## Integrative analysis examples

In this document we’ll review a few approaches to using genome-scale data of different types to reason about certain focused questions.

### TF binding and expression co-regulation in yeast

An example of integrative analysis can be found in a paper of Lee and Rinaldi in connection with the regulatory program of the yeast cell cycle. There are two key experimental components:

• Protein binding patterns: based on ChIP-chip experiments, we can determine the gene promoter regions to which transcription factors bind.

• Expression patterns: based on timed observations of gene expression in a yeast colony we can identify times at which groups of genes reach maximal expression.

Figure 5 of the paper indicates that the Mbp1 transcription factor played a role in regulating expression in the transition from G1 to S phases of the cell cycle. The ChIP-chip data is in the harbChIP package.

library(harbChIP)
data(harbChIP)
harbChIP

## ExpressionSet (storageMode: lockedEnvironment)
## assayData: 6230 features, 204 samples
##   element names: exprs, se.exprs
## protocolData: none
## phenoData
##   sampleNames: A1 (MATA1) ABF1 ... ZMS1 (204 total)
##   varLabels: txFac
## featureData
##   featureNames: YAL001C YAL002W ... MRH1 (6230 total)
##   fvarLabels: ID PLATE ... REV_SEQ (12 total)
## experimentData: use 'experimentData(object)'
##   pubMedIds: 15343339
## Annotation:


This is a well-documented data object, and we can read the abstract of the paper directly.

abstract(harbChIP)

## [1] "DNA-binding transcriptional regulators interpret the genome's regulatory code by binding to specific sequences to induce or repress gene expression. Comparative genomics has recently been used to identify potential cis-regulatory sequences within the yeast genome on the basis of phylogenetic conservation, but this information alone does not reveal if or when transcriptional regulators occupy these binding sites. We have constructed an initial map of yeast's transcriptional regulatory code by identifying the sequence elements that are bound by regulators under various conditions and that are conserved among Saccharomyces species. The organization of regulatory elements in promoters and the environment-dependent use of these elements by regulators are discussed. We find that environment-specific use of regulatory elements predicts mechanistic models for the function of a large population of yeast's transcriptional regulators."


Let’s find MBP1 and assess the distribution of reported binding affinity measures. The sample names of the ExpressionSet (structure used for convenience even though the data are not expression data) are the names of the proteins “chipped” onto the yeast promoter array.

mind = which(sampleNames(harbChIP)=="MBP1")
qqnorm(exprs(harbChIP)[,mind], main="MBP1 binding")


The shape of the qq-normal plot is indicative of a strong departure from Gaussianity in the distribution of binding scores, with a very long right tail. We’ll focus on the top five genes.

topb = featureNames(harbChIP)[ order(
exprs(harbChIP)[,mind], decreasing=TRUE)[1:5] ]
topb

## [1] "YDL101C" "YPR075C" "YCR064C" "YCR065W" "YGR109C"

library(org.Sc.sgd.db)

##

select(org.Sc.sgd.db, keys=topb, keytype="ORF",
columns="COMMON")

## 'select()' returned 1:many mapping between keys and columns

##       ORF        SGD                               COMMON
## 1 YDL101C S000002259                                 DUN1
## 2 YDL101C S000002259 serine/threonine protein kinase DUN1
## 3 YPR075C S000006279                                 OPY2
## 4 YCR064C S000000660                                 <NA>
## 5 YCR065W S000000661                                 HCM1
## 6 YGR109C S000003341                                 CLB6
## 7 YGR109C S000003341                   B-type cyclin CLB6


Our conjecture is that these genes will exhibit similar expression trajectories, peaking well within the first half of cell cycle for the yeast strain studied.

We will subset the cell cycle expression data from the yeastCC package to a colony whose cycling was synchronized using alpha pheromone.

