Data management
10 June 2021
Package
ORFik 1.12.3
Contents
1 Introduction
Welcome to the introduction of data management with ORFik experiment. This vignette will walk you through how to work with large amounts of sequencing data effectively in ORFik.
ORFik is an R package containing various functions for analysis of RiboSeq, RNASeq, RCP-seq, TCP-seq, Chip-seq and Cage data, we advice you to read ORFikOverview vignette, before starting this one.
1.1 Motivation
NGS libraries are becoming more and more numerous. As a bioinformatician / biologist you often work on multi-library experiments, like 6 libraries of RNA-seq and 6 Ribo-seq libraries, split on 3 conditions with 2 replicates each. Then make some plots or statistics. A lot of things can go wrong when you scale up from just 1 library to many, or even to multiple experiments.
Another problem is also that annotations like gff and fasta files combined with the NGS data, must be separately loaded. Making it possible to use wrong annotation for the NGS data.
1.2 What is an ORFik experiment?
It is an object that simplify and error correct your NGS workflow, creating a single R object that stores and controls all results relevant to a specific experiment. It contains following important parts:
- filepaths and info for each library in the experiment (for multiple files formats: bam, bed, wig, ofst, ..)
- genome annotation files of the experiment (fasta genome, index, gtf, txdb)
- organism name (for automatic GO, sequence analysis..)
- description and author information (list.experiments(), show all experiments you have made with ORFik, easy to find and load them later)
- API: ORFik supports a rich API for using the experiment,like outputLibs(experiment, type = “wig”) will load all libraries converted to wig format into R, loadTxdb(experiment) will load the txdb (gtf) of experiment, transcriptWindow() will automatically plot metacoverage of all libraries in the experiment, countTable(experiment) will load count tables, etc..)
- Safety: It is also a safety in that it verifies your experiments contain no duplicate, empty or non-accessible files. Making it almost impossible to load the wrong data. In addition it has other safety checks; comparing chromosome naming of libraries and annotation, making sure there is no mixing of chr1 vs 1 as name for chromosome 1 etc.
2 creating an ORFik experiment
Let’s say we have a human experiment, containing annotation files (.gtf and .fasta genome) + Next generation sequencing libraries (NGS-data); RNA-seq, ribo-seq and CAGE.
An example of how to make the experiment will now be shown:
First load ORFik
library(ORFik)In a normal experiment, you would usually have only bam files from alignment of your experiment to start with (and split this into 3 experiments, 1 for RNA-seq, 1 for Ribo-seq and 1 for CAGE), but to simplify this for you to replicate we use the ORFik example data.
2.1 A minimal experiment
The minimal amount of information you need to make an ORFik experiment is:
- A folder with NGS data
- A path to transcriptome annotation (gtf->slow or txdb->faster)
- A path to genome (fasta)
- A name for the experiment
# 1. Pick directory (normally a folder with your aligned bam files)
NGS.dir <- system.file("extdata", "", package = "ORFik")
# 2. .gff/.gtf location
txdb <- system.file("extdata", "annotations.gtf", package = "ORFik")
# 3. fasta genome location
fasta <- system.file("extdata", "genome.fasta", package = "ORFik")
# 4. Pick an experiment name
exper.name <- "ORFik_example_human"
list.files(NGS.dir)## [1] "QC_STATS" "annotations.gtf" "cage-seq-heart.bed.bgz"
## [4] "features.rdata" "genome.fasta" "genome.fasta.fai"
## [7] "pshifted" "ribo-seq-heart.bed.bgz" "ribo-seq.bam"
## [10] "ribo-seq.bam.bai" "rna-seq-heart.bed.bgz"Experiments are created by all accepted files from a folder (file extension given by type argument, default: bam, bed, wig), so remember to keep your experiment folder clean of other NGS libraries not related to the experiment.
