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###################################################### Peter A. Combs and Hunter B. Fraser

Department of Biology, Stanford University

This repository contains the analysis code for Combs and Fraser 2017 (bioRxiv preprint; currently submitted for review). Raw and processed data files available from the Gene Expression Omnibus.

Very briefly, the data analyses in these scripts do two things:

1. Process RNA-seq data from cryosliced D. melanogaster x simulans hybrid embryos. We are looking for genes with spatially varying allele-specific expression (svASE) is different in one part of the embryo compared to the other.

2. Perform modeling of the cis-regulatory input functions of genes using a modeling approach inspired in large part by Ilsley, et al (2013). Using genome alignments and motif searches, we can then make inferences about which cis-regulatory changes actually produced the spatially varying ASE that we observed in part 1.

Almost all of the code is written in either Python 3 or Snakemake. Known dependencies include:

• SRA Tools
• Snakemake
• Bowtie2 (Reference gDNA alignment)
• STAR (RNA-seq alignment)
• Cufflinks (RNA-seq quantification)
• Bedtools
• Samtools
• ASEr (https://github.com/thefraserlab/aser)
• Hornet (https://github.com/thefraserlab/hornet)
• Various python modules
  • pysam
  • progressbar
  • numpy/scipy/pandas/matplotlib
  • svgwrite
  • pyemd
  • BioPython
  • statsmodels

You should be able to go from raw reads to summary data tables by doing:

snakemake

Though you probably want to run this on a pretty high-powered machine---or, ideally, a compute cluster. The basic steps for a single sample are:

1. Download reads from SRA
2. Map reads to the D. melanogaster genome (the resulting file is called assigned_dmelR.bam for various historical reasons)
3. Calculate absolute abundances using Cufflinks
4. Filter out potentially ambiguous/mismapped reads using Hornet to implement the WASP pipeline.
5. Count allele-specific reads for each gene using ASEr

Then, all of the expresion and ASE data are combined into summary tables.

We found that fitting either a logistic or gaussian function to the data found all of the genes whose allele-specific expression had spatial patterns.