIT0_noVsyesHyperalgesia",

"MRIT1_noVsyesHyperalgesia",

"NoHyperalgesia_MRIT0vsT1",

"YesHyperalgesia_MRIT0vsT1",

"T0_noVsyesHyperalgesia",

"T1__noVsyesHyperalgesia",

"NoHyperalgesia_T0vsT1",

"YesHyperalgesia_T0vsT1"

),

"columns.pairing"=c(

"Sample_B1"="Sample_B1_ID",

"Sample_MRI"="Sample_MRI_ID",

"MRIT0_noVsyesAllodynia"="MRIT0_noVsyesAllodynia_ID",

"MRIT1_noVsyesAllodynia"="MRIT1_noVsyesAllodynia_ID",

"NoAllodynia_MRIT0vsT1"="NoAllodynia_MRIT0vsT1_ID",

"YesAllodynia_MRIT0vsT1"="YesAllodynia_MRIT0vsT1_ID",

"T0_noVsyesAllodynia"="T0_noVsyesAllodynia_ID",

"T1__noVsyesAllodynia"="T1__noVsyesAllodynia_ID",

"NoAllodynia_T0vsT1"="NoAllodynia_T0vsT_ID",

"YesAllodynia_T0vsT1"="YesAllodynia_T0vsT1_ID",

"MRIT0_noVsyesHyperalgesia"="MRIT0_noVsyesHyperalgesia_ID",

"MRIT1_noVsyesHyperalgesia"="MRIT1_noVsyesHyperalgesia_ID",

"NoHyperalgesia_MRIT0vsT1"="NoHyperalgesia_MRIT0vsT1_ID",

"YesHyperalgesia_MRIT0vsT1"="YesHyperalgesia_MRIT0vsT1_ID",

"T0_noVsyesHyperalgesia"="T0_noVsyesHyperalgesia_ID",

"T1__noVsyesHyperalgesia"="T1__noVsyesHyperalgesia_ID",

"NoHyperalgesia_T0vsT1"="NoHyperalgesia_T0vsT1_ID",

"YesHyperalgesia_T0vsT1"="YesHyperalgesia_T0vsT1_ID"

),# End sample pairing

enforce.memory.management=TRUE

)

#End Option

The sample sheet is arranged thus:

