- Install Python 2.7.x, NumPy 1.8.0 and SciPy 0.15.1
- Install pysam 0.8.4
- Install STAR_2.5.2b for the run without alignment files provided. The star index of human and mouse can be downloaded through the following link http://www.mimg.ucla.edu/faculty/xing/custom_track/shaofang/SQUID/STARhg19.tgz http://www.mimg.ucla.edu/faculty/xing/custom_track/shaofang/SQUID/STARmm10.tgz
- Install kallisto 0.43.0 for the run without FPKM files provided. The kallisto index of human and mouse can be downloaded throught the following link http://www.mimg.ucla.edu/faculty/xing/custom_track/shaofang/SQUID/hg19_Ensemble74 http://www.mimg.ucla.edu/faculty/xing/custom_track/shaofang/SQUID/mm10_Ensemble78
- Install cufflinks 2.2.1 for the fun without FPKM and fastq files provided.
- Install DEXSeq 1.16.10 to run differential spliced intron analysis
The source code can be directly called from Python.
Run SQUID with provided fastq files
python ../SQUID.py --GTF ./test.gtf --fastq ./test_R1_1.fq:./test_R1_2.fq,./test_R2_1.fq:./test_R2_2.fq,./control_R1_1.fq:./control_R1_2.fq,./control_R2_1.fq:./control_R2_2.fq --check_len true --index_kallisto ./kallisto/test --index_star ./star --anchor 8 --length 100 --lib first --read P --Cal All --c1 0.05 --p 1 --Comparison ./Comparison --analysis U -o ./bam1 --resume true
Please note that this run require high RAM due to STAR alignment
Run SQUID with provided alignment files and fastq files
python ../SQUID.py --GTF ./test.gtf --fastq ./test_R1_1.fq:./test_R1_2.fq,./test_R2_1.fq:./test_R2_2.fq,./control_R1_1.fq:./control_R1_2.fq,./control_R2_1.fq:./control_R2_2.fq --align ./test_R1.bam,./test_R2.bam,./control_R1.bam,./control_R2.bam --check_len true --index_kallisto ./kallisto/test --anchor 8 --length 100 --lib first --read P --Cal All --c1 0.05 --p 1 --Comparison ./Comparison --analysis U -o ./bam2
Run SQUID with provided alignment files and FPKM files
python ../SQUID.py --GTF ./test.gtf --align ./test_R1.bam,./test_R2.bam,./control_R1.bam,./control_R2.bam --FPKM transcript_exp.txt --anchor 8 --length 100 --lib unstrand --read P --Cal All --c1 0.05 --p 1 --Comparison ./Comparison --analysis U -o ./bam3
--align:
s1.bam/s1.sam[,s2.bam/s2.sam]. Mapping results for all of samples in bam/sam format. Different samples are separated by commas
--GTF:
The gtf file
--fastq:
s1_1.fq[:s1_2.fq][,s1_1.fq[:s2_2.fq],...]. The raw sequencing reads in fastq or fastq format that is required to call kallisto to calculate FPKM value
--index_star:
The path to the star index that is required to do the alignment using STAR
--FPKM:
A file providing the FPKM value for each sample, the first column is transcript ID with the following column being the FPKM value for each sample. If it is not provided, kallisto will be called to calculate FPKM value
--index_kallisto:
The path to the kallisto index that is required to run kallisto from raw reads
--o/--output:
The output directory. The default is current directory
--check_len:
Whether to generate new fastq files to with equal read length. The default value is false
--l:
Estimated average fragment length. The parameter to run kallisto with default value of 200
--s:
Estimated standard deviation of fragment length. The parameter to run kallisto with default value of 100
--update:
Whether to update the attributes of introns using spliced reads. The default is false
--lib:
The library type with choices of unstrand/first/second. The details are explained in the parameter of library-type in tophat2. The default is unstrand
--read:
The sequencing strategy of producing reads with choices of P(paired end) or S (single end). The default is P
--length:
The read length of sequencing reads in nucleotide. The default length is 100
--anchor:
The anchor length in nucleotide. The program will only count reads spanning junctions with at least this anchor length on each side. The default is 8
--Cal:
Which part of the program user choose to run, the choices are All/count/DSI. All means run the whole program, count means only run the PI value calculation part, DSI means only run the differential analysis of spliced introns. The default is All
