L. panamensis 20190914: Annotation data.

1 Annotation version: 20190914

1.1 Genome annotation input

There are a few methods of importing annotation data into R. The following are two attempts, the second is currently being used in these analyses.

1.1.1 AnnotationHub

AnnotationHub is a newer service and has promise to be an excellent top-level resource for gathering annotation data.

tt <- sm(library(AnnotationHub))
ah <- sm(AnnotationHub())
orgdbs <- query(ah, "OrgDB")
annot_lm <- query(ah, c("OrgDB", "Friedlin"))
lm_name <- names(annot_lm)
annot_lm <- sm(annot_lm[[lm_name[[2]]]])
txtdbs <- query(ah, "TxDb")

## AH48429 appears to be panamensis
##annot_lp <- annot_lp[["AH48429"]]
annot_lp <- query(ah, c("OrgDB", "panamensis"))
lp_name <- names(annot_lp)
annot_lp <- sm(annot_lp[[lp_name[[2]]]])

1.2 OrganismDb

Since this document was originally written, I have made substantial changes to how I create, load, and manipulate the eupathdb annotation data. As a result, this needs to be significantly reworked.

AnnotationHub is the new and fancier version of what OrganismDb does. Keith already made these for the parasites though, lets try and use one of those.

library(EuPathDB)
panamensis_entry <- get_eupath_entry("panamensis", webservice="tritrypdb")
testing_panamensis <- make_eupath_orgdb(panamensis_entry)

braziliensis_entry <- get_eupath_entry("braziliensis", webservice="tritrypdb")
testing_braziliensis <- make_eupath_orgdb(braziliensis_entry)

donovani_entry <- get_eupath_entry("donovani", webservice="tritrypdb")
testing_donovani <- make_eupath_orgdb(donovani_entry)

mexicana_entry <- get_eupath_entry("mexicana", webservice="tritrypdb")
testing_mexicana <- make_eupath_orgdb(mexicana_entry)

major_entry <- get_eupath_entry("major", webservice="tritrypdb")
testing_major <- make_eupath_orgdb(major_entry)

crith_entry <- get_eupath_entry("Crit", webservice="tritrypdb")
testing_crith <- make_eupath_orgdb(crith_entry)

Assuming the above packages got created, we may load them and extract the annotation data.

library(EuPathDB)

## Loading required package: GenomeInfoDbData

## 
## This is EuPathDB version 1.6.0
##  Read 'EuPathDB()' to get started.

## 
## Attaching package: 'EuPathDB'

## The following objects are masked from 'package:hpgltools':
## 
##     download_uniprot_proteome, get_kegg_orgn,
##     load_kegg_annotations, load_orgdb_annotations, load_orgdb_go,
##     load_uniprot_annotations, orgdb_from_ah

tritryp_metadata <- download_eupath_metadata(webservice="tritrypdb")

## Bioconductor version 3.10 (BiocManager 1.30.8), ?BiocManager::install for
##   help

major_entry <- get_eupath_entry("major", webservice="tritrypdb")

## Found the following hits: Leishmania major strain Friedlin, Leishmania major strain LV39c5, Leishmania major strain SD 75.1, choosing the first.

## Using: Leishmania major strain Friedlin.

major_names <- get_eupath_pkgnames(major_entry)
major_names$orgdb

## org.Lmajor.Friedlin.v45.eg.db

wanted_fields <- c("annot_cds_length", "annot_chromosome", "annot_gene_entrez_id",
                   "annot_gene_name", "annot_strand", "gid", "go_go_id",
                   "go_go_term_name", "go_ontology",
                   "interpro_description" ,"interpro_e_value", "type_gene_type")

lm_org <- sm(EuPathDB::load_orgdb_annotations(major_names$orgdb,
                                              keytype="gid",
                                              fields=wanted_fields))

panamensis_entry <- get_eupath_entry("panamensis", webservice="tritrypdb")

## Found the following hits: Leishmania panamensis MHOM/COL/81/L13, Leishmania panamensis strain MHOM/PA/94/PSC-1, choosing the first.