Sample_ID

Sample_Plate

Sample_Name

Sample_Group

Sample_Group_ID

Sample_B1

Sample_B1_ID

Sample_MRI

Sample_MRI_ID

AMP_Plate

Sample_Well

Sentrix_Position

Sentrix_ID

ID

Age

Sex

Allodynia_sort

Allodynia_sort_ID

MRIT0_noVsyesAllodynia

MRIT0_noVsyesAllodynia_ID

MRIT1_noVsyesAllodynia

MRIT1_noVsyesAllodynia_ID

NoAllodynia_MRIT0vsT1

NoAllodynia_MRIT0vsT1_ID

YesAllodynia_MRIT0vsT1

YesAllodynia_MRIT0vsT1_ID

T0_noVsyesAllodynia

T0_noVsyesAllodynia_ID

T1__noVsyesAllodynia

T1__noVsyesAllodynia_ID

NoAllodynia_T0vsT1

NoAllodynia_T0vsT1_ID

YesAllodynia_T0vsT1

YesAllodynia_T0vsT1_ID

Hyperalgesia_sorting

Hyperalgesia_sorting_ID

MRIT0_noVsyesHyperalgesia

MRIT0_noVsyesHyperalgesia_ID

MRIT1_noVsyesHyperalgesia

MRIT1_noVsyesHyperalgesia_ID

NoHyperalgesia_MRIT0vsT1

NoHyperalgesia_MRIT0vsT1_ID

YesHyperalgesia_MRIT0vsT1

YesHyperalgesia_MRIT0vsT1_ID

T0_noVsyesHyperalgesia

T0_noVsyesHyperalgesia_ID

T1__noVsyesHyperalgesia

T1__noVsyesHyperalgesia_ID

NoHyperalgesia_T0vsT1

NoHyperalgesia_T0vsT1_ID

YesHyperalgesia_T0vsT1

YesHyperalgesia_T0vsT1_ID

1007B1T0

0

1007B1T0

B1T0

1007

B1T0

1007

0

E02

R01C01

4E+09

1007

22

F

Yes_allodynia

1007

B1T0_Yes_allodynia

1007

B1T0_Yes_allodynia

1007

Yes_hyperalgesia

1007

B1T0_Yes_hyperalgesia

1007

B1T0_Yes_hyperalgesia

1007

1007B1T1

0

1007B1T1

B1T1

1007

B1T1

1007

0

F02

R02C01

4E+09

1007

22

F

Yes_allodynia

1007

B1T1_Yes_allodynia

1007

B1T1_Yes_allodynia

1007

Yes_hyperalgesia

1007

B1T1_Yes_hyperalgesia

1007

B1T1_Yes_hyperalgesia

1007

1007MRIT0

0

1007MRIT0

MRIT0

1007

MRIT0

1007

0

A04

R01C01

4E+09

1007

22

F

Yes_allodynia

1007

MRIT0_Yes_allodynia

1007

MRIT0_Yes_allodynia

1007

Yes_hyperalgesia

1007

MRIT0_Yes_hyperalgesia

1007

MRIT0_Yes_hyperalgesia

1007

Basically, for any differential contrast there is an ID column that has a patient ID. If I run it as above I get an error:

Error in `[.data.frame`(annotations.all, , columns.pairs[cc]) :

undefined columns selected

If I run with only a single contrast and one set of pairing info, the logger will tell me:

Invalid pairing information

I take it I am doing something wrong with my sample sheet, but cant' seem to figure it out. I have ran the pipeline without the sample pairing and it runs fine, but with the pairing info the run time is vastly increased and then it stops at the diff step (the exploratory step works, if with a long run time).

Session info

R version 3.2.3 (2015-12-10)

Platform: x86_64-w64-mingw32/x64 (64-bit)

Running under: Windows 7 x64 (build 7601) Service Pack 1

locale:

[1] LC_COLLATE=English_United States.1252 LC_CTYPE=English_United States.1252 LC_MONETARY=English_United States.1252 LC_NUMERIC=C

[5] LC_TIME=English_United States.1252

attached base packages:

[1] grid stats4 parallel stats graphics grDevices utils datasets methods base

other attached packages:

[1] IlluminaHumanMethylation450kmanifest_0.4.0 RnBeads.hg19_1.2.0 RnBeads_1.2.1 plyr_1.8.3

[5] methylumi_2.16.0 minfi_1.16.0 bumphunter_1.10.0 locfit_1.5-9.1

[9] iterators_1.0.8 foreach_1.4.3 Biostrings_2.38.4 XVector_0.10.0

[13] SummarizedExperiment_1.0.2 lattice_0.20-33 FDb.InfiniumMethylation.hg19_2.2.0 org.Hs.eg.db_3.2.3

[17] RSQLite_1.0.0 DBI_0.3.1 TxDb.Hsapiens.UCSC.hg19.knownGene_3.2.2 GenomicFeatures_1.22.12

[21] AnnotationDbi_1.32.3 reshape2_1.4.1 scales_0.3.0 Biobase_2.30.0

[25] illuminaio_0.12.0 matrixStats_0.50.1 limma_3.26.7 gridExtra_2.0.0

[29] gplots_2.17.0 ggplot2_2.0.0 fields_8.3-6 maps_3.0.2

[33] spam_1.3-0 ff_2.2-13 bit_1.1-12 cluster_2.0.3

[37] RColorBrewer_1.1-2 MASS_7.3-45 GenomicRanges_1.22.4 GenomeInfoDb_1.6.3

[41] IRanges_2.4.7 S4Vectors_0.8.11 BiocGenerics_0.16.1

loaded via a namespace (and not attached):

[1] nlme_3.1-124 bitops_1.0-6 tools_3.2.3 doRNG_1.6 nor1mix_1.2-1 KernSmooth_2.23-15 colorspace_1.2-6

[8] base64_1.1 preprocessCore_1.32.0 pkgmaker_0.22 labeling_0.3 rtracklayer_1.30.2 caTools_1.17.1 hexbin_1.27.1

[15] genefilter_1.52.1 quadprog_1.5-5 stringr_1.0.0 digest_0.6.9 Rsamtools_1.22.0 siggenes_1.44.0 GEOquery_2.36.0

[22] mclust_5.1 BiocParallel_1.4.3 gtools_3.5.0 RCurl_1.95-4.7 magrittr_1.5 futile.logger_1.4.1 Rcpp_0.12.3