--Comparison:
A file providing the sample pairs to calculate the differential RI level.The format should be column 1(name of comparions), column 2 (sample 1 order in the align files,replicates separated by commas), column 3 (sample 2 order in the align files,replicates separated by commas), column 4 (optional, if present as 'pool', the replicates are combined together in rMATS calculation). If absent, the step of calculation of differential spliced introns will be skipped
--analysis:
Type of rMATS analysis to perform. analysisType is either P or U. P is for paired analysis and U is for unpaired analysis. Default is U
--c1:
The cutoff of splicing difference of rMATS run using Junction method. The cutoff used in the null hypothesis test for differential splicing. The default is 0.0001
--p:
The number of threads used to run rMATS. The default is 1;
--F_deltaPI:
The cutoff of delta to output differential spliced introns.The default is 0.05;
--F_FDR:
The cutoff of combined FDR to output differential spliced introns.The default is 0.1;
--resume:
Whether to resume previous run. The default is false.
PI_Junction:
Inclusion counts divided by the sum of inclusion and skipping junction counts
PI_Density:
The observed counts divided by the expected counts of the intron
Notes: $n denotes the number of samples provided in SQUID run
###Result### The folder contains the final output of four types of files: intron_PI.txt, Diff_$comparison_intron_PI.txt, Decrease_$comparison_intron_PI.txt,Increase_$comparison_intron_PI.txt.
$comparison denotes the label of comparison performed
intron_PI.txt is the file containing the info of annotated introns
Diff_$comparison_intron_PI.txt containing the differential spliced introns info of all annotated introns
Decrease_$comparison_intron_PI.txt containing the differential spliced introns info of the introns with decreased PI in sample2
Increase_$comparison_intron_PI.txt containing the differential spliced introns info of the introns with increased PI in sample2
Common columns in all files:
Intron_id: Intron Id representing the chromosome position, start and end
Gene_id: Gene id of intron residing genes
Strand: Strand of intron residing genes
Chr: Chromosome name of introns
Start: Start coordinate of introns
End: End coordinate of introns
Annotated: Whether this intron was annotated in the gtf file as retained intron event
Attributes: One of the four intron types, U/E/I/EI
Inclusion_counts: Inclusion counts separated by commas
Skipping_counts: Skipping counts separated by commas
Inclusion_length: Effective inclusion length
Skipping_length: Effective skipping length
PI_Junction: PI_Junction value separated by commas
Observed_counts: Observed counts separated by commas
Expected_counts: Expected counts separated by commas
PI_Density: PI_Density separated by commas
Extra columns in Diff_$comparison_intron_PI.txt, Decrease_$comparison_intron_PI.txt,Increase_$comparison_intron_PI.txt.
PValue_rMATS: p-value from rMATS
FDR_rMATS: FDR from rMATS
PValue_DEXSeq: p-value from DEXSeq
FDR_DEXSeq: FDR from DEXSeq
Combined_FDR: FDR of rank product test of rMATS and DEXSeq
###test### A folder contains test files to run the program
###log.SQUID### Log file for running SQUID pipeline
###gtf_files### An intermediate folder contains different types of gtf files to run the program. Use mouse genome as examples.
Mus_musculus.Ensembl.GRCm38.78.gtf: the ensemble gtf files. This file should be provided by user.
Exon_Mus_musculus.Ensembl.GRCm38.78.gtf: the gtf file contains exons only
Intron_Mus_musculus.Ensembl.GRCm38.78.gtf: the gtf file contains intron only
Intron_Annotated_Mus_musculus.Ensembl.GRCm38.78.gtf: the gtf file contains the attributes whether the intron was annotated as retained introns in the original gtf files
Intron_attri_Mus_musculus.Ensembl.GRCm38.78.gtf: the gtf file contains the attributes whether the intron was overlapped with Exon and whether the intron is overlapped with other intron.
