---
title: "VAR-Seq Workflow Template"
author: "Author: First Last Name"
date: "Last update: `r format(Sys.time(), '%d %B, %Y')`"
output:
BiocStyle::html_document:
toc_float: true
code_folding: show
package: systemPipeR
vignette: |
%\VignetteEncoding{UTF-8}
%\VignetteIndexEntry{WF: VAR-Seq Template}
%\VignetteEngine{knitr::rmarkdown}
fontsize: 14pt
bibliography: bibtex.bib
---
```{css, echo=FALSE}
pre code {
white-space: pre !important;
overflow-x: scroll !important;
word-break: keep-all !important;
word-wrap: initial !important;
}
```
```{r style, echo = FALSE, results = 'asis'}
BiocStyle::markdown()
options(width = 60, max.print = 1000)
knitr::opts_chunk$set(
eval = as.logical(Sys.getenv("KNITR_EVAL", "TRUE")),
cache = as.logical(Sys.getenv("KNITR_CACHE", "TRUE")),
tidy.opts = list(width.cutoff = 60), tidy = TRUE
)
```
```{r setup, echo=FALSE, message=FALSE, warning=FALSE, eval=TRUE}
suppressPackageStartupMessages({
library(systemPipeR)
})
```
# Introduction
## Overview
This workflow template is for analyzing VAR-Seq data. It is provided by
[systemPipeRdata](https://bioconductor.org/packages/devel/data/experiment/html/systemPipeRdata.html),
a companion package to [systemPipeR](https://www.bioconductor.org/packages/devel/bioc/html/systemPipeR.html) [@H_Backman2016-bt].
Similar to other `systemPipeR` workflow templates, a single command generates
the necessary working environment. This includes the expected directory
structure for executing `systemPipeR` workflows and parameter files for running
command-line (CL) software utilized in specific analysis steps. For learning
and testing purposes, a small sample (toy) data set is also included (mainly
FASTQ and reference genome files). This enables users to seamlessly run the
numerous analysis steps of this workflow from start to finish without the
requirement of providing custom data. After testing the workflow, users have
the flexibility to employ the template as is with their own data or modify it
to suit their specific needs. For more comprehensive information on designing
and executing workflows, users want to refer to the main vignettes of
[systemPipeR](https://www.bioconductor.org/packages/devel/bioc/vignettes/systemPipeR/inst/doc/systemPipeR.html)
and
[systemPipeRdata](https://www.bioconductor.org/packages/devel/data/experiment/vignettes/systemPipeRdata/inst/doc/systemPipeRdata.html).
The `Rmd` file (`systemPipeVARseq.Rmd`) associated with this vignette serves a dual purpose. It acts
both as a template for executing the workflow and as a template for generating
a reproducible scientific analysis report. Thus, users want to customize the text
(and/or code) of this vignette to describe their experimental design and
analysis results. This typically involves deleting the instructions how to work
with this workflow, and customizing the text describing experimental designs,
other metadata and analysis results.
## Experimental design
Typically, the user wants to describe here the sources and versions of the
reference genome sequence along with the corresponding annotations. The standard
directory structure of `systemPipeR` (see [here](https://www.bioconductor.org/packages/devel/bioc/vignettes/systemPipeR/inst/doc/systemPipeR.html#3_Directory_structure)),
expects the input data in a subdirectory named `data`
and all results will be written to a separate `results` directory. The Rmd source file
for executing the workflow and rendering its report (here `systemPipeVARseq.Rmd`) is
expected to be located in the parent directory.
The test (toy) data set used by this template ([SRP010938](http://www.ncbi.nlm.nih.gov/sra/?term=SRP010938))
contains 18 paired-end (PE) read sets from *Arabidposis thaliana*
[@Howard2013-fq]. To minimize processing time during testing, each FASTQ
file has been reduced to 90,000-100,000 randomly sampled PE reads that
map to the first 100,000 nucleotides of each chromosome of the *A.
thaliana* genome. The corresponding reference genome sequence (FASTA) and
its GFF annotation files have been reduced to the same genome regions. This way the entire
test sample data set is less than 200MB in storage space. A PE read set has been
chosen here for flexibility, because it can be used for testing both types
of analysis routines requiring either SE (single end) reads or PE reads.
To use their own VAR-Seq and reference genome data, users want to move or link the
data to the designated `data` directory and execute the workflow from the parent directory
using their customized `Rmd` file. Beginning with this template, users should delete the provided test
data and move or link their custom data to the designated locations.
Alternatively, users can create an environment skeleton (named `new` [here](https://www.bioconductor.org/packages/devel/data/experiment/vignettes/systemPipeRdata/inst/doc/new.html)) or
build one from scratch. To perform a VAR-Seq analysis with new FASTQ files
from the same reference genome, users only need to provide the FASTQ files and
an experimental design file called 'targets' file that outlines the experimental
design. The structure and utility of targets files is described in `systemPipeR's`
main vignette [here](https://www.bioconductor.org/packages/devel/bioc/vignettes/systemPipeR/inst/doc/systemPipeR.html#4_The_targets_file).
## Workflow steps
The default analysis steps included in this VAR-Seq workflow template are listed below. Users
can modify the existing steps, add new ones or remove steps as needed.
__Default analysis steps__
1. Read preprocessing
+ Quality filtering (trimming)
+ FASTQ quality report
2. Alignments: _`BWA`_ (or any other DNA aligner)
3. Alignment stats
4. Variant calling
+ with `GATK`, substeps 1-10
+ with `BCFtools`, 1 substep
5. Filter variants
+ Filter variants called by `GATK`
+ Filter variants called by `BCFtools`
6. Annotate filtered variants
7. Combine annotation results among samples
8. Calculate Summary statistics of variants
9. Plot Venn diagram of variants
10. Plot variants of interests
## Load workflow environment {#load-wf}
The environment for this VAR-Seq workflow is auto-generated below with the
`genWorkenvir` function (selected under `workflow="varseq"`). It is fully populated
with a small test data set, including FASTQ files, reference genome and annotation data. The name of the
resulting workflow directory can be specified under the `mydirname` argument.
