---
author:
  - name: Sonali Arora
    affiliation: Fred Hutchinson Cancer Research Center, 1100 Fairview Ave. N., P.O. Box 19024, Seattle, WA, USA 98109-1024
    email: sarora@fredhutch.org
  - name: Martin Morgan
    affiliation: Roswell Park Cancer Institute, Elm and Carlton St, Buffalo, NY 14263
    email: martin.morgan@roswellpark.org
title: "Introduction to Bioconductor for Sequence Data"
date: "3 June 2015"
vignette: >
  %\VignetteIndexEntry{Introduction to Bioconductor for Sequence Data}
  %\VignetteEngine{knitr::rmarkdown}
  %\VignetteEncoding{UTF-8}
output: 
  BiocStyle::html_document
---

```{r style, echo=FALSE, message=FALSE, warning=FALSE, results="asis"}
options(width=100)
knitr::opts_chunk$set(message = FALSE, error = FALSE, warning = FALSE,
fig.width=6, fig.height=4)
BiocStyle::markdown()
```

```{r, echo=FALSE, results="hide", warning=FALSE}
suppressPackageStartupMessages({
   library(GenomicRanges)
   library(GenomicAlignments) 
   library(Biostrings)
   library(Rsamtools)
   library(ShortRead)
   library(BiocParallel)
   library(rtracklayer)
   library(VariantAnnotation)
   library(AnnotationHub)
   library(BSgenome.Hsapiens.UCSC.hg19)
   library(RNAseqData.HNRNPC.bam.chr14)
})
ah = AnnotationHub()

```
**R version**: `r R.version.string`

**Bioconductor version**: `r BiocInstaller::biocVersion()`

**Package**: `r packageVersion("sequencing")`

# Abstract

_Bioconductor_ enables the analysis and comprehension of high-
throughput genomic data. We have a vast number of packages that allow
rigorous statistical analysis of large data while keeping
technological artifacts in mind. Bioconductor helps users place their
analytic results into biological context, with rich opportunities for
visualization. Reproducibility is an important goal in _Bioconductor_
analyses. Different types of analysis can be carried out using
_Bioconductor_, for example

- Sequencing : RNASeq, ChIPSeq, variants, copy number.. 
- Microarrays: expression, SNP, ...
- Domain specific analysis : Flow cytometry, Proteomics ..
 
For these analyses, one typically imports and works with diverse
sequence-related file types, including fasta, fastq, BAM, gtf, bed,
and wig files, among others. _Bioconductor_ packages support import,
common and advanced sequence manipulation operations such as trimming,
transformation, and alignment including quality assessment.

# Sequencing Resources

Here is a illustrative description elaborating the different
file types at various stages in a typical analysis, with the 
package names (in pink boxes) that one will use for each stage. 

<p><img src="sequencepkg.png" width="500" height="400"/></p>


The following packages illustrate the diversity of functionality
available; all are in the release version of _Bioconductor_.

* `r Biocpkg("IRanges")` and `r Biocpkg("GenomicRanges")` for
  range-based (e.g., chromosomal regions) calculation, data
  manipulation, and general-purpose data representation.
  `r Biocpkg("Biostrings")` for DNA and amino acid sequence
  representation, alignment, pattern matching (e.g., primer removal),
  and data manipulation of large biological sequences or sets of
  sequences. `r Biocpkg("ShortRead")` for working with FASTQ files of
  short reads and their quality scores.

* `r Biocpkg("Rsamtools")` and `r Biocpkg("GenomicAlignments")` for
  aligned read (BAM file) I/O and data manipulation.
  `r Biocpkg("rtracklayer")` for import and export of diverse data
  formats (e.g., BED, WIG, bigWig, GTF, GFF) and manipualtion of
  tracks on the UCSC genome browser.

* `r Biocpkg("BSgenome")` for accessing and manipulating curated whole-genome
  representations.  `r Biocpkg("GenomicFeatures")` for annotation of sequence
  features across common genomes, `r Biocpkg("biomaRt")` for access to Biomart
  databases.

* `r Biocpkg("SRAdb")`  for querying and retrieving data from the 
  Sequence Read Archive.

_Bioconductor_ packages are organized by
[biocViews](http://bioconductor.org/packages/biocViews). Some of the
entries under
[Sequencing](http://bioconductor.org/packages/biocViews.html#__Sequencing)
and other terms, and representative packages, include:

* [RNASeq](http://bioconductor.org/packages/biocViews.html#__RNASeq),
   e.g., `r Biocpkg("edgeR")`, `r Biocpkg("DESeq2")`,
   `r Biocpkg("edgeR")`, `r Biocpkg("derfinder")`, and
   `r Biocpkg("QuasR")`.