[29] munsell_0.4.2 stringi_1.0-1 zlibbioc_1.16.0 gdata_2.17.0 splines_3.2.3 multtest_2.26.0 annotate_1.48.0

[36] beanplot_1.2 igraph_1.0.1 rngtools_1.2.4 corpcor_1.6.8 codetools_0.2-14 biomaRt_2.26.1 mixOmics_5.2.0

[43] futile.options_1.0.0 XML_3.98-1.3 lambda.r_1.1.7 gtable_0.1.2 reshape_0.8.5 xtable_1.8-2 survival_2.38-3

[50] GenomicAlignments_1.6.3 registry_0.3 ellipse_0.3-8 rgl_0.95.1441

Cameron Lassiter

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Feb 23, 2016, 9:30:28 AM2/23/16

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I updated RNbeads and am still not getting the paired analysis. It would seem that the issue is the the paired info is not making it into rnb.set

rnb.options(

+ filtering.sex.chromosomes.removal=FALSE,

+

+ covariate.adjustment.columns=c("Sex", "Age"),

+ enforce.memory.management = FALSE,

+ identifiers.column="Sample_ID",

+

+ differential.comparison.columns=c(

+

+ "NoAllodynia_T0vsT1"

+

+ ),

+

+ columns.pairing=c(

+

+ "NoAllodynia_T0vsT1"="XXXX"

+ )# End sample pairing

+ )

> #End Option

>

> ## Data import

> data.source <- c(idat.dir, sample.annotation)

> result <- rnb.run.import(data.source = data.source, dir.reports = report.dir)

2016-02-23 09:24:02 0.9 STATUS STARTED Loading Data

2016-02-23 09:24:02 0.9 INFO Number of cores: 1

2016-02-23 09:24:02 0.9 INFO Loading data of type "idat.dir"

2016-02-23 09:24:02 0.9 STATUS STARTED Loading Data from IDAT Files

2016-02-23 09:24:03 0.9 INFO Added column barcode to the provided sample annotation table

2016-02-23 09:24:11 1.5 STATUS COMPLETED Loading Data from IDAT Files

2016-02-23 09:24:50 0.7 STATUS Loaded data from C:/Users/camer_000/Desktop/R Working Directory/RNBEADS/idat/idat

2016-02-23 09:24:51 1.2 STATUS Predicted gender for the loaded samples

2016-02-23 09:24:51 0.9 STATUS Added data loading section to the report

2016-02-23 09:24:51 0.9 STATUS Loaded 5 samples and 485577 sites

2016-02-23 09:24:51 0.9 INFO Output object is of type RnBeadRawSet

2016-02-23 09:24:51 0.9 STATUS COMPLETED Loading Data

> rnb.set <- result$rnb.set

> rnb.sample.groups(rnb.set)

$Sample_Group

$Sample_Group$B1T0

[1] 2 4

$Sample_Group$B1T1

[1] 1 3 5

$Sentrix_ID

$Sentrix_ID$`3999119037`

[1] 1 2 3

$Sentrix_ID$`3999119083`

[1] 4 5

$Sex

$Sex$F

[1] 1 4 5

$Sex$M

[1] 2 3

$NoAllodynia_T0vsT1

$NoAllodynia_T0vsT1$B1T0_No_allodynia

[1] 2 4

$NoAllodynia_T0vsT1$B1T1_No_allodynia

[1] 1 3 5

$`Predicted Gender`

$`Predicted Gender`$female

[1] 1 4 5

$`Predicted Gender`$male

[1] 2 3

attr(,"paired")

[1] FALSE FALSE FALSE FALSE FALSE

> pheno(rnb.set)

Sample_ID Sample_Name Sample_Group Sample_Well Sentrix_Position Sentrix_ID Age Sex T0_noVsyesAllodynia T0_noVsyesAllodynia_ID T1__noVsyesAllodynia

1 2011B1T1 2011B1T1 B1T1 D02 R06C02 3999119037 18 F <NA> <NA> B1T1_No_allodynia

2 2006B1T0 2006B1T0 B1T0 G01 R01C02 3999119037 41 M B1T0_No_allodynia X2006 <NA>

3 2006B1T1 2006B1T1 B1T1 H01 R02C02 3999119037 41 M <NA> <NA> B1T1_No_allodynia

4 1031B1T0 1031B1T0 B1T0 A05 R03C02 3999119083 24 F B1T0_No_allodynia X1031 <NA>

5 1031B1T1 1031B1T1 B1T1 B05 R04C02 3999119083 24 F <NA> <NA> B1T1_No_allodynia

T1__noVsyesAllodynia_ID NoAllodynia_T0vsT1 NoAllodynia_T0vsT1_ID barcode Predicted Male Probability Predicted Gender