The default `NULL` uses the name of the chosen workflow. An error is issued if
a directory of the same name and path exists already. After this, the user’s R
session needs to be directed into the resulting `varseq` directory (here with
`setwd`).
```{r generate_workenvir, eval=FALSE}
library(systemPipeRdata)
genWorkenvir(workflow = "varseq", mydirname = "varseq")
setwd("varseq")
```
### Input data: `targets` file
The `targets` file defines the input files (e.g. FASTQ or BAM) and sample
comparisons used in a data analysis workflow. It can also store any number of
additional descriptive information for each sample. The following shows the first
four lines of the `targets` file used in this workflow template.
```{r load_targets_file, eval=TRUE}
targetspath <- system.file("extdata", "targetsPE.txt", package = "systemPipeR")
targets <- read.delim(targetspath, comment.char = "#")
targets[1:4,-c(5,6)]
```
To work with custom data, users need to generate a _`targets`_ file containing
the paths to their own FASTQ files. [Here](https://www.bioconductor.org/packages/devel/bioc/vignettes/systemPipeR/inst/doc/systemPipeR.html#4_The_targets_file) is a detailed description of the structure and
utility of `targets` files.
# Quick start {#quick-start}
After a workflow environment has been created with the above `genWorkenvir`
function call and the corresponding R session directed into the resulting directory (here `varseq`),
the `SPRproject` function is used to initialize a new workflow project instance. The latter
creates an empty `SAL` workflow container (below `sal`) and at the same time a
linked project log directory (default name `.SPRproject`) that acts as a
flat-file database of a workflow. Additional details about this process and
the SAL workflow control class are provided in `systemPipeR's` main vignette
[here](https://www.bioconductor.org/packages/devel/bioc/vignettes/systemPipeR/inst/doc/systemPipeR.html#11_Workflow_control_class)
and [here](https://www.bioconductor.org/packages/devel/bioc/vignettes/systemPipeR/inst/doc/systemPipeR.html#5_Detailed_tutorial).
Next, the `importWF` function imports all the workflow steps outlined in the
source Rmd file of this vignette (here `systemPipeVARseq.Rmd`) into the `SAL` workflow container.
An overview of the workflow steps and their status information can be returned
at any stage of the loading or run process by typing `sal`.
```{r project_varseq, eval=FALSE}
library(systemPipeR)
sal <- SPRproject()
sal <- importWF(sal, file_path = "systemPipeVARseq.Rmd", verbose = FALSE)
sal
```
After loading the workflow into `sal`, it can be executed from start to finish
(or partially) with the `runWF` command. Running the workflow will only be
possible if all dependent CL software is installed on a user's system. Their
names and availability on a system can be listed with `listCmdTools(sal,
check_path=TRUE)`. For more information about the `runWF` command, refer to the
help file and the corresponding section in the main vignette
[here](https://www.bioconductor.org/packages/devel/bioc/vignettes/systemPipeR/inst/doc/systemPipeR.html#61_Overview).
Running workflows in parallel mode on computer clusters is a straightforward
process in `systemPipeR`. Users can simply append the resource parameters (such
as the number of CPUs) for a cluster run to the `sal` object after importing
the workflow steps with `importWF` using the `addResources` function. More
information about parallelization can be found in the corresponding section at
the end of this vignette [here](#paralellization) and in the main vignette
[here](https://www.bioconductor.org/packages/devel/bioc/vignettes/systemPipeR/inst/doc/systemPipeR.html#63_Parallel_evaluation).
```{r run_varseq, eval=FALSE}
sal <- runWF(sal)
```
Workflows can be visualized as topology graphs using the `plotWF` function.
```{r plot_varseq, eval=FALSE}
plotWF(sal)
```
```{r varseq-toplogy, eval=TRUE, warning= FALSE, echo=FALSE, out.width="100%", fig.align = "center", fig.cap= "Toplogy graph of VAR-Seq workflow.", warning=FALSE}
knitr::include_graphics("results/plotwf_varseq.png")
```
Scientific and technical reports can be generated with the `renderReport` and
`renderLogs` functions, respectively. Scientific reports can also be generated
with the `render` function of the `rmarkdown` package. The technical reports are
based on log information that `systemPipeR` collects during workflow runs.
```{r report_varseq, eval=FALSE}
# Scientific report
sal <- renderReport(sal)
rmarkdown::render("systemPipeVARseq.Rmd", clean = TRUE, output_format = "BiocStyle::html_document")
# Technical (log) report
sal <- renderLogs(sal)
```
The `statusWF` function returns a status summary for each step in a `SAL` workflow instance.
```{r status_varseq, eval=FALSE}
statusWF(sal)
```
# Workflow steps
The data analysis steps of this workflow are defined by the following workflow code chunks.
They can be loaded into `SAL` interactively, by executing the code of each step in the
R console, or all at once with the `importWF` function used under the Quick start section.
R and CL workflow steps are declared in the code chunks of `Rmd` files with the
`LineWise` and `SYSargsList` functions, respectively, and then added to the `SAL` workflow
container with `appendStep<-`. Their syntax and usage is described
[here](https://www.bioconductor.org/packages/devel/bioc/vignettes/systemPipeR/inst/doc/systemPipeR.html#52_Constructing_workflows).
## Required packages and resources
The first step loads the `systemPipeR` package.