###fq### An intermediate folder contains all of the fastq with equal read length files
###align### An intermediate folder contains all of the alignment files
###counts### An intermediate folder contains all of the count files
count_intron.txt: a file contains the counts for all of the introns
column 1: Intron Id representing the chromosome position, start and end.
column 2: Gene id
column 3: Strand
column 4: Comma separated logical values to denote Whether this intron was intron (E) or intron (I) based on read info
column 5: Chromosome name
column 6: Start coordinate
column 7: End coordinate
column 8: Inclusion counts at 5' splice sites for sample 1
column 9: Skipping counts at 5' splice sites for sample 1
column 10: Inclusion counts at 3' splice sites for sample 1
column 11: Skipping counts at 3' splice sites for sample 1
column 12: Skipping counts of the intron for sample 1
column 13: counts lying in the intron for sample 1
column 14~6*(n+1)+1: more counts for samples 2~n
count_exon.txt: a file contains the counts for all of the exon in each gene
column 1: Gene id
column 2~ n+1: Gene counts in samples 2~n
column n+2: Gene strand
column n+3: The chromosome of the gene residing
count_all_Density.txt: a file contains the observed counts and expected counts for all of the introns
column 1: Intron id representing the chromosome position, start and end.
column 2: The length of introns
column 3~n+2: The observed counts
column n+3~2n+2: The expected counts
Total.txt: a file contains total number of unique reads in each sample
column 1~n: Total number of unique reads in samples 1~n
###FPKM An intermediate optional folder contains the result of FPKM result and gene expression files for the squid run without gene expression file provided.
kallisto_$n
The result of kallisto of each sample
transcript_exp.txt
The file is FPKM file that contains FPKM value for each gene.
column 1: Gene ID
column 2~n+1: FPKM value for samples
###rMATS_files### An intermediate folder contains all of the rMATS input and output files
###DEXSeq_files### An intermediate folder contains all of the DEXSeq input and output files
Yi Xing xingyi@email.chop.edu
Shaofang Li shaofangli@cau.edu.cn
If you found a bug or mistake in this project, we would like to know about it. Before you send us the bug report though, please check the following:
- Are you using the latest version? The bug you found may already have been fixed.
- Check that your input is in the correct format and you have selected the correct options.
- Please reduce your input to the smallest possible size that still produces the bug; we will need your input data to reproduce the problem, and the smaller you can make it, the easier it will be.
Copyright (C) 2016 University of California, Los Angeles (UCLA) Shaofang Li, Yi Xing
Authors: Shaofang Li, Yi Xing
This program is free software: you can redistribute it and/or modify it under the terms of the GNU General Public License as published by the Free Software Foundation, either version 3 of the License, or (at your option) any later version.
This program is distributed in the hope that it will be useful, but WITHOUT ANY WARRANTY; without even the implied warranty of MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the GNU General Public License for more details.
You should have received a copy of the GNU General Public License along with this program. If not, see http://www.gnu.org/licenses/.
Q: How much RAM does SQUID require?
A: It depends on the input you used in the SQUID. If the input is alignment files and FPKM files, 4G is enough for the human genome. If FPKM is not provided, FPKM will be generated by Kallisto which may takes 1G extra RAM. If alignment files is not provided, STAR will generate alignment files that may takes up to 40G to for the human genome.
Q: Are the observation counts always larger than the expected counts?
A: There is a small portion of introns that have larger observation counts and PI_Density is assigned as 1 in these introns.
Q: Does the index in the comparison file 0-based or 1-based?
A: The index in the comparison file is 1-based.
Q: Can SQUID resume previous run?
A: Yes, SQUID will resume previous run by setting parameter resume as true. Please delete the intermediate files that you’d like to regenerate.
Q: Do the reads in the fastq files have to be the same length?
A: No. If the reads in the fastq files do not have equal length, please set parameter check_len to true to generate new fastq files with equal length.