library(yeastCC)
data(spYCCES)
alp = spYCCES[, spYCCES$syncmeth=="alpha"] par(mfrow=c(1,1)) plot(exprs(alp)[ topb[1], ]~alp$time, lty=1,
type="l", ylim=c(-1.5,1.5), lwd=2, ylab="Expression",
xlab="Minutes elapsed")
for (i in 2:5) lines(exprs(alp)[topb[i],]~alp$time, lty=i, lwd=2) legend(75,-.5, lty=1:10, legend=topb, lwd=2, cex=.6, seg.len=4)  We have the impression that at least three of these genes reach peak expression roughly together near times 20 and 80 minutes. There is considerable variability. A data filtering and visualization pattern is emerging by which genes bound by a given transcription factor can be assessed for coregulation of expression. We have not entered into the assessment of statistical significance, but have focused on how the data types are brought together. ### TF binding and genome-wide DNA-phenotype associations in humans Genetic epidemiology has taken advantage of high-throughput genotyping (mostly using genotyping arrays supplemented with model-based genotype imputation) to develop the concept of “genome-wide association study” (GWAS). Here a cohort is assembled and individuals are distinguished in terms of disease status or phenotype measurement, and the genome is searched for variants exhibiting statistical association with disease status or phenotypic class or value. An example of a GWAS result can be seen with the gwascat package, which includes selections from the NHGRI GWAS catalog, which has recently moved to EBI-EMBL. library(gwascat) data(gwrngs19) gwrngs19[100]  ## gwasloc instance with 1 records and 35 attributes per record. ## Extracted: Mon Sep 8 13:08:13 2014 ## Genome: hg19 ## Excerpt: ## GRanges object with 1 range and 35 metadata columns: ## seqnames ranges strand | Date.Added.to.Catalog PUBMEDID ## <Rle> <IRanges> <Rle> | <character> <integer> ## 100 chr11 118729391 * | 08/05/2014 24390342 ## First.Author Date Journal ## <character> <character> <character> ## 100 Okada Y 12/25/2013 Nature ## Link ## <character> ## 100 http://www.ncbi.nlm.nih.gov/pubmed/24390342 ## Study ## <character> ## 100 Genetics of rheumatoid arthritis contributes to biology and drug discovery. ## Disease.Trait ## <character> ## 100 Rheumatoid arthritis ## Initial.Sample.Size ## <character> ## 100 up to 14,361 European ancestry cases, up to 42,923 European ancestry controls, up to 4,873 East Asian ancestry cases, up to 17,642 East Asian ancestry controls ## Replication.Sample.Size ## <character> ## 100 up to 3,775 European ancestry cases, up to 5,801 European ancestry controls, up to 6,871 East Asian ancestry cases, up to 6,392 East Asian ancestry controls ## Region Chr_id Chr_pos.hg38 Reported.Gene.s. Mapped_gene ## <character> <character> <numeric> <character> <character> ## 100 11q23.3 11 118858682 CXCR5 SETP16 - CXCR5 ## Upstream_gene_id Downstream_gene_id Snp_gene_ids ## <character> <character> <character> ## 100 649925 643 ## Upstream_gene_distance Downstream_gene_distance ## <character> <character> ## 100 24.12 25.08 ## Strongest.SNP.Risk.Allele SNPs Merged Snp_id_current ## <character> <character> <character> <character> ## 100 rs10790268-G rs10790268 0 10790268 ## Context Intergenic Risk.Allele.Frequency p.Value Pvalue_mlog ## <character> <character> <character> <numeric> <numeric> ## 100 Intergenic 1 0.81 1e-15 15 ## p.Value..text. OR.or.beta X95..CI..text. ## <character> <numeric> <character> ## 100 1.14 [1.11-1.18] ## Platform..SNPs.passing.QC. CNV ## <character> <character> ## 100 Affymetrix & Illumina [up to 9,739,303] (Imputed) N ## num.Risk.Allele.Frequency ## <numeric> ## 100 0.81 ## ------- ## seqinfo: 23 sequences from hg19 genome  mcols(gwrngs19[100])[,c(2,7,8,9,10,11)]  ## DataFrame with 1 row and 6 columns ## PUBMEDID ## <integer> ## 100 24390342 ## Study ## <character> ## 100 Genetics of rheumatoid arthritis contributes to biology and drug discovery. ## Disease.Trait ## <character> ## 100 Rheumatoid arthritis ## Initial.Sample.Size ## <character> ## 100 up to 14,361 European ancestry cases, up to 42,923 European ancestry controls, up to 4,873 East Asian ancestry cases, up to 17,642 East Asian ancestry controls ## Replication.Sample.Size ## <character> ## 100 up to 3,775 European ancestry cases, up to 5,801 European ancestry controls, up to 6,871 East Asian ancestry cases, up to 6,392 East Asian ancestry controls ## Region ## <character> ## 100 11q23.3  ## <integer> ## 24390342 ## <character> ## Genetics of rheumatoid arthritis contributes to biology and drug discovery. ## <character> ## Rheumatoid arthritis ## <character> ## up to 14,361 European ancestry cases, up to 42,923 European ancestry controls, up to 4,873 East Asian ancestry cases, up to 17,642 East Asian ancestry controls ## <character> ## up to 3,775 European ancestry cases, up to 5,801 European ancestry controls, up to 6,871 East Asian ancestry cases, up to 6,392 East Asian ancestry controls ## <character> ## 11q23.3  This shows the complexity involved in recording information about a replicated genome-wide association finding. There are many fields recorded, by the key elements are the name and location of the SNP, and the phenotype to which it is apparently linked. In this case, we are talking about rheumatoid arthritis. We will now consider the relationship between ESRRA binding in B-cells and phenotypes for which GWAS associations have been reported. It is tempting to proceed as follows. We simply compute overlaps between the binding peak regions and the catalog GRanges. library(ERBS) data(GM12878) fo = findOverlaps(GM12878, gwrngs19) fo  ## Hits object with 55 hits and 0 metadata columns: ## queryHits subjectHits ## <integer> <integer> ## [1] 12 2897 ## [2] 28 11889 ## [3] 28 17104 ## [4] 39 3975 ## [5] 84 9207 ## ... ... ... ## [51] 1869 14660 ## [52] 1869 14661 ## [53] 1869 16004 ## [54] 1869 16761 ## [55] 1869 16793 ## ------- ## queryLength: 1873 / subjectLength: 17254  sort(table(gwrngs19$Disease.Trait[
subjectHits(fo) ]), decreasing=TRUE)[1:5]