```{r load_SPR, message=FALSE, eval=FALSE, spr=TRUE}
# Some samples in the test dataset do not work well in VARseq, and VARseq workflow
# takes long time to process each sample. To better test and speed up the test workflow,
# sample set is reduced to the first 13 samples.
# Please REMOVE the next two lines in your real analysis
cat(crayon::red$bold("Some samples in targets are removed for test workflow. Please change the template to disable this in your real analysis.\n"))
writeLines(readLines("targetsPE.txt")[1:13], "targetsPE.txt")
cat(crayon::blue$bold("To use this workflow, following R packages are expected:\n"))
cat(c("'GenomicFeatures", "VariantAnnotation", "GenomicFeatures", "ggbio", "ggplot2'\n"), sep = "', '")
###pre-end
appendStep(sal) <- LineWise(
code = {
library(systemPipeR)
},
step_name = "load_SPR"
)
```
## FASTQ quality report
The following `seeFastq` and `seeFastqPlot` functions generate and plot a series of useful
quality statistics for a set of FASTQ files including per cycle quality box
plots, base proportions, base-level quality trends, relative k-mer
diversity, length, and occurrence distribution of reads, number of reads
above quality cutoffs and mean quality distribution. The results are
written to a png file named `fastqReport.png`.
This is the pre-trimming fastq report. Another post-trimming fastq report step
is not included in the default. It is recommended to run this step first to
decide whether the trimming is needed.
Please note that initial targets files are being used here. In this case,
it has been added to the first step, and later, we used the function `getColumn`
to extract a named vector.
```{r fastq_report_pre, eval=FALSE, message=FALSE, spr=TRUE}
appendStep(sal) <- LineWise(
code = {
targets <- read.delim("targetsPE.txt", comment.char = "#")
updateColumn(sal, step = "load_SPR", position = "targetsWF") <- targets
fq_files <- getColumn(sal, "load_SPR", "targetsWF", column = 1)
fqlist <- seeFastq(fastq = fq_files, batchsize = 10000, klength = 8)
png("./results/fastqReport.png", height = 162, width = 288 * length(fqlist))
seeFastqPlot(fqlist)
dev.off()
},
step_name = "fastq_report_pre",
dependency = "load_SPR"
)
```
Figure 1: FASTQ quality report for 18 samples
## Read preprocessing
### Read trimming with Trimmomatic
Next, we need to populate the object created with the first step in the
workflow. Here, an example of how to perform this task using parameters template
files for trimming FASTQ files with [Trimmomatic](http://www.usadellab.org/cms/?page=trimmomatic)
software [@Bolger2014-yr].
For this step, the `SYSargsList` function has been used to build the command-line
and append to `sal` object. For more details of all the features and utilities,
please consult the [main vignette](http://www.bioconductor.org/packages/release/bioc/vignettes/systemPipeR/inst/doc/systemPipeR.html).
If GATK (default) is used for variant calling, any type of fastq trimming is
strongly depreciated. GATK have internal function to handle low quality posistions.
```{r trimmomatic, eval=FALSE, spr=TRUE}
appendStep(sal) <- SYSargsList(
step_name = "trimmomatic",
targets = "targetsPE.txt",
wf_file = "trimmomatic/trimmomatic-pe.cwl",
input_file = "trimmomatic/trimmomatic-pe.yml",
dir_path = "param/cwl",
inputvars = c(
FileName1 = "_FASTQ_PATH1_",
FileName2 = "_FASTQ_PATH2_",
SampleName = "_SampleName_"
),
dependency = c("fastq_report_pre"),
run_step = "optional"
)
```
### Preprocessing with _`preprocessReads`_ function
The function _`preprocessReads`_ allows to apply predefined or custom
read preprocessing functions to all FASTQ files referenced in a
_`SYSargsList`_ container, such as quality filtering or adaptor trimming
routines. Internally, _`preprocessReads`_ uses the _`FastqStreamer`_ function from
the _`ShortRead`_ package to stream through large FASTQ files in a
memory-efficient manner. The following example performs adaptor trimming with
the _`trimLRPatterns`_ function from the _`Biostrings`_ package.
Here, we are appending this step at the _`SYSargsList`_ object created previously.
All the parameters are defined on the _`preprocessReads/preprocessReads-pe.yml`_ file.
```{r preprocessing, message=FALSE, eval=FALSE, spr=TRUE}
appendStep(sal) <- SYSargsList(
step_name = "preprocessing",
targets = "targetsPE.txt", dir = TRUE,
wf_file = "preprocessReads/preprocessReads-pe.cwl",
input_file = "preprocessReads/preprocessReads-pe.yml",
dir_path = "param/cwl",
inputvars = c(
FileName1 = "_FASTQ_PATH1_",
FileName2 = "_FASTQ_PATH2_",
SampleName = "_SampleName_"
),
dependency = c("fastq_report_pre"),
run_step = "optional"
)
```
After the trimming step, the `outfiles` files can be used to generate the new
targets files containing the paths to the trimmed FASTQ files. The new targets
information can be used for the next workflow step instance, _e.g._ running the
NGS alignments with the trimmed FASTQ files.
The following example shows how one can design a custom read _'preprocessReads'_
function using utilities provided by the _`ShortRead`_ package, and then run it
in batch mode with the _'preprocessReads'_ function. For here, it is possible to
replace the function used on the `preprocessing` step and modify the `sal` object.
Because it is a custom function, it is necessary to save the part in the R object,
and internally the `preprocessReads.doc.R` is loading the function. If the R object
is saved with a different name (here `"param/customFCT.RData"`), please replace
that accordingly in the `preprocessReads.doc.R`.
Please, note that this step is not added to the workflow, here just for demonstration.
First, we defined the function in the workflow:
```{r custom_preprocessing_function, eval=FALSE}
appendStep(sal) <- LineWise(
code = {
filterFct <- function(fq, cutoff = 20, Nexceptions = 0) {
qcount <- rowSums(as(quality(fq), "matrix") <= cutoff, na.rm = TRUE)
# Retains reads where Phred scores are >= cutoff with N exceptions
fq[qcount <= Nexceptions]
}
save(list = ls(), file = "param/customFCT.RData")
},
step_name = "custom_preprocessing_function",
dependency = "preprocessing"
)
```
After, we can edit the input parameter:
```{r editing_preprocessing, message=FALSE, eval=FALSE}
yamlinput(sal, "preprocessing")$Fct
yamlinput(sal, "preprocessing", "Fct") <- "'filterFct(fq, cutoff=20, Nexceptions=0)'"
yamlinput(sal, "preprocessing")$Fct ## check the new function
cmdlist(sal, "preprocessing", targets = 1) ## check if the command line was updated with success
```
## FASTQ quality after Trimming
This is the post-trimming fastq quality report. If the trimming step is included, it is
recommended to add this step to compare trimming of fastq before and after.