1 X2011 B1T1_No_allodynia X2011 3999119037_R06C02 2.091507e-05 female

2 <NA> B1T0_No_allodynia X2006 3999119037_R01C02 9.992526e-01 male

3 X2006 B1T1_No_allodynia X2006 3999119037_R02C02 9.995426e-01 male

4 <NA> B1T0_No_allodynia X1031 3999119083_R03C02 8.356714e-06 female

5 X1031 B1T1_No_allodynia X1031 3999119083_R04C02 5.003652e-06 female

Any info would be helpful.

Thanks,

Cameron

Fabian

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Feb 24, 2016, 3:21:32 AM2/24/16

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Hi Cameron,
sorry for the late reply. It's kind of hard to see the exact column assignment from the post you made. Would it be possible to send the table in some separated format, so that I could look into it?

Best regards,
Fabian

Cameron Lassiter

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Feb 24, 2016, 10:04:41 AM2/24/16

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Hi Fabian,

Attached is a short version of the sample sheet. I think it may help as pairing for YesAllodynia_T0vsT1 makes it in but not for NoAllodynia_T0vsT1. I've been completely baffled by it. Thanks for looking into it for me

Thanks,

Cameron

#Set to run vanilla analysis

rnb.options(

filtering.sex.chromosomes.removal=FALSE,

covariate.adjustment.columns=c("Sex", "Age"),

enforce.memory.management = FALSE,

identifiers.column="Sample_ID",

differential.comparison.columns=c(

"NoAllodynia_T0vsT1",

"YesAllodynia_T0vsT1"

),

columns.pairing=c(

"NoAllodynia_T0vsT1"="NoAllodynia_T0vsT1_ID",

"YesAllodynia_T0vsT1" = "YesAllodynia_T0vsT1_ID"

)# End sample pairing

)

#End Option

master_sample_sheet_IDs_allo_test2.csv

Fabian

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Feb 24, 2016, 10:35:22 AM2/24/16

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Hi Cameron,
after having a closer look, it seems, that you specified everything correctly. What precisely is the error you get? Could you send the log output and the traceback() information?

Cameron Lassiter

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Feb 24, 2016, 11:09:58 AM2/24/16

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Fabian,

So, I have a big data set ~ 160 arrays. I think the error I got the first time was due to an out of date dependence. Anyway updating bioconductor and R seems to have made it go away (at least with smaller test sets). Now what I am seeing is that my sample pairing information is not accepted by rnb.sample.groups(). The logger just informs me that the sample ID pairing is invalid during the differential (so no error or traceback). The log is attached, no log errors or anything in it at the import step, however the ID info is missing by rnb.sampe.group.

Thanks,

Cameron

Cameron Lassiter

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Feb 24, 2016, 11:10:29 AM2/24/16

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logger_test.txt

Cameron Lassiter

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Feb 24, 2016, 2:06:28 PM2/24/16

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Hi Fabian,

So I spoke to quickly before, I reran the full analysis and still get the error (below with traceback).

Attached is also the logger from this run and the sample sheet used.

Thanks,

Cameron

rnb.run.differential(rnb.set, report.dir)

Error in `[.data.frame`(annotations.all, , columns.pairs[cc]) :

undefined columns selected

10: stop("undefined columns selected")

9: `[.data.frame`(annotations.all, , columns.pairs[cc])

8: annotations.all[, columns.pairs[cc]]

7: FUN(X[[i]], ...)