## Using: Leishmania panamensis MHOM/COL/81/L13.

panamensis_names <- get_eupath_pkgnames(panamensis_entry)
panamensis_names$orgdb

## org.Lpanamensis.MHOMCOL81L13.v45.eg.db

lp_org <- sm(EuPathDB::load_orgdb_annotations(panamensis_names$orgdb,
                                    keytype="gid",
                                    fields=wanted_fields))

braziliensis_names <- get_eupath_pkgnames("braziliensis")
braziliensis_names$orgdb
old_name <- "org.Lbraziliensis.MHOMBR75M2903.v38.eg.db"
lb_org <- sm(load_orgdb_annotations(old_name, keytype="gid", fields=wanted_fields))

##donovani_names <- get_eupath_pkgnames("donovani")
##donovani_names$orgdb
##ld_org <- load_orgdb_annotations(donovani_names$orgdb, keytype="gid", fields=wanted_fields)
##
##mexicana_names <- get_eupath_pkgnames("mexicana")
##mexicana_names$orgdb
##lmex_org <- load_orgdb_annotations(mexicana_names$orgdb, keytype="gid", fields=wanted_fields)
##
##fasciculata_names <- get_eupath_pkgnames("rithidia", metadata=tritryp_metadata)
##fasciculata_names$orgdb
##cf_org <- load_orgdb_annotations(fasciculata_names$orgdb, keytype="gid", fields=wanted_fields)

1.3 Read a gff file

In contrast, it is possible to load most annotations of interest directly from the gff files used in the alignments. More in-depth information for the human transcriptome may be extracted from biomart.

## The old way of getting genome/annotation data
lp_gff <- "reference/lpanamensis.gff"
lb_gff <- "reference/lbraziliensis.gff"
hs_gff <- "reference/hsapiens.gtf"

lp_fasta <- "reference/lpanamensis.fasta.xz"
lb_fasta <- "reference/lbraziliensis.fasta.xz"
hs_fasta <- "reference/hsapiens.fasta.xz"

lp_annotations <- sm(load_gff_annotations(lp_gff, type="gene"))
rownames(lp_annotations) <- paste0("exon_", lp_annotations$web_id, ".1")

lb_annotations <- sm(load_gff_annotations(lb_gff, type="gene"))
hs_gff_annot <- sm(load_gff_annotations(hs_gff, id_col="gene_id"))

hs_annotations <- sm(load_biomart_annotations())$annotation
hs_annotations$ID <- hs_annotations$geneID
rownames(hs_annotations) <- make.names(hs_annotations[["ensembl_gene_id"]], unique=TRUE)

lp_size_dist <- plot_histogram(lp_annotations[["width"]])
lp_size_dist

hs_size_dist <- plot_histogram(hs_annotations[["cds_length"]])
hs_size_dist +
  ggplot2::scale_x_continuous(limits=c(0, 20000))

## Warning: Removed 103681 rows containing non-finite values (stat_bin).

## Warning: Removed 103681 rows containing non-finite values (stat_density).

## Warning: Removed 2 rows containing missing values (geom_bar).

1.4 Getting ontology data

Annotation for gene ontologies may be gathered from a similarly large number of sources. The following are a couple.