```{r fastq_report_pos, eval=FALSE, message=FALSE, spr=TRUE}
appendStep(sal) <- LineWise(
code = {
fq_files <- getColumn(sal, "preprocessing", "outfiles", column = 1) ## get outfiles path
fqlist <- seeFastq(fastq = fq_files, batchsize = 10000, klength = 8)
png("./results/fastqReport_pos.png", height = 18, width = 4 * length(fqlist))
seeFastqPlot(fqlist)
dev.off()
},
step_name = "fastq_report_pos",
dependency = "trimmomatic",
run_step = "optional"
)
```
## Read mapping with `BWA-MEM`
The NGS reads of this project are aligned against the reference genome
sequence using the highly variant tolerant short read aligner `BWA-MEM`
[@Li2013-oy; @Li2009-oc]. The parameter settings of the aligner are
defined in the `param/cwl/gatk/bwa-pe.cwl`.
This test code uses untrimmed fastq files since the demo data is minimal and
limited. However, it is best to test with `FASTQ quality report` function provided
above to verify your real data first.
### Build index and dictionary files for BWA and GATK
Build the index and dictionary files for BWA and GATK to run.
```{r bwa_index, eval=FALSE, spr=TRUE}
appendStep(sal) <- SYSargsList(
step_name = "bwa_index",
dir = FALSE, targets = NULL,
wf_file = "gatk/workflow_bwa-index.cwl",
input_file = "gatk/gatk.yaml",
dir_path = "param/cwl",
dependency = "load_SPR"
)
```
Create reference `fasta` dictionary.
```{r fasta_index, eval=FALSE, spr=TRUE}
appendStep(sal) <- SYSargsList(
step_name = "fasta_index",
dir = FALSE, targets = NULL,
wf_file = "gatk/workflow_fasta_dict.cwl",
input_file = "gatk/gatk.yaml",
dir_path = "param/cwl",
dependency = "bwa_index"
)
```
Create dictionary index.
```{r faidx_index, eval=FALSE, spr=TRUE}
appendStep(sal) <- SYSargsList(
step_name = "faidx_index",
dir = FALSE, targets = NULL,
wf_file = "gatk/workflow_fasta_faidx.cwl",
input_file = "gatk/gatk.yaml",
dir_path = "param/cwl",
dependency = "fasta_index"
)
```
### Mapping reads with BWA
```{r bwa_alignment, eval=FALSE, spr=TRUE}
appendStep(sal) <- SYSargsList(
step_name = "bwa_alignment",
targets = "targetsPE.txt",
wf_file = "gatk/workflow_bwa-pe.cwl",
input_file = "gatk/gatk.yaml",
dir_path = "param/cwl",
inputvars = c(
FileName1 = "_FASTQ_PATH1_",
FileName2 = "_FASTQ_PATH2_",
SampleName = "_SampleName_"
),
dependency = c("faidx_index")
)
```
## Read and alignment stats
The following provides an overview of the number of reads in each sample
and how many of them aligned to the reference.
```{r align_stats, eval=FALSE, spr=TRUE}
appendStep(sal) <- LineWise(
code = {
bampaths <- getColumn(sal, step = "bwa_alignment", "outfiles", column = "samtools_sort_bam")
fqpaths <- getColumn(sal, step = "bwa_alignment", "targetsWF", column = "FileName1")
read_statsDF <- alignStats(args = bampaths, fqpaths = fqpaths, pairEnd = TRUE)
write.table(read_statsDF, "results/alignStats.xls", row.names = FALSE, quote = FALSE, sep = "\t")
},
step_name = "align_stats",
dependency = "bwa_alignment",
run_step = "optional"
)
```
## Create symbolic links for viewing BAM files in IGV
The `symLink2bam` function creates symbolic links to view the BAM alignment files in a
genome browser such as IGV. The corresponding URLs are written to a file
with a path specified under `urlfile` in the `results` directory.
```{r bam_urls, eval=FALSE, spr=TRUE}
appendStep(sal) <- LineWise(
code = {
bampaths <- getColumn(sal, step = "bwa_alignment", "outfiles", column = "samtools_sort_bam")
symLink2bam(
sysargs = bampaths, htmldir = c("~/.html/", "somedir/"),
urlbase = "http://cluster.hpcc.ucr.edu/~tgirke/",
urlfile = "./results/IGVurl.txt"
)
},
step_name = "bam_urls",
dependency = "bwa_alignment",
run_step = "optional"
)
```
## Variant calling
The following performs variant calling with `GATK` and `BCFtools` on a single
machine by `runWF` function for each sample sequentially. If a cluster compute
is available, running in parallel mode on a compute cluster can be performed by
`runWF`, making available the resources and choose `run_session = "compute"`.
Not all users have a cluster system, so here to demonstrate an example of variant calling
workflow, only single-machine commands are shown. For cluster jobs, please refer
to our main [vignette](http://www.bioconductor.org/packages/release/bioc/vignettes/systemPipeR/inst/doc/systemPipeR.html).
In addition, the user would choose only one variant caller here rather than
running several ones. However, the workflow manager allows keeping multiple
options available for running the analysis.
### Variant calling with `GATK`
The following steps are based on `GATK 4.1.1.0` [Best Practice](https://software.broadinstitute.org/gatk/best-practices/).
There are 10 individual steps where the user can choose where to jump in and where to skip.