6: lapply(names(columns.pairs), FUN = function(cc) {

annotations.all[, columns.pairs[cc]]

})

5: lapply(names(columns.pairs), FUN = function(cc) {

annotations.all[, columns.pairs[cc]]

})

4: rnb.sample.groups(x, columns = pheno.cols, columns.pairs = columns.pairs)

3: get.comparison.info(x, pheno.cols = pheno.cols, region.types = region.types,

pheno.cols.all.pairwise = pheno.cols.all.pairwise, columns.pairs = columns.pairs,

columns.adj = columns.adj, adjust.sva = adjust.sva, pheno.cols.adjust.sva = pheno.cols.adjust.sva,

adjust.celltype = adjust.celltype)

2: rnb.execute.computeDiffMeth(rnb.set, cmp.cols, region.types = reg.types,

n.perm = permutations, covg.thres = rnb.getOption("filtering.coverage.threshold"),

pheno.cols.all.pairwise = rnb.getOption("differential.comparison.columns.all.pairwise"),

columns.pairs = rnb.getOption("columns.pairing"), columns.adj = rnb.getOption("covariate.adjustment.columns"),

adjust.sva = rnb.getOption("differential.adjustment.sva"),

pheno.cols.adjust.sva = rnb.getOption("inference.targets.sva"),

adjust.celltype = rnb.getOption("differential.adjustment.celltype"),

skip.sites = !rnb.getOption("analyze.sites"), disk.dump = disk.dump,

disk.dump.dir = disk.dump.dir)

1: rnb.run.differential(rnb.set, report.dir)

logger_test.txt

master_sample_sheet_IDs.csv

Fabian

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Feb 25, 2016, 10:13:43 AM2/25/16

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From your initial post It looks like you specified "NoAllodynia_T0vsT_ID" in the columns.pairing option. This column does not exist in your annotation table that you posted in your last post.

Am Donnerstag, 18. Februar 2016 19:10:35 UTC+1 schrieb Cameron Lassiter:

Cameron Lassiter

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Feb 25, 2016, 10:56:08 AM2/25/16

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Sorry about the typo, but after fixing the typo, I still get in the logger warnings:

STARTED Retrieving comparison info

2016-02-25 10:33:19 0.6 WARNING Invalid pairing information for column NoAllodynia_MRIT0vsT1 . --> Treating as unpaired.

2016-02-25 10:33:19 0.6 WARNING Invalid pairing information for column YesAllodynia_MRIT0vsT1 . --> Treating as unpaired.

2016-02-25 10:33:20 0.6 WARNING Invalid pairing information for column NoAllodynia_T0vsT1 . --> Treating as unpaired.

2016-02-25 10:33:20 0.6 WARNING Invalid pairing information for column YesHyperalgesia_MRIT0vsT1 . --> Treating as unpaired.

2016-02-25 10:33:20 0.6 WARNING Invalid pairing information for column NoHyperalgesia_T0vsT1 . --> Treating as unpaired.

2016-02-25 10:33:20 0.6 STATUS COMPLETED Retrieving comparison info

Only three of the 8 make it into the rnb.sample.group(). Even if the sample pairing is inputed wrong shouldn't the info from pheno() be added anyway? Even if I try to do it explicitly (with master_sample_sheet_IDs.csv):

> rnb.sample.groups(rnb.set, columns = c(

+ "MRIT0_noVsyesAllodynia",

+ "MRIT1_noVsyesAllodynia",

+ "NoAllodynia_MRIT0vsT1",

+ "YesAllodynia_MRIT0vsT1",

+ "T0_noVsyesAllodynia",

+ "T1__noVsyesAllodynia",

+ "NoAllodynia_T0vsT1",

+ "YesAllodynia_T0vsT1",

+ "MRIT0_noVsyesHyperalgesia",

+ "MRIT1_noVsyesHyperalgesia",

+ "NoHyperalgesia_MRIT0vsT1",

+ "YesHyperalgesia_MRIT0vsT1",

+ "T0_noVsyesHyperalgesia",

+ "T1__noVsyesHyperalgesia",

+ "NoHyperalgesia_T0vsT1",

+ "YesHyperalgesia_T0vsT1"

+ ), columns.pairs = c(

+

+ "NoAllodynia_MRIT0vsT1"="NoAllodynia_MRIT0vsT1_ID",

+ "YesAllodynia_MRIT0vsT1"="YesAllodynia_MRIT0vsT1_ID",

+

+ "NoAllodynia_T0vsT1"="NoAllodynia_T0vsT1_ID",

+ "YesAllodynia_T0vsT1"="YesAllodynia_T0vsT1_ID",

+

+ "NoHyperalgesia_MRIT0vsT1"="NoHyperalgesia_MRIT0vsT1_ID",

+ "YesHyperalgesia_MRIT0vsT1"="YesHyperalgesia_MRIT0vsT1_ID",

+

+ "NoHyperalgesia_T0vsT1"="NoHyperalgesia_T0vsT1_ID",

+ "YesHyperalgesia_T0vsT1"="YesHyperalgesia_T0vsT1_ID"