* [ChIPSeq](http://bioconductor.org/packages/biocViews.html#__ChIPSeq),
  e.g.,`r Biocpkg("DiffBind")`, `r Biocpkg("csaw")`, `r Biocpkg("ChIPseeker")`,
  `r Biocpkg("ChIPQC")`.

* [SNPs](http://bioconductor.org/packages/biocViews.html#__SNP) and
  other variants, e.g., `r Biocpkg("VariantAnnotation")`,
  `r Biocpkg("VariantFiltering")`, `r Biocpkg("h5vc")`.

* [CopyNumberVariation](http://bioconductor.org/packages/biocViews.html#__CopyNumberVariation)
  e.g., `r Biocpkg("DNAcopy")`, `r Biocpkg("crlmm")`, `r Biocpkg("fastseg")`.

* [Microbiome](http://bioconductor.org/packages/biocViews.html#__Microbiome)
  and metagenome sequencing, e.g., `r Biocpkg("metagenomeSeq")`,
  `r Biocpkg("phyloseq")`, `r Biocpkg("DirichletMultinomial")`.

# Ranges Infrastructure

Many _Bioconductor_ packages rely heavily on the *IRanges* /
*GenomicRanges* infrastructure. Thus we will begin with a quick
introduction to these and then cover different file types.

The `r Biocpkg("GenomicRanges")` package allows us to associate a
range of chromosome coordinates with a sequence name (e.g.,
chromosome) and a strand. Such genomic ranges are very useful for
describing both data (e.g., the coordinates of aligned reads, called
ChIP peaks, SNPs, or copy number variants) and annotations (e.g., gene
models, Roadmap Epigenomics regulatory elements, known clinically
relevant variants from dbSNP).  `GRanges` is an object representing a
vector of genomic locations and associated annotations.  Each element
in the vector is comprised of a sequence name, a range, a strand,
and optional metadata (e.g. score, GC content, etc.).

```[r}
library(GenomicRanges)
GRanges(seqnames=Rle(c('chr1', 'chr2', 'chr3'), c(3, 3, 4)),
      IRanges(1:10, width=5), strand='-',
      score=101:110, GC = runif(10))
```

Genomic ranges can be created 'by hand', as above, but are often the
result of importing data (e.g., via
`GenomicAlignments::readGAlignments()`) or annotation (e.g., via
`GenomicFeatures::select()` or `rtracklayer::import()` of BED, WIG,
GTF, and other common file formats).  Use `help()` to list the help
pages in the `r Biocpkg("GenomicRanges")` package, and `vignettes()`
to view and access available vignettes.

```{r eval=FALSE}
help(package="GenomicRanges")
vignette(package="GenomicRanges")
```

Some of the common operations on `GRanges` include
`findOverlaps(query, subject)` and `nearest(query, subject)`, which
identify the ranges in `query` that overlap ranges in `subject`, or
the range in `subject` nearest to `query. These operations are useful
both in data analysis (e.g., counting overlaps between aligned reads
and gene models in RNAseq) and comprehension (e.g., annotating genes
near ChIP binding sites).

# DNA /amino acid sequence from FASTA files

`r Biocpkg("Biostrings")` classes (e.g., `DNAStringSet`) are used to
represent DNA or amino acid sequences. In the example below we will
construct a DNAString and show some manipulations.

```{r message=FALSE}
library(Biostrings)
d <- DNAString("TTGAAAA-CTC-N")
length(d)  #no of letters in the DNAString
```

We will download all _Homo sapiens_ cDNA sequences from the FASTA file
'Homo_sapiens.GRCh38.cdna.all.fa' from Ensembl using
`r Biocpkg("AnnotationHub")`.

```{r eval=FALSE}
library(AnnotationHub)
ah <- AnnotationHub()
```

This file is downloaded as a FaFile object

```{r}
ah2 <- query(ah, c("fasta", "homo sapiens", "Ensembl"))
fa <- ah2[["AH18522"]]
fa
```

The sequences in the file can be read in using
`readDNAStringSet()` from the `r Biocpkg("Biostrings")` package.
The sequences are returned as a _DNAStringSet_ object.

```{r}
readDNAStringSet(path(fa))
```

Alternatively, we can use tools from the `r Biocpkg("Rsamtools")` package
to selectively load a subset of the sequences only.

```{r}
library(Rsamtools)
idx <- scanFaIndex(fa)
idx
```

The index is returned as a _GRanges_ object. Then we use `getSeq()` to
load only the sequences indicated by `param`.

```{r}
long <- idx[width(idx) > 82000]
getSeq(fa, param=long)
```

`r Biocpkg("BSgenome")` packages inside _Bioconductor_ contain whole
genome sequences as distributed by ENSEMBL, NCBI and others.  In this
next example we will load the whole genome sequence for _Homo sapiens_
from UCSC's `hg19` build, and calculate the GC content across
chromosome 14.

```{r}
library(BSgenome.Hsapiens.UCSC.hg19)

chr14_range = GRanges("chr14", IRanges(1, seqlengths(Hsapiens)["chr14"]))
chr14_dna <- getSeq(Hsapiens, chr14_range)
letterFrequency(chr14_dna, "GC", as.prob=TRUE)
```

# Reads from FASTQ files

`r Biocpkg("ShortRead")` package from _Bioconductor_ can be used for
working with fastq files. Here we illustrate a quick example where one
can read in multiple fasta files, collect some statistics and generate
a report about the same.