All scripts are located at `param/cwl/gatk`. `BQSR` (Base Quality Score Recalibration)
and `VQSR` (Variant Quality Score Recalibration) are very specific
to a limited species like human, so this workflow does not support these steps.
### Step1: `fastq` to `ubam`
Convert `fastq` files to `bam` files to prepare for the following step. It is very
important to specific your sequencing platform, default is `illumina`. User need
to change `param/cwl/gatk/gatk_fastq2ubam.cwl` if the platform is different. Platform information
is needed for the variant caller in later steps to correct calling parameters.
```{r fastq2ubam, eval=FALSE, spr=TRUE}
appendStep(sal) <- SYSargsList(
step_name = "fastq2ubam",
targets = "targetsPE.txt",
wf_file = "gatk/workflow_gatk_fastq2ubam.cwl",
input_file = "gatk/gatk.yaml",
dir_path = "param/cwl",
inputvars = c(
FileName1 = "_FASTQ_PATH1_",
FileName2 = "_FASTQ_PATH2_",
SampleName = "_SampleName_"
),
dependency = c("faidx_index")
)
```
### Step2: Merge `bam` and `ubam`
This step merges a `bam` and `ubam` and creates a third `bam` file that contains
alignment information and remaining information that was removed by the aligner like `BWA`.
The removed information is essential for variant statistics calculation. Previous steps are
recommended, but variant calling can still be performed without these steps.
```{r merge_bam, eval=FALSE, spr=TRUE}
appendStep(sal) <- SYSargsList(
step_name = "merge_bam",
targets = c("bwa_alignment", "fastq2ubam"),
wf_file = "gatk/workflow_gatk_mergebams.cwl",
input_file = "gatk/gatk.yaml",
dir_path = "param/cwl",
inputvars = c(
bwa_men_sam = "_bwasam_",
ubam = "_ubam_",
SampleName = "_SampleName_"
),
rm_targets_col = c("preprocessReads_1", "preprocessReads_2"),
dependency = c("bwa_alignment", "fastq2ubam")
)
```
### Step3: Sort `bam` files by genomic coordinates
Sort `bam` files by genomic coordinates.
```{r sort, eval=FALSE, spr=TRUE}
appendStep(sal) <- SYSargsList(
step_name = "sort",
targets = "merge_bam",
wf_file = "gatk/workflow_gatk_sort.cwl",
input_file = "gatk/gatk.yaml",
dir_path = "param/cwl",
inputvars = c(merge_bam = "_mergebam_", SampleName = "_SampleName_"),
rm_targets_col = c(
"bwa_men_sam", "ubam", "SampleName_fastq2ubam",
"Factor_fastq2ubam", "SampleLong_fastq2ubam",
"Experiment_fastq2ubam", "Date_fastq2ubam"
),
dependency = c("merge_bam")
)
```
### Step4: Mark duplicates
Mark PCR artifacts in sequencing. A `duplicate_metrics` file will also be produced
by this step, but will not be used for the next step. This file is just for the user
to check duplicates status summary.
```{r mark_dup, eval=FALSE, spr=TRUE}
appendStep(sal) <- SYSargsList(
step_name = "mark_dup",
targets = "sort",
wf_file = "gatk/workflow_gatk_markduplicates.cwl",
input_file = "gatk/gatk.yaml",
dir_path = "param/cwl",
inputvars = c(sort_bam = "_sort_", SampleName = "_SampleName_"),
rm_targets_col = c("merge_bam"),
dependency = c("sort")
)
```
### Step5: Fixing tags
Takes the `bam` from the last step and calculates the NM, MD, and UQ tags.
These tags are important for variant calling and filtering.
This step is recommended but can be skipped.
```{r fix_tag, eval=FALSE, spr=TRUE}
appendStep(sal) <- SYSargsList(
step_name = "fix_tag",
targets = "mark_dup",
wf_file = "gatk/workflow_gatk_fixtag.cwl",
input_file = "gatk/gatk.yaml",
dir_path = "param/cwl",
inputvars = c(mark_bam = "_mark_", SampleName = "_SampleName_"),
rm_targets_col = c("sort_bam"),
dependency = c("mark_dup")
)
```
Up till this step, sample preprocess is done. All analysis ready `BAM` files and
their index `.bai` files are created. Individual and cohort calling by
`HaplotypeCaller` is performed from the next step.
### Step6: HaplotypeCaller `gvcf`
The `HaplotypeCaller` is running a **gvcf** mode in this step. G stands for 'genomic'.
The file not only contains variant sites information but also non-variant sites information;
thus, at the following step, the cohort caller can use this information to validate the true variants.
```{r hap_caller, eval=FALSE, spr=TRUE}
appendStep(sal) <- SYSargsList(
step_name = "hap_caller",
targets = "fix_tag",
wf_file = "gatk/workflow_gatk_haplotypecaller.cwl",
input_file = "gatk/gatk.yaml",
dir_path = "param/cwl",
inputvars = c(fixtag_bam = "_fixed_", SampleName = "_SampleName_"),
rm_targets_col = c("mark_bam"),
dependency = c("fix_tag")
)
```
### Step7: Import all `gvcfs`
It is recommended to import all **gvcfs** to a
[TileDB](https://github.com/Intel-HLS/GenomicsDB/wiki) database for fast cohort
variant calling at the following step. Note: if you are working with non-diploid data,
use `CombineGVCFs` function from `GATK` and change the `gvcf_db_folder` parameter
in `param/cwl/gatk/gatk.yaml` to be your combined **gvcf** file path.
**Important**: Make sure all samples' `*.g.vcf.gz` files are in the results folder,
also the `tbi index` files also should be there.
```{r import, eval=FALSE, spr=TRUE}
appendStep(sal) <- SYSargsList(
step_name = "import",
targets = NULL, dir = FALSE,
wf_file = "gatk/workflow_gatk_genomicsDBImport.cwl",
input_file = "gatk/gatk.yaml",
dir_path = "param/cwl",
dependency = c("hap_caller")
)
```
### Step8: Cohort calling of `gvcf`
Assess variants by information from all `gvcfs`. A collective `vcf` called
`samples.vcf.gz` is created by default naming.
