OVERVIEW

SQUID is designed to detect transcriptomic structural variations from RNA-seq alignment.

INSTALLATION

SQUID requires boost, Gurobi Optimizer, bamtools.

Boost

Download boost from (http://www.boost.org), and decompress. For Max and Unix/Linux, you don't need to build or compile anything, but just change the BOOST variable in Makefile into the decompressed boost path.

Gurobi Optimizer

Download Gurobi Optimizer from (http://www.gurobi.com/downloads/download-center), and get the license. Change GUROBI variable in Makefile into the path of gurobi folder (the folder having include and lib in it). You may need to change LDLIBS of gurobi into the version you download, for example, -lgurobi65 if you download gurobi version 65*, or -lgurobi70 if you download version 70*.

Gurobi doesn't support gcc5 currently. If you are using gcc5, you need to add -D_GLIBCXX_USE_CXX11_ABI=0 to CXXFLAGS in Makefile

Bamtools

Download bamtools from (https://github.com/pezmaster31/bamtools). Follow the instructions from (https://github.com/pezmaster31/bamtools/wiki/Building-and-installing) to build bamtools. Change the BAMTOOLS variable in Makefile into the path of bamtools directory (the folder having include and lib in it).

Compile SQUID

Go into SQUID folder and make.

cd squid
make

On Mac, you need to additionly run the following command to dynamicly linking dependent libraries:

export DYLD_LIBRARY_PATH=<bamtools_folder>/lib
export DYLD_LIBRARY_PATH=<gurobi_folder>/lib

USAGE

squid [options] -b <Input_BAM> -o <Output_Prefix>

SQUID supports the following options:

Parameters	Default value	Data type	Description
-c		string
-f		string
-pt	0	bool	Phred type: 0 for Phred33, 1 for Phred64
-pl	10	int	Maximum Length of continuous low Phred score to filter alignment
-pm	4	int	Threshold to count as low Phred score
-mq	1	int	Minimum mapping quality
-dp	50000	int	Maximum paired-end aligning distance to be count as concordant alignment
-di	20	int	Maximum distance of segment indexes to be count as read-through
-w	5	int	Minimum edge weight
-G	0	bool	Whether or not output graph file (0 for not outputing, 1 for outputing)
-CO	0	bool	Whether or not output ordering of connected components (0 for not outputing, 1 for outputing)
-TO	0	bool	Whether or not output ordering of all segments (0 for not outputing, 1 for outputing)
-RG	0	bool	Whether or not output rearranged genome sequence (0 for not outputing, 1 for outputing)

OUTPUT SPECIFICATION

• <Output_Prefix>_sv.txt: a list of predicted TSV in bedpe format. This is the main output of SQUID. All positions in the file are 0-based. Each columns represents:
  • chr1: chromosome name of the first breakpoint.
  • start1: starting position of the segment of the first breakpoint, or the predicted breakpoint position if strand1 is "-".
  • end1: ending position of the segment of the first breakpoint, or the predicted breakpoint position if strand1 is "+".
  • chr2: chromosome name of the second breakpoint.
  • start2: starting position of the segment of the second breakpoint, or the predicted breakpoint position if strand2 is "-".
  • end2: ending position of the segment of the second breakpoint, or the predicted breakpoint position if strand2 is "+".
  • name: TSV is not named yet, this column shows with dot.
  • score: Edge weight, or weighted number of reads supporting this TSV.
  • strand1: strand of the first segment in TSV.
  • strand2: strand of the second segment in TSV. example record:

  17 38135881 38136308 17 38137195 38137773 . 5480 + +

  This means the right end (position 38136308) of segment 38135881-38137195 on chr17 is connected to the right end (position 38137773) of segment 38137195-38137773 also on chr17. And the score of this TSV is 5480.

  5 176370330 176370489 8 128043988 128044089 . 328 - +

  This means the left end (position 176370330) of segment 176370330-176370489 on chr5 is connected with the right end (position 128044089) of segment 128043988-128044089 on chr8.
• <Output_Prefix>_graph.txt: genome segment graph, will be output only if -G is set to 1. It has two types of records, nodes (or segment) and edges.
  • node: for each node, the following information are included. ID, start position, end position, label for connected component.
  • edge: for each edge, the following information are included. ID, node id for the first segment, strand of the first segment, node id for the second segment, strand for the second segment, edge weight.
• <Output_Prefix>_component_pri.txt: ordering of each connected component by ILP, will be output only if -CO is set to 1. In this file, ordering of each connected component will be output into a line. Each segment is represented by its id, with a "-" in the front if the ordering suggests the segment should be using its reverse strand.
• <Output_Prefix>_component.txt: ordering of the entire genome segments, will be output only is -TO is set to 1. In this file, each newly generated chromosome will be output into one line. Other spefications are the same as <Output_Prefix>_component_pri.txt.
• <Output_Prefix>_genome.fa: sequence of rearranged genome sequence, where each chromosome corresponds to one line in <Output_Prefix>_component.txt.

EXAMPLE WORKFLOW

Suppose you have the alignment BAM file, and chimeric BAM file generated by STAR (https://github.com/alexdobin/STAR), run SQUID with:

squid -b alignment.bam -c chimeric.bam -o squidout

Or a combined BAM file of both concordant and discordant alignments generated by BWA (http://bio-bwa.sourceforge.net/) or SpeedSeq (https://github.com/hall-lab/speedseq), run SQUID with

squid --bwa -b combined_alignment.bam -o squidout

An example can be run be downloading the sample data (sampledata.tgz) from (https://cmu.box.com/s/e9u6alp73rfdhfve2a51p6v391vweodq) into example folder, and decompress it with

tar -xzvf sampledata.tgz

Run SQUID command in example/SQUIDcommand.sh. Or if you want to test the workflow of STAR and SQUID, make sure STAR is in your path, and run bash script example/STARnSQUIDcommand.sh.

cd example
./SQUIDcommand.sh
./STARnSQUIDcommand.sh