+

+ )

+

+ )

$MRIT0_noVsyesAllodynia

$MRIT0_noVsyesAllodynia$MRIT0_No_allodynia

[1] 7 48 59 62 76 86 94 118 130 136 140

$MRIT0_noVsyesAllodynia$MRIT0_Yes_allodynia

[1] 3 17 23 30 34 42 55 70 82 98 102 106 110 114 126

$MRIT1_noVsyesAllodynia

$MRIT1_noVsyesAllodynia$MRIT1_No_allodynia

[1] 8 49 60 63 77 87 95 119 131 137

$MRIT1_noVsyesAllodynia$MRIT1_Yes_allodynia

[1] 4 18 31 35 43 56 71 83 99 103 107 111 115 127

$NoAllodynia_MRIT0vsT1

$NoAllodynia_MRIT0vsT1$MRIT0_No_allodynia

[1] 7 48 59 62 76 86 94 118 130 136 140

$NoAllodynia_MRIT0vsT1$MRIT1_No_allodynia

[1] 8 49 60 63 77 87 95 119 131 137

$YesAllodynia_MRIT0vsT1

$YesAllodynia_MRIT0vsT1$MRIT0_Yes_allodynia

[1] 3 17 23 30 34 42 55 70 82 98 102 106 110 114 126

$YesAllodynia_MRIT0vsT1$MRIT1_Yes_allodynia

[1] 4 18 31 35 43 56 71 83 99 103 107 111 115 127

$T0_noVsyesAllodynia

$T0_noVsyesAllodynia$B1T0_No_allodynia

[1] 5 9 11 19 36 46 50 57 61 66 74 78 84 88 90 92 116 122 128 134 138 141 143

$T0_noVsyesAllodynia$B1T0_Yes_allodynia

[1] 1 13 15 21 24 28 32 38 40 44 53 68 80 96 100 104 108 112 124 132 145

$T1__noVsyesAllodynia

$T1__noVsyesAllodynia$B1T1_No_allodynia

[1] 6 10 12 20 37 47 51 52 58 67 75 79 85 89 91 93 117 123 129 135 139 142 144

$T1__noVsyesAllodynia$B1T1_Yes_allodynia

[1] 2 14 16 22 25 29 33 39 41 45 54 69 81 97 101 105 109 113 125 133 146

$NoAllodynia_T0vsT1

$NoAllodynia_T0vsT1$B1T0_No_allodynia

[1] 5 9 11 19 36 46 50 57 61 66 74 78 84 88 90 92 116 122 128 134 138 141 143

$NoAllodynia_T0vsT1$B1T1_No_allodynia

[1] 6 10 12 20 37 47 51 52 58 67 75 79 85 89 91 93 117 123 129 135 139 142 144

$YesAllodynia_T0vsT1

B1T0_Yes_allodynia B1T1_Yes_allodynia

1007 1 2

1020 13 14

1022 15 16

1048 21 22

1054 24 25

1060 28 29

1062 32 33

1081 38 39

2003 40 41

2004 44 45

2017 53 54

2032 68 69

2044 80 81

2056 96 97

2057 100 101

2058 104 105

2063 108 109

2065 112 113

2071 124 125

2074 132 133

2083 145 146

$MRIT0_noVsyesHyperalgesia

$MRIT0_noVsyesHyperalgesia$MRIT0_No_hyperalgesia

[1] 7 62 86 114 118 130 136

$MRIT0_noVsyesHyperalgesia$MRIT0_Yes_hyperalgesia

[1] 3 17 23 30 34 42 48 55 59 70 76 82 94 98 102 106 110 140

$MRIT1_noVsyesHyperalgesia

$MRIT1_noVsyesHyperalgesia$MRIT1_No_hyperalgesia

[1] 8 63 87 115 119 131 137

$MRIT1_noVsyesHyperalgesia$MRIT1_Yes_hyperalgesia

[1] 4 18 31 35 43 49 56 60 71 77 83 95 99 103 107 111

$NoHyperalgesia_MRIT0vsT1

MRIT0_No_hyperalgesia MRIT1_No_hyperalgesia

1009 7 8

2025 62 63

2045 86 87

2065 114 115

2066 118 119

2072 130 131

2075 136 137

$YesHyperalgesia_MRIT0vsT1

$YesHyperalgesia_MRIT0vsT1$MRIT0_Yes_hyperalgesia

[1] 3 17 23 30 34 42 48 55 59 70 76 82 94 98 102 106 110 140

$YesHyperalgesia_MRIT0vsT1$MRIT1_Yes_hyperalgesia

[1] 4 18 31 35 43 49 56 60 71 77 83 95 99 103 107 111

$T0_noVsyesHyperalgesia

$T0_noVsyesHyperalgesia$B1T0_No_hyperalgesia

[1] 5 61 84 88 112 116 122 128 134

$T0_noVsyesHyperalgesia$B1T0_Yes_hyperalgesia

