estimateOffTargets: Estimate primer off-targets using BLAST
In TAPseq: Targeted scRNA-seq primer design for TAP-seq

Description Usage Arguments Details Value Functions Examples

Functions to use BLAST to align TAP-seq primers against a genome and chromosome reference to estimate potential off-target binding sites.

createBLASTDb(
  genome,
  annot,
  blastdb,
  standard_chromosomes = TRUE,
  tx_id = "transcript_id",
  tx_name = "transcript_name",
  gene_name = "gene_name",
  gene_id = "gene_id",
  title = "TAP-seq_GT_DB",
  verbose = FALSE,
  makeblastdb = getOption("TAPseq.makeblastdb")
)

blastPrimers(
  object,
  blastdb,
  max_mismatch = 0,
  min_aligned = 0.75,
  primer_targets = c("transcript_id", "transcript_name", "gene_id", "gene_name"),
  tmpdir = tempdir(),
  blastn = getOption("TAPseq.blastn")
)

## S4 method for signature 'TsIO'
blastPrimers(
  object,
  blastdb,
  max_mismatch = 0,
  min_aligned = 0.75,
  primer_targets = c("transcript_id", "transcript_name", "gene_id", "gene_name"),
  tmpdir = tempdir(),
  blastn = getOption("TAPseq.blastn")
)

## S4 method for signature 'TsIOList'
blastPrimers(
  object,
  blastdb,
  max_mismatch = 0,
  min_aligned = 0.75,
  primer_targets = c("transcript_id", "transcript_name", "gene_id", "gene_name"),
  tmpdir = tempdir(),
  blastn = getOption("TAPseq.blastn")
)

`genome`	A `BSgenome` (or `DNAStringSet`) object containing the sequences of all chromosomes to obtain genome and transcript sequences.
`annot`	A `GRanges` object containing all exons of transcripts to be considered.
`blastdb`	TAP-seq BLAST database created with `createBLASTDb`.
`standard_chromosomes`	(logical) Specifies whether only standard chromosomes should be included in output genome sequences (e.g. chr1-22, chrX, chrY, chrM for homo sapiens).
`tx_id, tx_name, gene_name, gene_id`	(character) Column names in annot metadata containing transcript id, transcript name, gene name and gene id information.
`title`	Optional title for BLAST database.
`verbose`	(logical) If `TRUE`, additional information from `makeblastdb` is printed to the console. Default: `FALSE`.
`makeblastdb`	Path to the `makeblastdb` executable. Usually this is inferred when loading/attaching the package.
`object`	A `TsIO` or `TsIOList` object containing designed primers.
`max_mismatch`	Maximum number of mismatches allowed for off-target hits (default: 0).
`min_aligned`	Minimum portion of the primer sequence starting from the 3' end that must align for off-target hits (default: 0.75).
`primer_targets`	Specifies what should be used to identify primer targets for off-target identification. I.e. to what does the `sequence_id` in TsIO objects refer? Can be a subset of `transcript_id`, `transcript_name`, `gene_id` or `gene_name`. By default all 4 are checked. Set to `NULL` to disable any off-target identification. See Details for more information.
`tmpdir`	Directory needed to store temporary files.
`blastn`	Path (character) to the `blastn` executable. Usually this is inferred when loading/attaching the package.

createBLASTDb creates a BLAST database containing genome and transcriptome sequences, which is required by blastPrimers. The created database contains both sequence files for BLAST and annotations to process the results.

Use blastPrimers to align designed TAP-seq primers against the created database to estimate off-target priming potential. Only hits where at least a specified portion of the sequence involving the 3' end of the primer aligns with not more than a certain number of mismatches are considered.

blastPrimers counts the number of genes in which a primer has 1) exonic hits or 2) intronic hits, or 3) the number of hits in intergenic regions of the genome. The exonic and intronic counts should be interptreted as: "In how many genes does a primer have exonic (or intronic) hits?".

If a BLAST hit falls in both intronic and exonic regions of a given gene (i.e. exonic for one transcript, intronic for another transcript), only the exonic hit is counted for that gene. If a primer has for instance 3 BLAST hits in one gene, 2 exonic and 1 intronic, then one exonic hit and one intronic hit is counted for that gene.

If sequence IDs of the designed primers (sequence_id) refer to the target gene/transcripts and can be found in the BLAST database annotations via primer_targets, then only off-target hits are counted. This is usually the case if input for primer design was produced from target gene annotations.

For createBLASTDb a directory containing the BLAST database. For blastPrimers a TsIO or TsIOList object with the number of potential off-targets added to the TAP-seq primer metadata.

createBLASTDb: Create a genome and transcriptome TAP-seq BLAST database
blastPrimers,TsIO-method: BLAST primers in a TsIO object
blastPrimers,TsIOList-method: BLAST primers in a TsIOList object

## Not run: 
library(BSgenome)

# human genome (hg38) BSgenome object
hg38 <- getBSgenome("BSgenome.Hsapiens.UCSC.hg38")

# get annotations for BLAST
annot_url <- paste0("ftp://ftp.ebi.ac.uk/pub/databases/gencode/Gencode_human/release_32/",
                    "gencode.v32.annotation.gtf.gz")
annot <- import(annot_url, format = "gtf")
blast_exons <- annot[annot$type == "exon" & annot$gene_type == "protein_coding"]

# build BLAST database
blastdb <- file.path(tempdir(), "blastdb")
createBLASTDb(genome = hg38, annot = blast_exons, blastdb = blastdb)

# chr11 primers example data (already contains off-targets, but we can overwrite them)
data("chr11_primers")
chr11_primers <- chr11_primers[1:3]  # only use a small subset for this example

# run blast to identify potential off-targets
chr11_primers <- blastPrimers(chr11_primers, blastdb = blastdb)
tapseq_primers(chr11_primers)

# allow 1 mismatch between primer and off-target
chr11_primers <- blastPrimers(chr11_primers, blastdb = blastdb, max_mismatch = 1)
tapseq_primers(chr11_primers)

## End(Not run)