# This experiment is intentionally malformed, so we first make only a template:
template <- create.experiment(dir = NGS.dir, # directory of the NGS files for the experiment
exper.name, # Experiment name
txdb = txdb, # gtf / gff / gff.db annotation
fa = fasta, # Fasta genome
organism = "Homo sapiens", # Scientific naming
saveDir = NULL, # Create template instead of ready experiment
)
# The experiment contains 3 main parts:
# 1. Annotation, organism, general info:
data.frame(template)[1:3, ]## X1 X2 X3 X4
## 1 name ORFik_example_human
## 2 gff /tmp/Rtmp3rd1iQ/Rinst2ffde860b8e3ae/ORFik/extdata/annotations.gtf
## 3 fasta /tmp/Rtmp3rd1iQ/Rinst2ffde860b8e3ae/ORFik/extdata/genome.fasta
## X5 X6
## 1
## 2 organism Homo sapiens
## 3# 2. NGS data set-up info:
data.frame(template)[4:8, 1:5]## X1 X2 X3 X4 X5
## 4 libtype stage rep condition fraction
## 5 CAGE heart
## 6 RFP heart
## 7 RFP
## 8 RNA heart# 3. NGS File paths:
data.frame(template)[4:8, 6]## [1] "filepath"
## [2] "/tmp/Rtmp3rd1iQ/Rinst2ffde860b8e3ae/ORFik/extdata/cage-seq-heart.bed.bgz"
## [3] "/tmp/Rtmp3rd1iQ/Rinst2ffde860b8e3ae/ORFik/extdata/ribo-seq-heart.bed.bgz"
## [4] "/tmp/Rtmp3rd1iQ/Rinst2ffde860b8e3ae/ORFik/extdata/ribo-seq.bam"
## [5] "/tmp/Rtmp3rd1iQ/Rinst2ffde860b8e3ae/ORFik/extdata/rna-seq-heart.bed.bgz"You see from the template, it excludes files with .bai or .fai, .rdata etc, and only using data of NGS libraries, defined by argument (type).
You can also see it tries to guess library types, stages, replicates, condition etc. It will also try to auto-detect paired end bam files.
2.2 Fixing or updating an experiment
Since every NGS file in a experiment must be a unique set of information columns ( there can not be 2 RNA-seq libraries from wildtype that are replicate1 etc), the create.experiment function will intentionally abort if it can not distinguish all the libraries in some way. (Example: It might find 2 files that are categorized as RNA-seq replicate 1, but the condtion: Wild type vs crispr mutant was not auto-detected), so the files would be non-unique.
To fix the things it did not find (a condition not specified, etc), there are 3 ways:
- Specify the values manually in create.experiment. Example: condition = rep(c(“WT”, “Mutant”), each = 3)
- Open the file in Excel / Libre office and edit
- Edit the template (only applies if object was not directly saved, like in this vignette)
Let’s update the template to have correct tissue-fraction in one of the samples.
template$X5[6] <- "heart_valve" # <- fix non unique row (tissue fraction is heart valve)
df <- read.experiment(template)# read experiment from templateNormally you read experiments saved to disc, if you made only a template, save it by doing:
save.experiment(df, file = "path/to/save/experiment.csv")You can then load the experiment whenever you need it.
3 The experiment object
To see the object, just show it like this:
df## experiment: ORFik_example_human with 3 library types and 4 runs
## libtype stage fraction
## 1: CAGE heart
## 2: RFP heart heart_valve
## 3: RFP
## 4: RNA heartYou see here that file paths are hidden, you can access them like this:
filepath(df, type = "default")## [1] "/tmp/Rtmp3rd1iQ/Rinst2ffde860b8e3ae/ORFik/extdata/cage-seq-heart.bed.bgz"
## [2] "/tmp/Rtmp3rd1iQ/Rinst2ffde860b8e3ae/ORFik/extdata/ribo-seq-heart.bed.bgz"
## [3] "/tmp/Rtmp3rd1iQ/Rinst2ffde860b8e3ae/ORFik/extdata/ribo-seq.bam"
## [4] "/tmp/Rtmp3rd1iQ/Rinst2ffde860b8e3ae/ORFik/extdata/rna-seq-heart.bed.bgz"If you have varying version of libraries, like p-shifted, bam, simplified wig files, you can get filepaths to different version with this function.
