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References

In this section I will describe how references (GTF/GFF2/GFF3) from either gencode or ENSEMBL can be read directly into R to create analysis-ready dataframe objects for later use. More specifically, entire genome references are quite large and take a long time to load so its advantagous to process and organize them ahead of time.

GENCODE V32 Example in R Lang.

In this subsection example code is shown on how to process and load references in R with the gencode v32 reference file (GTF).


library(rtracklayer) # this library used to read the GFF filetype
library(data.table)

### 1. 
# read in GTF
gtf_file = './gencode.v32.primary_assembly.annotation.gtf'
print(paste0('loading reference: ', gtf_file))
reference <- import.gff2(gtf_file) # columns=cols, tags=character(0) # GRanges object with 2900491 ranges and 21 metadata columns:
reference =  as.data.table(reference) # [1] 2900491      26
reference = reference %>% rowwise() %>% dplyr::mutate(ensembl_gene_id_trim = str_split(gene_id, "[.]")[[1]][1] ) # extract ENSEMBL id from full id w/ transcript id

### 2. protein coding gene reference 
gencode32_protein_coding = reference %>% dplyr::filter(type=='gene' &  gene_type=='protein_coding' ) # A tibble: 19,994 × 27 genes 
gencode32_protein_coding = gencode32_protein_coding %>% dplyr::select(where(~all(!is.na(.)))) # drop emtpy cols

### 3.  protein coding gene reference WITH lncRNAs INCLUDED  
gencode32_protein_coding_lncRNA = reference %>% dplyr::filter(type=='gene' & (gene_type=='protein_coding' | gene_type=='lncRNA')) # A tibble: 36,843 x 27 genes 
gencode32_protein_coding_lncRNA = gencode32_protein_coding_lncRNA %>% dplyr::select(where(~all(!is.na(.)))) # drop emtpy cols

### Z. bonus mito frame (for quickly loading in sc QC)
# gencode32_mito = reference %>% dplyr::filter(type=='gene' &  seqnames=='chrM') # A tibble: 37 × 27 genes
gencode32_mito2 = reference %>% dplyr::filter(type=='gene' & stringr::str_detect(gene_name, "^MT-")) # A tibble: 37 × 27 genes
gencode32_mito2 = gencode32_mito2 %>% dplyr::select(where(~all(!is.na(.)))) # drop emtpy cols