SliceSeq code

This is a collection of code that has evolved, poorly, to do various generic and specific analyses for SliceSeq, which at the time of writing, consists of pooling a carrier RNA with the total RNA from each of the slices, then computationally un-pooling the resulting RNA-seq reads, or just sequencing the sample of interest with no carrier.

I make no claims to suitability for any purpose, and all code (unless otherwise noted) is released under the CRAPL v0 license. Please contact me directly (peter.combs@berkeley.edu) for any data I've generated, as it's most likely too large to fit on github anyways.

The Fall2012 and Spring2013 branches are used for RNA-seq analysis of sliced single Drosophila melanogaster embryos, in particular for the submission of the paper "Sequencing mRNA from cryo-siced Drosophila embryos to determine genome-wide spatial patterns of gene expression".

I'm attempting to use a makefile as much as possible (See, for example, this blog post). Thus, to do "everything", for some minimal definition of "everything", simply run:

$ ./configure
$ make

The data for that paper is available at the Gene Expression Omnibus, under accession GSE43506 Dependencies

Software dependencies, at time of writing, include:

• STAR RNA-Seq for mapping. Selected primarily for speed over Bowtie/Tophat, with no obvious major differences in mapping results. Version 2.3.0.1
• Cufflinks for abundance estimation (i.e. FPKM values for each gene). Version 2.1.1

As much as possible, these data files will be publicly available, standardized sets. Known data dependencies are:

• FASTA and GFF files for all species.
• journal.pbio.1000590.s002.txt, the supplementary data file from Lott, et al 2011
• RunConfig.cfg A tab-delimited file indicating, for each sample, the carrier species and sequencing index, among other statistics. Please contact me for my copy if there's any trouble guessing what you need.

AssignReads2.py

Utility to demultiplex reads from a pooled RNA-seq sample. Given a list of accepted_hits.bam files from Tophat, this will assign the reads into bam files for uniquely mapping to either species, or to an ambiguous.bam file, all in the same directory as the original bam file. There is a variable ambig_threshold that determines what is counted as uniquely mapping, and is currently set to 3, meaning that reads require 4 or more mismatches to prefer one species over another.

CheckCoverage.py

Utility to estimate the relative level of PCR Duplication found in a sample. By looking at the number of unique read positions in low-to-moderately expressed genes, and comparing to a simulated model, a Badness score can be calculated, which roughly corresponds to the number of reads per fragment. A perfect Badness score would be 1, with higher scores indicating a higher level of PCR duplication in the sample.

Usage:

$ python CheckCoverage.py GTF-file bamfile.bam [bamfile.bam ...]

The GTF file works best when using a FlyBase derived file, and assumes the following order of annotation types:

• mRNA: Should have both the FBtr ID and the FBgn ID in the annotation field

• exon: One or more exons per transcript, containingi the FBtr ID in the annotation field

• CDS: Used by this program as a signal that there are no more exons for this transcript. If there are, it will confuse the program.

PointClouds.py

This is a utility class for reading Point Cloud files from the Berkeley Drosophila Transcription Network (http://bdtnp.lbl.gov/Fly-Net/bioimaging.jsp?w=vpcFormat). Thus far, it's only been tested on VirtualEmbryo files, but it should also work on single embryo point clouds. Metadata is loaded into the PointCloudReader object as variables. Example usage:

from PointClouds import PointCloudReader
pcr = PointCloudReader(open('D_mel.vpc'))
bcddata = []
for line in pcr:
    bcddata.append(line[pcr.column.index('bcd__1')])

which loads all of the data from the first timepoint with Bicoid into the bcddata list.