##
##        Rheumatoid arthritis             LDL cholesterol
##                           7                           6
##          Cholesterol, total Lipid metabolism phenotypes
##                           4                           4
##            Bipolar disorder
##                           3


The problem with this is that gwrngs19 is a set of records of GWAS hits. There are cases of SNP that are associated with multiple phenotypes, and there are cases of multiple studies that find the same result for a given SNP. It is easy to get a sense of the magnitude of the problem using reduce.

length(gwrngs19)-length(reduce(gwrngs19))

## [1] 3659


So our strategy will be to find overlaps with the reduced version of gwrngs19 and then come back to enumerate phenotypes at unique SNPs occupying binding sites.

fo = findOverlaps(GM12878, reduce(gwrngs19))
fo

## Hits object with 28 hits and 0 metadata columns:
##        queryHits subjectHits
##        <integer>   <integer>
##    [1]        12        3120
##    [2]        28       12162
##    [3]        39        7588
##    [4]        84        9947
##    [5]       257        7797
##    ...       ...         ...
##   [24]      1564       11567
##   [25]      1635        4554
##   [26]      1669       12996
##   [27]      1709        8057
##   [28]      1869          83
##   -------
##   queryLength: 1873 / subjectLength: 13595

ovrngs = reduce(gwrngs19)[subjectHits(fo)]
#phset = lapply( ovrngs, function(x)
#  unique( gwrngs19[ which(gwrngs19 %over% x) ]$Disease.Trait ) ) phset = vector("list", length(ovrngs)) for (i in 1:length(ovrngs)) { phset[[i]] = unique( gwrngs19[ which(gwrngs19 %over% ovrngs[i]) ]$Disease.Trait )
}
sort(table(unlist(phset)), decreasing=TRUE)[1:5]

##
## Rheumatoid arthritis     Bipolar disorder   Cholesterol, total
##                    4                    2                    2
##      HDL cholesterol      Type 1 diabetes
##                    2                    2


What can explain this observation? We see that there are commonly observed DNA variants in locations where ESRRA tends to bind. Do individuals with particular genotypes of SNPs in these areas have higher risk of disease because the presence of the variant allele interferes with ESRRA function and leads to arthritis or abnormal cholesterol levels? Or is this observation consistent with the play of chance in our work with these data? We will examine this in the exercises.

### Harvesting GEO for families of microarray archives

The NCBI Gene Expression Omnibus is a basic resource for integrative bioinformatics. The Bioconductor GEOmetadb package helps with discovery and characterization of GEO datasets.

The GEOmetadb database (in August 2019) is a 503MB download that decompresses to 8.2 GB of SQLite. Once you have acquired the GEOmetadb.sqlite file using the getSQLiteFile function, you can create a connection and start interrogating the database locally. Here we use an environment variable to establish the location of the database. Use your operating system environment variables to emulate this.

library(RSQLite)
dbListTables(lcon)


You will see

 [1] "gds"               "gds_subset"        "geoConvert"
[4] "geodb_column_desc" "gpl"               "gse"
[7] "gse_gpl"           "gse_gsm"           "gsm"
[10] "metaInfo"          "sMatrix"


We will build a query that returns all the GEO GSE entries that have the phrase “pancreatic cancer” in their titles. Because GEO uses uninformative labels for array platforms, we will retrieve a field that records the Bioconductor array annotation package name so that we know what technology was in use. We’ll tabulate the various platforms used.

vbls = "gse.gse, gse.title, gpl.gpl, gpl.bioc_package"
req1 = " from gse join gse_gpl on gse.gse=gse_gpl.gse"
req2 = " join gpl on gse_gpl.gpl=gpl.gpl"
goal = " where gse.title like '%pancreatic%cancer%'"
quer = paste0("select ", vbls, req1, req2, goal)
lkpc = dbGetQuery(lcon, quer)
dim(lkpc)
table(lkpc\$bioc_package)


For the August 2019 image you will see


hgu133a                    hgu133a2
3                           3
hgu133b                 hgu133plus2
3                          30
hgu219                   hgug4110b
1                           1
HsAgilentDesign026652   huex10sttranscriptcluster
2                           4
hugene10sttranscriptcluster IlluminaHumanMethylation27k
1                           2
illuminaHumanv4 mogene10sttranscriptcluster
12                           1
mouse430a2
11


We won’t insist that you take the GEOmetadb.sqlite download/expansion, but if you do, variations on the query string constructed above can assist you with targeted identification of GEO datasets for analysis and reinterpretation.