[1] 1 9 11 13 15 19 21 24 26 28 32 36 38 40 44 46 50 53 57 64 66 68 72 74 78 80 90 92 96 100 104 108 120 132 138 141 143 145 147 149

$T1__noVsyesHyperalgesia

$T1__noVsyesHyperalgesia$B1T1_No_hyperalgesia

[1] 6 85 89 113 117 123 129 135

$T1__noVsyesHyperalgesia$B1T1_Yes_hyperalgesia

[1] 2 10 12 14 16 20 22 25 27 29 33 37 39 41 45 47 51 54 58 65 67 69 73 75 79 81 91 93 97 101 105 109 121 133 139 142 144 146 148 150

$NoHyperalgesia_T0vsT1

$NoHyperalgesia_T0vsT1$B1T0_No_hyperalgesia

[1] 5 61 84 88 112 116 122 128 134

$NoHyperalgesia_T0vsT1$B1T1_No_hyperalgesia

[1] 6 85 89 113 117 123 129 135

$YesHyperalgesia_T0vsT1

B1T0_Yes_hyperalgesia B1T1_Yes_hyperalgesia

1007 1 2

1012 9 10

1015 11 12

1020 13 14

1022 15 16

1031 19 20

1048 21 22

1054 24 25

1059 26 27

1060 28 29

1062 32 33

1080 36 37

1081 38 39

2003 40 41

2004 44 45

2005 46 47

2006 50 51

2017 53 54

2023 57 58

2028 64 65

2029 66 67

2032 68 69

2037 72 73

2040 74 75

2042 78 79

2044 80 81

2052 90 91

2053 92 93

2056 96 97

2057 100 101

2058 104 105

2063 108 109

2067 120 121

2074 132 133

2076 138 139

2077 141 142

2079 143 144

2083 145 146

2084 147 148

2087 149 150

attr(,"paired")

[1] FALSE FALSE FALSE FALSE FALSE FALSE FALSE TRUE FALSE FALSE TRUE FALSE FALSE FALSE FALSE TRUE

Fabian

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Feb 25, 2016, 11:27:27 AM2/25/16

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I noticed that in the columns that fail to recognize the pairing information, you have uneven group sizes between the two groups that are compared. RnBeads' pairing assumption is that for each individual you have one sample in each group. Hence, the group sizes should be equal. Could you check again, if this is the case or samples are removed somehow?

Am Donnerstag, 18. Februar 2016 19:10:35 UTC+1 schrieb Cameron Lassiter:

Cameron Lassiter

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Feb 25, 2016, 11:49:39 AM2/25/16

to Epigenomics forum

Hi Fabian,

Yes, that seems to have been the issue. Everything is getting pulled now. I think it will run the rest of the way without any issues.

However, while I have your ear:

How are the linear models in RnBeads actually built with sample pairing? Does it use a blocking design, mean parameterization, duplicateCorrelation(), etc? i have HTA data from these patients also, and would like to make sure the linear models are the same.

At any rate, thank you for the help, RnBeads is easily the most comprehensive methylation package in R.

Thanks again for all the help,

Cameron Lassiter

Fabian

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Feb 26, 2016, 5:26:38 AM2/26/16

to Epigenomics forum

Glad I could help.
Paired analysis enters the limma analysis in a very simple fashion: the pair information is simply added as a covariate. See p42 of the limma user guide:
http://www.bioconductor.org/packages/release/bioc/vignettes/limma/inst/doc/usersguide.pdf

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