## Try using biomart
hs_go_biomart <- sm(load_biomart_go())
## or the org.Hs.eg.db sqlite database
tt <- sm(library("Homo.sapiens"))
hs <- Homo.sapiens
##hs_go_ensembl <- load_orgdb_go(hs, hs_annotations$geneID)
##dim(hs_go_biomart)
##dim(hs_go_ensembl)
##hs_goids <- hs_go_biomart

## While testing, I called this desc, that will need to change.
##lp_tooltips <- make_tooltips(lp_annotations)
##lb_tooltips <- make_tooltips(lb_annotations)

lp_lengths <- lp_annotations[, c("ID", "width")]
lb_lengths <- lb_annotations[, c("ID", "width")]
hs_lengths <- hs_annotations[, c("ensembl_gene_id", "cds_length")]
colnames(hs_lengths) <- c("ID", "width")

lp_goids <- read.csv(file="reference/lpan_go.txt.xz", sep="\t", header=FALSE)
lb_goids <- read.csv(file="reference/lbraz_go.txt.xz", sep="\t", header=FALSE)
colnames(lp_goids) <- c("ID","GO","ont","name","source","tag")
colnames(lb_goids) <- c("ID","GO","ont","name","source","tag")

2 Putting the pieces together

The macrophage experiment has samples across 2 contexts, the host and parasite. The following block sets up one experiment for each. If you open the all_samples-species.xlsx files, you will note immediately that a few different attempts were made at ascertaining the most likely experimental factors that contributed to the readily apparent batch effects.

2.1 The human transcriptome mappings

Keep in mind that if I change the experimental design with new annotations, I must therefore regenerate the following.

hs_final_annotations <- hs_annotations
hs_final_annotations <- hs_final_annotations[, c("ensembl_transcript_id", "ensembl_gene_id", "cds_length",
                                                 "hgnc_symbol", "description", "gene_biotype")]
colnames(hs_final_annotations)[3] <- "length"
note <- "New experimental design factors by snp added 2016-09-20"

hs_expt <- sm(create_expt("sample_sheets/all_samples-combined.xlsx",
                          gene_info=hs_final_annotations,
                          file_column="humanfile",
                          notes=note))
knitr::kable(head(hs_expt$design, n=1))

	sampleid	pathogenstrain	experimentname	tubelabel	alias	condition	batch	anotherbatch	snpclade	snpcladev2	snpcladev3	pathogenstrain.1	label	donor	time	pctmappedparasite	pctcategory	state	sourcelab	expperson	pathogen	host	hostcelltype	noofhostcells	infectionperiodhpitimeofharvest	moiexposure	parasitespercell	pctinf	rnangul	rnaqcpassed	libraryconst	libqcpassed	index	descriptonandremarks	observation	lowercaseid	humanfile	parasitefile	bcftable	salmonreads	hssalmonmapped	hssalmonmaprate	lpsalmonmapped	lpsalmonmaprate	tophatpairs	hstophataligned	hstophatpct	hstophatmulti	hstophatdiscordant	hstophatconcordantpct	lptophataligned	lptophatpct	lptophatmulti	lptophatdiscordant	lpconcordantpct	variantpositions	file
HPGL0241	HPGL0241	none	macrophage	TM130-Nil (Blue label)	Nil	uninf	a	a	undef	undef	undef	none	uninf_1	d130	undef	undef	0	uninfected	Ade	Adriana	none	Human	Human macs	Max 2 mill	2h - 24h chase period	NA	unknown	unknown	468	Y	Wanderson	Y	1	Uninfected human macrophages	NA	hpgl0241	preprocessing/hpgl0241/outputs/tophat_hsapiens/accepted_paired.count.xz	undef	undef	46628648	26156539	0.561	NA	NA	46319335	40905961	0.8831	1374099	1430888	0.8522	NA	NA	NA	NA	NA	NA	null

cds_entries <- fData(hs_expt)
cds_entries <- cds_entries[["gene_biotype"]] == "protein_coding"
hs_cds_expt <- hs_expt
hs_cds_expt$expressionset <- hs_cds_expt$expressionset[cds_entries, ]
new_cds_entries <- fData(hs_cds_expt)

2.2 The parasite transcriptome mappings

parasite_expt <- sm(create_expt("sample_sheets/all_samples-combined.xlsx",
                             gene_info=lp_annotations, file_column="parasitefile"))
knitr::kable(head(parasite_expt$design, n=3),
             caption="The first three rows of the parasite experimental design.")