```{r call_variants, eval=FALSE, spr=TRUE}
appendStep(sal) <- SYSargsList(
step_name = "call_variants",
targets = NULL, dir = FALSE,
wf_file = "gatk/workflow_gatk_genotypeGVCFs.cwl",
input_file = "gatk/gatk.yaml",
dir_path = "param/cwl",
dependency = c("import")
)
```
### Step9: Cohort hard filter variants
Variant Quality Score Recalibration (VQSR) is not included in this workflow.
Variants are hard filtered together.
See this [Post](https://gatkforums.broadinstitute.org/gatk/discussion/2806/howto-apply-hard-filters-to-a-call-set) for parameters for hard filtering. Change these settings in `param/cwl/gak/gatk_variantFiltration.sh` if needed. VQSR requires a large quantity of
samples to be training data before you can do filtering. Read this
[post](https://gatk.broadinstitute.org/hc/en-us/articles/360035531612-Variant-Quality-Score-Recalibration-VQSR-) for more information.
```{r filter, eval=FALSE, spr=TRUE}
appendStep(sal) <- SYSargsList(
step_name = "filter",
targets = NULL, dir = FALSE,
wf_file = "gatk/workflow_gatk_variantFiltration.cwl",
input_file = "gatk/gatk.yaml",
dir_path = "param/cwl",
dependency = c("call_variants")
)
```
### Step10: Extract variant
After cohort calling, filtering, all variants for all samples are stored in one big file.
Extract variants for each sample and save them separately (only variants that have
passed the filters are stored).
```{r create_vcf, eval=FALSE, spr=TRUE}
appendStep(sal) <- SYSargsList(
step_name = "create_vcf",
targets = "hap_caller",
wf_file = "gatk/workflow_gatk_select_variant.cwl",
input_file = "gatk/gatk.yaml",
dir_path = "param/cwl",
inputvars = c(SampleName = "_SampleName_"),
dependency = c("hap_caller", "filter")
)
```
## Variant calling with `BCFtools`
Alternative option with `BCFtool`:
The following runs the variant calling with `BCFtools`. This tool takes `BWA`
aligned `BAM` files, sort, mark duplicates by `samtools` and finally call variants
by `BCFtools`.
**For legacy reasons we keep this option.**
```{r create_vcf_BCFtool, eval=FALSE, spr=TRUE}
appendStep(sal) <- SYSargsList(
step_name = "create_vcf_BCFtool",
targets = "bwa_alignment", dir = TRUE,
wf_file = "workflow-bcftools/workflow_bcftools.cwl",
input_file = "workflow-bcftools/bcftools.yml",
dir_path = "param/cwl",
inputvars = c(bwa_men_sam = "_bwasam_", SampleName = "_SampleName_"),
rm_targets_col = c("preprocessReads_1", "preprocessReads_2"),
dependency = "bwa_alignment",
run_step = "optional"
)
```
Variant calling ends here. Downstream analysis starts from the next section.
## Inspect VCF file
Scripts of downstream analysis are stored in `param/cwl/varseq_downstream`.
**optional**: This step is not included in the default workflow. After successfully
execute the entire workflow, users may load individual vcf files to R for
other analysis like below.
VCF files can be imported into R with the `readVcf` function.
Both `VCF` and `VRanges` objects provide convenient data structure for
working with variant data (_e.g._ SNP quality filtering).
This step is not included in the default workflow steps, but can be useful to
inspect individual sample's raw variants.
```{r inspect_vcf, eval=FALSE}
library(VariantAnnotation)
vcf_raw <- getColumn(sal, "create_vcf")
vcf <- readVcf(vcf_raw[1], "A. thaliana")
vcf
vr <- as(vcf, "VRanges")
vr
```
## Filter variants
The function `filterVars` filters VCF files based on user definable
quality parameters. It sequentially imports each VCF file into R, applies the
filtering on an internally generated `VRanges` object and then writes
the results to a new subsetted VCF file. The filter parameters are passed on to
the corresponding argument as a character string. The function applies this
filter to the internally generated `VRanges` object using the standard
subsetting syntax for two dimensional objects such as: `vr[filter, ]`.
### Filter variants called by `GATK`
The below example filters for variants that are supported by `>=x`
reads and >=80% of them support the called variants. In addition, all
variants need to pass `>=x` of the soft filters recorded in the VCF
files generated by GATK. Since the toy data used for this workflow is
very small, the chosen settings are unreasonabley relaxed. A more
reasonable filter setting is given in the line below (here commented
out).
There is already some cohort filtering in GATK step 10. Some additional hard
filtering is provided here. This step is included here, but in a real analysis,
you may skip this step.
For real samples, use following filters:
`filter <- "totalDepth(vr) >= 20 & (altDepth(vr) / totalDepth(vr) >= 0.8)"`
```{r filter_vcf, eval=FALSE, spr=TRUE}
appendStep(sal) <- LineWise(
code = {
vcf_raw <- getColumn(sal, "create_vcf")
library(VariantAnnotation)
filter <- "totalDepth(vr) >= 2 & (altDepth(vr) / totalDepth(vr) >= 0.8)"
vcf_filter <- suppressWarnings(filterVars(vcf_raw, filter, organism = "A. thaliana", out_dir = "results/vcf_filter"))
# dump the filtered path variable to running enviornment so
# other sysArg steps can get its values
updateColumn(sal, 'create_vcf', "outfiles") <- data.frame(vcf_filter=vcf_filter)
},
step_name = "filter_vcf",
dependency = "create_vcf"
)
```
### Filter variants called by `BCFtools`
The following shows how to filter the VCF files generated by `BCFtools` using
similar parameter settings as in the previous filtering of the GATK
results.
