ChopStitch 1.0.0.

Exon annotation and splice graph construction using transcriptome assembly and whole genome sequencing data

ChopStitch is a new method for finding putative exons and constructing splice graphs using an assembled transcriptome and whole genome shotgun sequencing (WGSS) data. ChopStitch identifies exon-exon boundaries in de novo assembled RNA-seq data with the help of a Bloom filter that represents the k-mer spectrum of WGSS reads. The algorithm also detects base substitutions in transcript sequences corresponding to sequencing or assembly errors, haplotype variations, or putative RNA editing events. The primary output of our tool is a FASTA file containing putative exons. Further, exon edges are interrogated for alternative exon-exon boundaries to detect transcript isoforms, which are reported as splice graphs in dot output format.

Requirements:

Install pip

Install requirements by running:

pip install -r requirements.txt

Install Graphviz version 2.4.0 command line tools

Install ChopStitch:

When installing ChopStitch from GitHub source the following tools are required:

• Autoconf
• Automake

To generate the configure script and make files:

./autogen.sh

To compile and install ChopStitch in /usr/local:

$ ./configure
$ make
$ sudo make install

To install ChopStitch in a specified directory:

$ ./configure --prefix=/opt/ChopStitch
$ make 
$ make install 

ChopStitch uses OpenMP for parallelization, which requires a modern compiler such as GCC 4.2 or greater. If you have an older compiler, it is best to upgrade your compiler if possible. If you have multiple versions of GCC installed, you can specify a different compiler:

$ ./configure CC=gcc-xx CXX=g++-xx 

For the best performance of ChopStitch, pass -O3 flag:

$ ./configure CFLAGS='-g -O3' CXXFLAGS='-g -O3' 

To run ChopStitch, its executables, CreateBloom and FindExons, should be found in your PATH. If you installed ChopStitch in /opt/ChopStitch, add /opt/ChopStitch/bin to your PATH:

$ PATH=/opt/ChopStitch/bin:$PATH

Run CreateBloom

Usage: CreateBloom [OPTION]... FILES...
Creates a Bloom filter (BF) to be used for FindExons.
Acceptable file formats: fastq, fasta, sam, bam, gz, bz, zip.

 Options:

  -t, --threads=N  use N parallel threads [1]
  -k, --kmer=N	the length of kmer [50]
  -d, --fpr1=N	primary BF fpr [0.01]
  -s, --fpr2=N	secondary BF fpr [0.01]
  -r, --ref	using FASTA reference as input instead of FASTQ reads. Don't use fpr2 in this case
      --help	display help and exit
      --version	output version information and exit
  `FILES`: input file or set of files seperated by space, in fasta, fastq, sam, and bam formats. The files can also be in compressed (`.gz`, `.bz2`, `.xz`) formats . A list of files containing file names in each row can be passed with `@` prefix.

Example:

./CreateBloom -t 32 -k 50 --fpr1 0.01 --fpr2 0.01 <FASTQ1> <FASTQ2>

To pass a list of files, list.in, as input:

./CreateBloom -t 32 -k 50 --fpr1 0.01 --fpr2 0.01 @list.in

To pass a reference fasta file as input:

./CreateBloom --ref -t 32 -k 50 --fpr1 0.01  <REFERENCE FASTA> 

Output:

Bfilter.bf : Bloom filter file

Bfilter.inf : Info file required for FindExons

Run FindExons

Find putative exons in TransAbySS Transcriptome assembly file Acceptable file formats: FASTA

  Options:
   -i, --input-bloom=FILE     load bloom filter from FILE
   -l, --leniency=N           leniency for exon-exon juction detection [10]
   -f, --lfactor=N            leniency calculated as ceil(FPR*lfactor*k)
   -s, --lsplicesignals=csv   Comma separated 3' splicesignals \n"
   -r, --rsplicesignals=csv   Comma separated 5' splicesignals \n"
       --allexons             Also output exons on either ends of contigs\n"
       --help	                display this help and exit
       --version	            output version information and exit

Example:

./FindExons -i Bfilter.bf <Transcriptome assembly file (TransABySS FASTA file)>
   
./FindExons -i Bfilter.bf -s AG,TG,AC,GC,GG -r GT,TT,AT  <Transcriptome assembly file (TransABySS FASTA file)>
   
./FindExons -i Bfilter.bf --allexons <Transcriptome assembly file (TransABySS FASTA file)>

Output: A FASTA file of exons with headers in this format -

>TranscriptName_startCoordinate_Endcoordinate

Run MakeSplicegraph.py with the putative exons FASTA file outputted by FindExons(confident-exons.fa)

Example:

python MakeSplicegraph.py -i <Putative exon in FASTA format> -o <Splicegraph-outputfile>

Run Graphviz ccomps to obtain Splice sub-graphs

Example:

ccomps <Splicegraph DOT file from MakeSplicegraph.py> -o <splicegraph_subgraph>

OR Run FindSubcomponents.py -w to generate a DOT file with Splicegraph sub-graphs

The script can also generate a file with mappings of transcripts to genes (denoted by random numbers) with the -m option By default, it generates a file with mappings of putative exons to genes (also denoted by random numbers)

Usage: FindSubcomponents.py [-h] -g DOTFILE [-m] [-w]

Find graph subcomponents and write output

optional arguments:
  -h, --help            show this help message and exit
  -g DOTFILE, --dotfile DOTFILE
                        Graph DOT file from MakeSplicegraph.py
  -m, --geneMap         Write a file with mappings of transcripts to genes
  -w, --writesplicesubgraphs
                        Write splice subgraphs to DOT file