The first three rows of the parasite experimental design.

	sampleid	pathogenstrain	experimentname	tubelabel	alias	condition	batch	anotherbatch	snpclade	snpcladev2	snpcladev3	pathogenstrain.1	label	donor	time	pctmappedparasite	pctcategory	state	sourcelab	expperson	pathogen	host	hostcelltype	noofhostcells	infectionperiodhpitimeofharvest	moiexposure	parasitespercell	pctinf	rnangul	rnaqcpassed	libraryconst	libqcpassed	index	descriptonandremarks	observation	lowercaseid	humanfile	parasitefile	bcftable	salmonreads	hssalmonmapped	hssalmonmaprate	lpsalmonmapped	lpsalmonmaprate	tophatpairs	hstophataligned	hstophatpct	hstophatmulti	hstophatdiscordant	hstophatconcordantpct	lptophataligned	lptophatpct	lptophatmulti	lptophatdiscordant	lpconcordantpct	variantpositions	file
HPGL0242	HPGL0242	s2271	macrophage	TM130-2271	Self-Healing	sh	a	a	white	whitepink	right	s2271	sh_2271	d130	undef	30	3	self_heal	Ade	Adriana	Lp	Human	Human macs	Max 2 mill	2h - 24h chase period	0.0486111111111111	unknown	unknown	276	Y	Wanderson	Y	8	Infected human macrophages.	NA	hpgl0242	preprocessing/hpgl0242/outputs/tophat_hsapiens/accepted_paired.count.xz	preprocessing/hpgl0242/outputs/tophat_lpanamensis/accepted_paired.count.xz	preprocessing/outputs/hpgl0242_parsed_count.txt	42742857	17945935	0.4199	8023463	0.1877	42612353	25394266	0.5959	869649	784620	0.5775	13117819	0.3078	350277	263923	0.3016	3930	null
HPGL0244	HPGL0244	s5433	macrophage	TM130-5433	Chronic	chr	a	a	blue_self	blue	left	s5433	chr_5433	d130	undef	15	1	chronic	Ade	Adriana	Lp	Human	Human macs	Max 2 mill	2h - 24h chase period	0.0486111111111111	unknown	unknown	261	Y	Wanderson	Y	27	Infected human macrophages	NA	hpgl0244	preprocessing/hpgl0244/outputs/tophat_hsapiens/accepted_paired.count.xz	preprocessing/hpgl0244/outputs/tophat_lpanamensis/accepted_paired.count.xz	preprocessing/outputs/hpgl0244_parsed_count.txt	47150925	25281958	0.5362	3761371	0.0798	46925604	36379602	0.7753	1070964	991929	0.7541	5755998	0.1227	154830	116414	0.1202	85981	null
HPGL0245	HPGL0245	s1320	macrophage	TM130-1320	Chronic	chr	a	a	multicolor	yellowbrownmulti	right	s1320	chr_1320	d130	undef	40	4	chronic	Ade	Adriana	Lp	Human	Human macs	Max 2 mill	2h - 24h chase period	0.0486111111111111	unknown	unknown	199	Y	Wanderson	Y	11	Infected human macrophages	NA	hpgl0245	preprocessing/hpgl0245/outputs/tophat_hsapiens/accepted_paired.count.xz	preprocessing/hpgl0245/outputs/tophat_lpanamensis/accepted_paired.count.xz	preprocessing/outputs/hpgl0245_parsed_count.txt	44501247	15904014	0.3574	10310518	0.2317	44384269	22909280	0.5162	673982	600480	0.5026	16414676	0.3698	441931	310092	0.3628	38072	null

3 Supplemental Table 1

Table S1 is going to be a summary of the metadata in all_samples-combined This may also include some of the numbers regarding mapping %, etc.