```{r filter_vcf_BCFtools, eval=FALSE, spr=TRUE}
appendStep(sal) <- LineWise(
code = {
vcf_raw <- getColumn(sal, step = "create_vcf_BCFtool",
position = "outfiles", column = "bcftools_call")
library(VariantAnnotation)
filter <- "rowSums(vr) >= 2 & (rowSums(vr[,3:4])/rowSums(vr[,1:4]) >= 0.8)"
vcf_filter_bcf <- suppressWarnings(filterVars(vcf_raw, filter, organism = "A. thaliana", out_dir = "results/vcf_filter_BCFtools", varcaller = "bcftools"))
updateColumn(sal, 'create_vcf', "outfiles") <- data.frame(vcf_filter_bcf=vcf_filter_bcf)
},
step_name = "filter_vcf_BCFtools",
dependency = "create_vcf_BCFtool",
run_step = "optional"
)
```
Check filtering outcome for one sample
This mini step can be used to compare `vcfs` files before and after filtering.
This can be used once the workflow has been run, and make sure "filter_vcf" is
done, since it is an optional step.
```{r check_filter, eval=FALSE}
copyEnvir(sal, "vcf_raw", globalenv())
copyEnvir(sal, "vcf_filter", globalenv())
length(as(readVcf(vcf_raw[1], genome = "Ath"), "VRanges")[, 1])
length(as(readVcf(vcf_filter[1], genome = "Ath"), "VRanges")[, 1])
```
## Annotate filtered variants
The function `variantReport` generates a variant report using
utilities provided by the `VariantAnnotation` package. The report for
each sample is written to a tabular file containing genomic context annotations
(_e.g._ coding or non-coding SNPs, amino acid changes, IDs of affected
genes, etc.) along with confidence statistics for each variant. The CWL
file `param/cwl/varseq_downstream/annotate.cwl` defines the paths to the input
and output files which are stored in a `SYSargs2` instance.
### Basics of annotating variants
This step can be run after running the default workflow, not included in the default.
Variants overlapping with common annotation features can be identified with `locateVariants`.
```{r annotate_basics, eval=FALSE}
library("GenomicFeatures")
# comment the next line if optional step "filter_vcf" is included
vcf_filter <- getColumn(sal, "create_vcf")
# uncomment the next line if optional step "filter_vcf" is included
# copyEnvir(sal, "vcf_filter", globalenv())
txdb <- loadDb("./data/tair10.sqlite")
vcf <- readVcf(vcf_filter[1], "A. thaliana")
locateVariants(vcf, txdb, CodingVariants())
```
Synonymous/non-synonymous variants of coding sequences are computed by the
`predictCoding` function for variants overlapping with coding regions.
```{r annotate_basics_non-synon, eval=FALSE}
fa <- FaFile("data/tair10.fasta")
predictCoding(vcf, txdb, seqSource = fa)
```
### Annotate filtered variants `GATK` or `BCFtools`
**required**
```{r annotate_vcf, eval=FALSE, spr=TRUE}
appendStep(sal) <- LineWise(
code = {
# get the filtered vcf path from R running environment
copyEnvir(sal, "vcf_filter", globalenv())
library("GenomicFeatures")
txdb <- loadDb("./data/tair10.sqlite")
fa <- FaFile("data/tair10.fasta")
vcf_anno <- suppressMessages(suppressWarnings(variantReport(vcf_filter, txdb = txdb, fa = fa, organism = "A. thaliana", out_dir = "results/vcf_anno")))
},
step_name = "annotate_vcf",
dependency = "filter_vcf"
)
```
View annotation result for single sample
```{r view_annotation, eval=FALSE}
copyEnvir(sal, "vcf_anno", globalenv())
read.delim(vcf_anno[1])[38:40, ]
```
## Combine annotation results among samples
To simplify comparisons among samples, the `combineVarReports`
function combines all variant annotation reports referenced in a
`SYSargs2` instance (here `args`). At the same time the function
allows to consider only certain feature types of interest. For instance, the
below setting `filtercol=c(Consequence="nonsynonymous")` will include
only nonsysynonymous variances listed in the `Consequence` column of
the annotation reports. To omit filtering, one can use the setting
`filtercol="All"`.
### Combine results
**required**
```{r combine_var, eval=FALSE, spr=TRUE}
appendStep(sal) <- LineWise(
code = {
combineDF <- combineVarReports(vcf_anno, filtercol = c(Consequence = "nonsynonymous"))
write.table(combineDF, "./results/combineDF_nonsyn.tsv", quote = FALSE, row.names = FALSE, sep = "\t")
},
step_name = "combine_var",
dependency = "annotate_vcf"
)
```
## Summary statistics of variants
The `varSummary` function counts the number of variants for each feature type
included in the annotation reports.
## Summary of variants
**required**
```{r summary_var, eval=FALSE, spr=TRUE}
appendStep(sal) <- LineWise(
code = {
write.table(varSummary(vcf_anno), "./results/variantStats.tsv", quote = FALSE, col.names = NA, sep = "\t")
},
step_name = "summary_var",
dependency = "combine_var"
)
```
## Venn diagram of variants
**Optional** but included in the default
The venn diagram utilities defined by the `systemPipeR` package can be used to
identify common and unique variants reported for different samples
and/or variant callers. The below generates a 3-way venn diagram
comparing 3 samples for each of the two variant callers.
```{r venn_diagram, eval=FALSE, spr=TRUE}
appendStep(sal) <- LineWise(
code = {
## make a list of first three samples
varlist <- sapply(names(vcf_anno[1:3]), function(x) as.character(read.delim(vcf_anno[x])$VARID))
vennset <- overLapper(varlist, type = "vennsets")
png("./results/vennplot_var.png")
vennPlot(list(vennset), mymain = "Venn Plot of First 3 Samples", mysub = "", colmode = 2, ccol = c("red", "blue"))
dev.off()
},
step_name = "venn_diagram",
dependency = "annotate_vcf"
)
```
Figure 2: Venn Diagram for 3 samples from GATK and BCFtools
## Plot variants programmatically
**Optional** but included in default
The following plots a selected variant with `ggbio`.