3.1 Loading data from experiment
3.1.1 Loading NGS data
There are 2 ways to load NGS data, the first one is to load data into an environment. By default all libraries are loaded into .GlobalEnv (global environment) with names decided by columns in experiment, to see what the names will be, do:
bamVarName(df) #This will be the names:## [1] "CAGE_heart" "RFP_heart_fheart_valve" "RFP"
## [4] "RNA_heart"Now let’s auto-load the libraries to the global environment
outputLibs(df)## Outputting libraries from: ORFik_example_humanThe second way is to load manually, more secure, but also more cumbersome.
files <- filepath(df, type = "default")
CAGE_loaded_manually <- fimport(files[1])If you use the auto-loading and you have multiple experiments, it might be a chance of non-unique naming, 2 experiments might have a library called cage. To be sure names are unique, we add the experiment name in the variable name:
df@expInVarName <- TRUE
bamVarName(df) #This will be the names:## [1] "ORFik_example_human_CAGE_heart"
## [2] "ORFik_example_human_RFP_heart_fheart_valve"
## [3] "ORFik_example_human_RFP"
## [4] "ORFik_example_human_RNA_heart"You see here that the experiment name, “ORFik” is in the variable name If you are only working on one experiment, you do not need to include the name, since there is no possibility of duplicate naming (the experiment class validates all names are unique).
Since we want NGS data names without “ORFik”, let’s remove the loaded libraries and load them again.
df@expInVarName <- FALSE
remove.experiments(df)## Removed loaded libraries from experiment:ORFik_example_humanoutputLibs(df) ## Outputting libraries from: ORFik_example_human3.1.2 Loading Annotation and specific regions
There is also many function to load specific parts of the annotation:
txdb <- loadTxdb(df) # transcript annotation## Import genomic features from the file as a GRanges object ... OK
## Prepare the 'metadata' data frame ... OK
## Make the TxDb object ... OKLet’s say we want to load all leaders, cds and 3’ UTRs that are longer than 30. With ORFik experiment this is easy:
txNames <- filterTranscripts(txdb, minFiveUTR = 30, minCDS = 30, minThreeUTR = 30)
loadRegions(txdb, parts = c("leaders", "cds", "trailers"), names.keep = txNames)The regions are now loaded into .GlobalEnv, only keeping transcripts from txNames.
3.2 Plotting with ORFik experiments
ORFik supports a myriad of plots for experiments. Lets make a plot with coverage over mrna, seperated by 5’ UTR, CDS and 3’ UTR in one of the ribo-seq libraries from the experiment
transcriptWindow(leaders, cds, trailers, df[3,])## RFP## [[1]]
##
## [[2]]
4 P-site shifting experiment
If your experiment consists of Ribo-seq, you want to do p-site shifting.
shiftFootprintsByExperiment(df[df$libtype == "RFP",])P-shifted ribo-seq will automaticly be stored as .wig (wiggle files for IGV and other genome browsers) and .ofst (ORFik serialized for R) files in a ./pshifted folder, relative to original libraries.
To validate p-shifting, use shiftPlots. Here is an example from Bazzini et al. 2014 I made.
df.baz <- read.experiment("zf_bazzini14_RFP")
shiftPlots(df.baz, title = "Ribo-seq, zebrafish, Bazzini et al. 2014")
p-site analysis
To see the shifts per library do:
shifts.load(df)To see the location of pshifted files:
filepath(df[df$libtype == "RFP",], type = "pshifted")To load p-shifted libraries, you can do:
outputLibs(df[df$libtype == "RFP",], type = "pshifted")4.1 Converting bam files to faster formats
Bam files are slow to load, and usually you don’t need all the information contained in a bam file.