Wanted columns:

• Sample ID: HPGLxxxx
• Donor Code: TM130 or PG1xx
• Cell Type: Macrophage or PBMC
• Infection Status: Infected or Uninfected
• Disease Outcome: Chronic or Self-Healing or NA
• Batch: A or B (macrophage); NA for PBMC
• Number of reads that passed Illumina filter
• Number of reads after trimming
• Number of reads mapped - human
• % reads mapped - human
• Number of reads mapped - L.panamensis
• % reads mapped - L.panamensis

Use the Tcruzi colors.

• A1 is a large title: “Macrophage Samples”
• Row 2 is the blue column headings
• 3-m contains Macrophage metadata
• m+1 is blank
• m+2 is a large title: “PBMC Samples”
• m+3-n contains PBMC metadata

4 End

At this point, we should have everything necessary to perform the various analyses of the 4 sub-experiments. So save the current data for reuse elsewhere.

The experimental design is available here.

message(paste0("This is hpgltools commit: ", get_git_commit()))

## If you wish to reproduce this exact build of hpgltools, invoke the following:

## > git clone http://github.com/abelew/hpgltools.git

## > git reset c2881f6d97e1ec981fd1481cf46d6bc875fac423

## This is hpgltools commit: Tue Oct 22 10:22:30 2019 -0400: c2881f6d97e1ec981fd1481cf46d6bc875fac423

this_save <- paste0(gsub(pattern="\\.Rmd", replace="", x=rmd_file), "-v", ver, ".rda.xz")
message(paste0("Saving to ", this_save))

## Saving to 01_annotation_20190914-v20190914.rda.xz

tmp <- saveme(filename=this_save)

## The savefile is: /mnt/sshfs/cbcbsub/fs/cbcb-lab/nelsayed/scratch/atb/rnaseq/lpanamensis_2016/savefiles/01_annotation_20190914-v20190914.rda.xz

## Renaming /mnt/sshfs/cbcbsub/fs/cbcb-lab/nelsayed/scratch/atb/rnaseq/lpanamensis_2016/savefiles/01_annotation_20190914-v20190914.rda.xz to /mnt/sshfs/cbcbsub/fs/cbcb-lab/nelsayed/scratch/atb/rnaseq/lpanamensis_2016/savefiles/01_annotation_20190914-v20190914.rda.xz.01.

## The save string is: con <- pipe(paste0('pxz > /mnt/sshfs/cbcbsub/fs/cbcb-lab/nelsayed/scratch/atb/rnaseq/lpanamensis_2016/savefiles/01_annotation_20190914-v20190914.rda.xz'), 'wb'); save(list=ls(all.names=TRUE, envir=globalenv()),
##      envir=globalenv(), file=con, compress=FALSE); close(con)

pander::pander(sessionInfo())

R version 3.6.1 (2019-07-05)

Platform: x86_64-pc-linux-gnu (64-bit)

locale: LC_CTYPE=en_US.UTF-8, LC_NUMERIC=C, LC_TIME=en_US.UTF-8, LC_COLLATE=en_US.UTF-8, LC_MONETARY=en_US.UTF-8, LC_MESSAGES=en_US.UTF-8, LC_PAPER=en_US.UTF-8, LC_NAME=C, LC_ADDRESS=C, LC_TELEPHONE=C, LC_MEASUREMENT=en_US.UTF-8 and LC_IDENTIFICATION=C

attached base packages: stats4, parallel, stats, graphics, grDevices, utils, datasets, methods and base