In this example, the input `BAM` file is from the `GATK` step 5, analysis ready bam.
You can use other aligned `BAMs` as well, but make sure they are indexed. The `VCF`
file is taken from `Inspect VCF file ` section or you can load your own vcf.
```{r plot_variant, eval=FALSE, spr=TRUE}
appendStep(sal) <- LineWise(
code = {
# get the filtered vcf path from R running environment
copyEnvir(sal, "vcf_filter", globalenv())
library(ggbio)
library(VariantAnnotation)
mychr <- "ChrM"
mystart <- 19000
myend <- 21000
bams <- getColumn(sal, "fix_tag")
vcf <- suppressWarnings(readVcf(vcf_filter["M6B"], "A. thaliana"))
ga <- readGAlignments(bams["M6B"], use.names = TRUE, param = ScanBamParam(which = GRanges(mychr, IRanges(mystart, myend))))
p1 <- autoplot(ga, geom = "rect")
p2 <- autoplot(ga, geom = "line", stat = "coverage")
p3 <- autoplot(vcf[seqnames(vcf) == mychr], type = "fixed") +
xlim(mystart, myend) +
theme(legend.position = "none", axis.text.y = element_blank(), axis.ticks.y = element_blank())
p4 <- autoplot(loadDb("./data/tair10.sqlite"), which = GRanges(mychr, IRanges(mystart, myend)), names.expr = "gene_id")
p1_4 <- tracks(Reads = p1, Coverage = p2, Variant = p3, Transcripts = p4, heights = c(0.3, 0.2, 0.1, 0.35)) + ylab("")
ggbio::ggsave(p1_4, filename = "./results/plot_variant.png", units = "in")
},
step_name = "plot_variant",
dependency = "filter_vcf"
)
```
Figure 3: Plot variants with programmatically.
## Workflow Information
```{r sessionInfo, eval=FALSE, spr=TRUE}
appendStep(sal) <- LineWise(
code = {
sessionInfo()
},
step_name = "sessionInfo",
dependency = "plot_variant")
```
# Running workflow
## Interactive job submissions in a single machine
For running the workflow, `runWF` function will execute all the steps store in
the workflow container. The execution will be on a single machine without
submitting to a queuing system of a computer cluster.
```{r runWF, eval=FALSE}
sal <- runWF(sal)
```
## Parallelization on clusters
Alternatively, the computation can be greatly accelerated by processing many files
in parallel using several compute nodes of a cluster, where a scheduling/queuing
system is used for load balancing.
The `resources` list object provides the number of independent parallel cluster
processes defined under the `Njobs` element in the list. The following example
will run 18 processes in parallel using each 4 CPU cores.
If the resources available on a cluster allow running all 18 processes at the
same time, then the shown sample submission will utilize in a total of 72 CPU cores.
Note, `runWF` can be used with most queueing systems as it is based on utilities
from the `batchtools` package, which supports the use of template files (_`*.tmpl`_)
for defining the run parameters of different schedulers. To run the following
code, one needs to have both a `conffile` (see _`.batchtools.conf.R`_ samples [here](https://mllg.github.io/batchtools/))
and a `template` file (see _`*.tmpl`_ samples [here](https://github.com/mllg/batchtools/tree/master/inst/templates))
for the queueing available on a system. The following example uses the sample
`conffile` and `template` files for the Slurm scheduler provided by this package.
The resources can be appended when the step is generated, or it is possible to
add these resources later, as the following example using the `addResources`
function:
```{r runWF_cluster, eval=FALSE}
# wall time in mins, memory in MB
resources <- list(conffile=".batchtools.conf.R",
template="batchtools.slurm.tmpl",
Njobs=18,
walltime=120,
ntasks=1,
ncpus=4,
memory=1024,
partition = "short"
)
sal <- addResources(sal, c("hisat2_mapping"), resources = resources)
sal <- runWF(sal)
```
## Visualize workflow
_`systemPipeR`_ workflows instances can be visualized with the `plotWF` function.
```{r plotWF, eval=FALSE}
plotWF(sal, rstudio = TRUE)
```
## Checking workflow status
To check the summary of the workflow, we can use:
```{r statusWF, eval=FALSE}
sal
statusWF(sal)
```
## Accessing logs report
_`systemPipeR`_ compiles all the workflow execution logs in one central location,
making it easier to check any standard output (`stdout`) or standard error
(`stderr`) for any command-line tools used on the workflow or the R code stdout.
```{r logsWF, eval=FALSE}
sal <- renderLogs(sal)
```
## Tools used
To check command-line tools used in this workflow, use `listCmdTools`, and use `listCmdModules`
to check if you have a modular system.
The following code will print out tools required in your custom SPR project in the report.
In case you are running the workflow for the first time and do not have a project yet, or you
just want to browser this workflow, following code displays the tools required by default.
```{r list_tools}
if(file.exists(file.path(".SPRproject", "SYSargsList.yml"))) {
local({
sal <- systemPipeR::SPRproject(resume = TRUE)
systemPipeR::listCmdTools(sal)
systemPipeR::listCmdModules(sal)
})
} else {
cat(crayon::blue$bold("Tools and modules required by this workflow are:\n"))
cat(c("trimmomatic/0.39", "samtools/1.14", "gatk/4.2.0.0", "bcftools/1.15",
"bwa/0.7.17"), sep = "\n")
}
```
## Report Session Info
This is the session information for rendering this report. To access the session information
of workflow running, check HTML report of `renderLogs`.
```{r report_session_info, eval=TRUE}
sessionInfo()
```
# Funding
This project was supported by funds from the National Institutes of
Health (NIH) and the National Science Foundation (NSF).
# References