Usually you convert to bed or wig files, but ORFik also support 2 formats for much faster loading and use of data.
4.1.1 ofst: ORFik serialized format
From the bam file store these columns as a serialized file: seqname, start, cigar, strand, score (number of identical replicates for that read).
This is the fastest format to use, loading time of 10GB Ribo-seq bam file reduced from minutes to ~ 1 second and ~ 20MB size.
4.1.2 bedo: bed ORFik file
From the bam file store these columns as text file: seqname, start, end (if not all widths are 1), strand, score (number of identical replicates for that read), size (size of cigar Ms according to reference)
The R object loaded from these files are GRanges, since cigar is not needed.
Loading time of 10GB Ribo-seq bam file reduced to ~ 10 seconds and ~ 100MB size.
4.1.3 bedoc: bed ORFik file with cigar
From the bam file store these columns as text file: seqname, cigar, start, strand, score (number of identical replicates for that read)
The R object loaded from these files are GAlignments or GAlignmentPairs, since cigar is needed.
Loading time of 10GB Ribo-seq bam file reduced to ~ 15 seconds and ~ 200MB size.
5 ORFik QC report
ORFik also support a full QC report for post alignment statistics, correlation plots, simplified libraries for plotting, meta coverage, ++.
5.1 General report
The default QC report:
QCreport(df)The plots and statistics are saved to disc. To see the statistics, you can do:
QCstats(df)5.2 Ribo-seq specific QC
In addition there is a QC report for Ribo-seq, with some addition analysis of read lengths and frames. This should only be run on when you have pshifted the reads.
RiboQC.plot(df)6 Using the ORFik experiment system in your script
Usually you want to do some operation on multiple data-sets. If ORFik does not include a premade function for what you want, you can make it yourself. If your data is in the format of an ORFik experiment, this operation is simple.
6.1 Looping over all libraries in experiment
There are 3 ways to run loops for the data:
- if you know you have enough memory to load all data at once
outputLibs(df, type = "pshifted") # Output all libraries, fastest way
libs <- bamVarName(df) # <- here are names of the libs that were outputed
cds <- loadRegion(df, "cds")
# parallel loop
bplapply(libs, FUN = function(lib, cds) {
return(entropy(cds, get(lib)))
}, cds = cds)- Not loading data to global environment
files <- filepath(df, type = "pshifted")
cds <- loadRegion(df, "cds")
# parallel loop
res <- bplapply(files, FUN = function(file, cds) {
return(entropy(cds, fimport(file)))
}, cds = cds)- No parallel evaluation
files <- filepath(df, type = "pshifted")
cds <- loadRegion(df, "cds")
# Single thread loop
lapply(files, FUN = function(file, cds) {
return(entropy(cds, fimport(file)))
}, cds = cds)6.1.1 Reformat output to data.table (merge column-wise)
Since the output from the above loops will output lists, a very fast conversion to data.table can be done with:
library(data.table)
outputLibs(df, type = "pshifted")
libs <- bamVarName(df) # <- here are names of the libs that were outputed
cds <- loadRegion(df, "cds")
# parallel loop
res <- bplapply(libs, FUN = function(lib, cds) {
return(entropy(cds, get(lib)))
}, cds = cds)
res.by.columns <- copy(res)
# Add some names and convert
names(res.by.columns) <- libs
data.table::setDT(res.by.columns) # Will give 1 column per library
res.by.columns # Now by columns6.1.2 Reformat output to data.table (merge row-wise)
To merge row-wise do:
res.by.rows <- copy(res)
# Add some names and convert
names(res.by.rows) <- libs
res.by.rows <- rbindlist(res.by.rows) # Will bind rows per library
res.by.columns # now melted row-wise7 Conclusion
ORFik contains a whole API for using the ORFik.experiment class to simplify coding over experiments. More examples of use shown in documentation and in the Annotation_Alignment and Ribo-seq pipeline vignettes.