CustomSelection: CustomSelection: a package for selecting reference genes from...

In KarenGoncalves/CustomSelection: Custom selected reference genes outperform pre-defined reference genes in transcriptomic analysis

CustomSelection: a package for selecting reference genes from RNAseq data

Description

The CustomSelection package provides four funtions: Counts_to_tpm, DAFS, gene_selection and customReferences.

Counts_to_tpm function

Transforms count data into Transcripts Per Million (TPM) data

With the matrix of counts and the size of the genes/transcripts, it calculates the TPM.

This function was modified from a gist from Slowkow (https://gist.github.com/slowkow/c6ab0348747f86e2748b).

Here, we do not calculate the effective length.

DAFS function

Calculates the threshold for a gene to be considered truly expressed

This function calculates the threshold for a gene to be considered truly expressed in each sample (columns of the expression data frame).

Modified from George and Chang (2014).

gene_selection fuction

Uses average TPM values and the covariance of TPM values to select reference genes from RNAseq data.

If counts_to_tpm and DAFS functions were already computed, this function will use their results to select the genes with lowest covariance, among those considered as expressed according to DAFS, as references.

custom_References function

Uses average TPM values and the covariance of TPM values to select reference genes from RNAseq data

This function uses the Counts_to_tpm and the DAFS function to select the reference genes.

After transforming the counts into TPM values, the tpm data frame is used as input for DAFS function.

We then select the genes with lowest covariance, among those considered as expressed according to DAFS (average expression higher than the cutoff), as references.

sample_counts dataset

Counts of 3 samples (4 replicates per sample) of Arabidopsis thaliana genes.

Transgenic Arabidopsis thaliana Columbia-0 plants expressing GFP alone (Control) or fused to a candidate secreted effector protein of the fungus Melampsora larici-populina (Mlp37347 or Mlp124499) were used for the transcriptome analysis.

RNA was extracted from pooled aerial tissue of 2-week-old soil-grown plants, doing four replicates per genotype. Libraries were generated using the TruSeq Stranded mRNA Library Prep kit (Illumina) and 100 ng of total RNA. The libraries were sequenced with Illumina HiSeq 4000 Sequencer paired-end reads of 100nt.

Trimmomatic (LEADING:4 TRAILING:4 SLIDINGWINDOW:4:20 MINLEN:20) and then the surviving paired reads were aligned to the TAIR10 assembly of the genome of A. thaliana with TopHat v2.0.14 in Galaxy (default options, with average mate inner distance varying for each replicate and standard deviation of distance between pairs of 50 base pairs).

Further analyses were done using R software v.3.2.5. Genomic ranges of Arabidopsis transcripts were obtained from Ensembl plants with GenomicFeatures and overlaps of sequencing reads with the transcripts were counted using GenomicAlignments, using options for paired-end reads and union mode.

ath_featureLength dataset

Length of Arabidopsis thaliana genes (TAIR10) obtained with the following code:

library(biomaRt)

ath <- useMart('plants_mart', host = "plants.ensembl.org", dataset = "athaliana_eg_gene")

gene_start_end = getBM(attributes = c('ensembl_gene_id', 'start_position', 'end_position'), mart = ath)

featureLength <- gene_start_end$end_position - gene_start_end$start_position

names(featureLength) <- gene_start_end$ensembl_gene_id

KarenGoncalves/CustomSelection documentation built on Oct. 24, 2023, 12:39 a.m.