`r Biocpkg("BiocParallel")` is another package from _Bioconductor_ which 
parallelizes this task and speeds up the process. 

```{r eval=FALSE}
## 1. attach ShortRead and BiocParallel
library(ShortRead)
library(BiocParallel)

## 2. create a vector of file paths
fls <- dir("~/fastq", pattern="*fastq", full=TRUE)

## 3. collect statistics
stats0 <- qa(fls)

## 4. generate and browse the report
if (interactive())
    browseURL(report(stats0))
```

Two useful functions in `r Biocpkg("ShortRead")` are `trimTails()` for
processing FASTQ files, and `FastqStreamer()` for iterating through
FASTQ files in manageable chunks (e.g., 1,000,000 records at a time).

# Aligned Reads from BAM files

The `r Biocpkg("GenomicAlignments")` package is used to input reads
aligned to a reference genome.

In this next example, we will read in a BAM file and specifically read
in reads supporting an apparent exon splice junction spanning position
19653773 of chromosome 14.

The package `r Biocexptpkg("RNAseqData.HNRNPC.bam.chr14_BAMFILES")`
contains 8 BAM files. We will use only the first BAM file. We will
load the software packages and the data package, construct a _GRanges_
with our region of interest, and use `summarizeJunctions()` to find
reads in our region of interest.

```{r}
## 1. load software packages
library(GenomicRanges)
library(GenomicAlignments)

## 2. load sample data
library('RNAseqData.HNRNPC.bam.chr14')
bf <- BamFile(RNAseqData.HNRNPC.bam.chr14_BAMFILES[[1]], asMates=TRUE)

## 3. define our 'region of interest'
roi <- GRanges("chr14", IRanges(19653773, width=1)) 

## 4. alignments, junctions, overlapping our roi
paln <- readGAlignmentsList(bf)
j <- summarizeJunctions(paln, with.revmap=TRUE)
j_overlap <- j[j %over% roi]

## 5. supporting reads
paln[j_overlap$revmap[[1]]]
```

For a detailed tutorial on working with BAM files do check out this
detailed [Overlap
Encodings](http://bioconductor.org/packages/release/bioc/vignettes/GenomicAlignments/inst/doc/OverlapEncodings.pdf)
vignette of GenomicAlignments.


# Called Variants from VCF files

VCF (Variant Call Files) describe SNP and other variants. The files
contain meta-information lines, a header line with column names, and
then (many!) data lines, each with information about a position in the
genome, and optional genotype information on samples for each
position.

Data are parsed into a VCF object with `readVcf()`
from `r Biocpkg("VariantAnnoation")`

```{r}
library(VariantAnnotation)
fl <- system.file("extdata", "chr22.vcf.gz", package="VariantAnnotation")
vcf <- readVcf(fl, "hg19")
```

An excellent workflow on working with Variants can be found
[here](http://bioconductor.org/help/workflows/variants/). In
particular it is possible to read in specific components of the VCF
file (e.g., `readInfo()`, `readGeno()`) and parts of the VCF at
specific genomic locations (using _GRanges_ and the `param = ScanVcfParam()`
argument to input functions).


# Genome Annotations from BED, WIG, GTF etc files

`r Biocpkg("rtracklayer")` import and export functions can read in
many common file types, e.g., BED, WIG, GTF, …, in addition to
querying and navigating the UCSC genome browser.

`r Biocpkg("rtracklayer")` contains a 'test' BED file which we will read in here

```{r}
library(rtracklayer)
test_path <- system.file("tests", package = "rtracklayer")
test_bed <- file.path(test_path, "test.bed")
  
test <- import(test_bed, format = "bed")
test
```

The file is returned to the user as a _GRanges_ instance.  A more
detailed tutorial can be found
[here](http://bioconductor.org/packages/devel/bioc/vignettes/rtracklayer/inst/doc/rtracklayer.pdf)

`r Biocpkg("AnnotationHub")` also contains a variety of genomic annotation files
(eg BED, GTF, BigWig) which use import() from rtracklayer 
behind the scenes. For a detailed tutorial the user is referred to
[Annotation workflow](http://bioconductor.org/help/workflows/annotation/Annotation_Resources/)
and [AnnotationHub HOW TO vignette](http://bioconductor.org/packages/devel/bioc/vignettes/AnnotationHub/inst/doc/AnnotationHub-HOWTO.html)


```{r}
sessionInfo()
```

