README

Below is a description of the directory structure of the uploaded data, scripts and outputs.
There are three input files (ASE_data_frame.csv,compiled_ddPCR_results.csv, dilution_data.csv). Input data is described in detail below, the methods of production are described in the associated manuscript.

The file "Howe_et_al_Rscript.Rmd" contains all code used to produce the results and figures within Howe et al. 2020. It is written in RMarkdown format, so that a more readable html output is produced. A more complete description of its contents is given within the file. In order to run the file, a number of R packages may need to be installed. The HTML output is also uploaded so that the results can be seen without needing to run the script.

The output files produced include a 'figures.pdf', where the figures produced for the manuscript are saved, and two supplementary tables (S4 and S5).

Files uploaded: 
input_data:
  |- ASE_data_frame.csv
  |- compiled_ddPCR_results.csv
  |- dilution_data.csv
scripts:
  |-Howe_et_al_Rscript.Rmd
outputs:
  |- figures.pdf *
  |- table_s4.csv *
  |- table_s5.csv *
  |-Howe_et_al_Rscript.html *

* indicates files generated by R script, they are also uploaded here to aid in usability.

Description of files and variables:
  ASE_data_frame.csv #data for allele-specific expression
    Qiye Li and Zongji Wang of BGI conducted the processing of the raw RNA and DNA data, to output a table of SNPs where the relative frequency of any given SNP-allele differed significantly between a paired DNA and RNA sample. This output table is used as the input here. Queries regarding the production of this table should be directed to them.

    Columns:
    - Chr #scaffold locus located upon
    - Locus #locus of SNP on scaffold
    - Reference # Base for reference allele
    - Caste #Caste of ants collected for sample
    - Colony #ant colony sample was taken from
    - Region #region within the gene that the SNP was located in
    - Gene #name of gene
    - BaseDNA1 # Base (A,T,C or G) of first DNA SNP-allele (order of alleles is arbitrary)
    - CountDNA1 #number of reads with BaseDNA1 that map to this locus
    - BaseDNA2 # Base (A,T,C or G) of second DNA SNP-allele (order of alleles is arbitrary)
    - CountDNA2 #number of reads with BaseDNA2 that map to this locus
    - BaseDNA3 # Base (A,T,C or G) of third DNA SNP-allele (order of alleles is arbitrary)
    - CountDNA3 #number of reads with BaseDNA3 that map to this locus
    - BaseRNA1 # Base (A,T,C or G) of first RNA SNP-allele (order of alleles is arbitrary)
    - CountRNA1 #number of reads with BaseRNA1 that map to this locus
    - BaseRNA2 # Base (A,T,C or G) of second RNA SNP-allele (order of alleles is arbitrary)
    - CountRNA2 #number of reads with BaseRNA2 that map to this locus
    - BaseRNA3 # Base (A,T,C or G) of third DNA SNP-allele (order of alleles is arbitrary)
    - CountRNA3 #number of reads with BaseRNA3 that map to this locus
    - BaseRNA4 # Base (A,T,C or G) of fourth DNA SNP-allele (order of alleles is arbitrary)
    - CountRNA4 #number of reads with BaseRNA4 that map to this locus
    - DNAreadsTotal #total number of DNA reads aligned to this locus
    - RNAreadsTotal	#total number of RNA reads aligned to this locus
    - DNAProp #proportion of BaseDNA1 of DNAreadsTotal
    - RNAProp #proportion of BaseRNA1 of RNAreadsTotal

    compiled_ddPCR_results.csv #data for gene specific ASE using generated from ddPCR experiments.
      Generated using DNA and RNA isolated from pooled bodies and heads of individuals collected from A. echinatior colonies
      Columns:
    - Sample #description of Sample,
    - Target	# gene that is targetted
    - Ch1+Ch2+	# number of droplets positive in both channel 1 and channel 2,
    - Ch1+Ch2-	# number of droplets positive for channel 1, but negative for 2
    - Ch1-Ch2+	# number of droplets positive for channel 2, but negative for 1
    - Ch1-Ch2-	# number of droplets positive for neither channel 1 nor 2
    - fraction	# Fraction of template that in channel 1.  (Ch1+Ch2+ + Ch1+Ch2-)/( Ch1+Ch2+ + Ch1+Ch2- + Ch1-Ch2-)
    - min	#lower bound of 95% confidence interval of fraction, based on poisson distribution of templates among droplets
    - max #upper bound of 95% confidence interval of fraction, based on poisson distribution of templates among droplets

        #notes: unfortunately data for number of droplets for MRJP3 is unavailable due to recording error, but fractions and CIs are present

  dilution_data.csv #Data for determining relative concentration of genes in samples of mixed DNA
    Columns:
    - Sample #sample name, contains concentration info
    - Target	#gene name
    - Concentration	# concentration of template per uL, based on number of positive droplets
    - CopiesPer20uLWell	# total number of template copies estimated per well
    - PoissonConfMax #  upper limit of 95% CI based on poisson distribution of templates among droplets
    - PoissonConfMin	 #  lower limit of 95% CI based on poisson distribution of templates among droplets
    - Positives	#number of positive droplets
    - Negatives #number of negative droplets

  Howe_et_al_Rscript.Rmd #R markdown script that runs all analyses presented in the manuscript

  Howe_et_al_Rscript.html #output of RMarkdown script in html format

  figures.pdf #figures output from the RScript

  table_s4.csv #supplementary table 4: the number of loci consistent with imprinting at different levels of alpha. Produced by Howe_et_al_Rscript.Rmd, but uploaded so script does not need to be run to generate.
    Columns:
    - Gene #name of gene
    - Chr #chromosome where gene lies
    - alpha_0 #number of loci consistent at alpha = 0
    - alpha_02 #number of loci consistent at alpha = 0.2
    - alpha_08 #number of loci consistent at alpha = 0.8
    - alpha_1 #number of loci consistent at alpha = 1
    - total #total number of loci with ASE in the gene

  table_s5.csv #supplementary table 5: the number of samples that were inconsistent with imprinting for each gene at varying levels of alpha. Produced by Howe_et_al_Rscript.Rmd, but uploaded so script does not need to be run to generate.
    Columns:
    - Gene #name of gene
    - Chr #chromosome where gene lies
    - Locus #location on chromosome
    - Complete_Paternal #number of samples inconsistent with exclusively patrigenic expression
    - Biased_Paternal #number of samples inconsistent with biased patrigenic expression
    - Biased_Maternal #number of samples inconsistent with biased matrigenic expression
    - Complete_Maternal #number of samples inconsistent with exclusively matrigenic expression