other attached packages: Homo.sapiens(v.1.3.1), TxDb.Hsapiens.UCSC.hg19.knownGene(v.3.2.2), org.Hs.eg.db(v.3.8.2), GO.db(v.3.8.2), OrganismDbi(v.1.27.1), GenomicFeatures(v.1.37.4), GenomicRanges(v.1.37.17), GenomeInfoDb(v.1.21.2), org.Lpanamensis.MHOMCOL81L13.v45.eg.db(v.2019.10), org.Lmajor.Friedlin.v45.eg.db(v.2019.10), AnnotationDbi(v.1.47.1), IRanges(v.2.19.17), S4Vectors(v.0.23.25), futile.logger(v.1.4.3), EuPathDB(v.1.6.0), GenomeInfoDbData(v.1.2.1), hpgltools(v.1.0), Biobase(v.2.45.1) and BiocGenerics(v.0.31.6)

loaded via a namespace (and not attached): colorspace(v.1.4-1), ellipsis(v.0.3.0), XVector(v.0.25.0), base64enc(v.0.1-3), bit64(v.0.9-7), interactiveDisplayBase(v.1.23.0), xml2(v.1.2.2), codetools(v.0.2-16), knitr(v.1.25), zeallot(v.0.1.0), jsonlite(v.1.6), Rsamtools(v.2.1.7), dbplyr(v.1.4.2), graph(v.1.63.0), shiny(v.1.4.0), BiocManager(v.1.30.8), compiler(v.3.6.1), httr(v.1.4.1), backports(v.1.1.5), assertthat(v.0.2.1), Matrix(v.1.2-17), fastmap(v.1.0.1), lazyeval(v.0.2.2), later(v.1.0.0), formatR(v.1.7), htmltools(v.0.4.0), prettyunits(v.1.0.2), tools(v.3.6.1), gtable(v.0.3.0), glue(v.1.3.1), dplyr(v.0.8.3), rappdirs(v.0.3.1), Rcpp(v.1.0.2), vctrs(v.0.2.0), Biostrings(v.2.53.2), rtracklayer(v.1.45.6), iterators(v.1.0.12), xfun(v.0.10), stringr(v.1.4.0), openxlsx(v.4.1.0.1), testthat(v.2.2.1), rvest(v.0.3.4), mime(v.0.7), lifecycle(v.0.1.0), XML(v.3.98-1.20), AnnotationHub(v.2.17.10), zlibbioc(v.1.31.0), scales(v.1.0.0), rBiopaxParser(v.2.25.0), hms(v.0.5.1), promises(v.1.1.0), SummarizedExperiment(v.1.15.9), RBGL(v.1.61.0), RColorBrewer(v.1.1-2), lambda.r(v.1.2.4), yaml(v.2.2.0), curl(v.4.2), memoise(v.1.1.0), pander(v.0.6.3), ggplot2(v.3.2.1), biomaRt(v.2.41.9), stringi(v.1.4.3), RSQLite(v.2.1.2), highr(v.0.8), foreach(v.1.4.7), zip(v.2.0.4), BiocParallel(v.1.19.4), rlang(v.0.4.1), pkgconfig(v.2.0.3), matrixStats(v.0.55.0), bitops(v.1.0-6), evaluate(v.0.14), lattice(v.0.20-38), purrr(v.0.3.3), GenomicAlignments(v.1.21.7), labeling(v.0.3), bit(v.1.1-14), tidyselect(v.0.2.5), AnnotationForge(v.1.27.4), magrittr(v.1.5), R6(v.2.4.0), RUnit(v.0.4.32), DelayedArray(v.0.11.8), DBI(v.1.0.0), pillar(v.1.4.2), biocViews(v.1.53.3), RCurl(v.1.95-4.12), tibble(v.2.1.3), crayon(v.1.3.4), futile.options(v.1.0.1), BiocFileCache(v.1.9.1), rmarkdown(v.1.16), progress(v.1.2.2), grid(v.3.6.1), data.table(v.1.12.6), blob(v.1.2.0), digest(v.0.6.22), xtable(v.1.8-4), tidyr(v.1.0.0), httpuv(v.1.5.2), openssl(v.1.4.1), munsell(v.0.5.0), AnnotationHubData(v.1.15.13) and askpass(v.1.1)