I have been learning tophat since lastweek. I will really appreciate if you give me tips.
1.
Let me describe one example first.
timepoint1: lane1.fastq, lane2.fastq, lane3.fastq, lane4.fastq (all of data come from plant1.)
timepoint2: lane1.fastq, lane2.fastq, lane3.fastq, lane4.fastq (all of data come from plant2.)
timepoint3: lane1.fastq, lane2.fastq, lane3.fastq, lane4.fastq (all of data come from plant3.)
I am thinking of run the below commands.
"tophat -o [output] -G [gff] [reference] t1_lane1.fastq",
"tophat -o [output] -G [gff] [reference] t1_lane2.fastq",
"tophat -o [output] -G [gff] [reference] t1_lane3.fastq",
"tophat -o [output] -G [gff] [reference] t1_lane4.fastq",
"tophat -o [output] -G [gff] [reference] t2_lane1.fastq",
"tophat -o [output] -G [gff] [reference] t2_lane2.fastq",
"tophat -o [output] -G [gff] [reference] t2_lane3.fastq",
"tophat -o [output] -G [gff] [reference] t2_lane4.fastq",
"tophat -o [output] -G [gff] [reference] t3_lane1.fastq",
"tophat -o [output] -G [gff] [reference] t3_lane2.fastq",
"tophat -o [output] -G [gff] [reference] t3_lane3.fastq",
"tophat -o [output] -G [gff] [reference] t3_lane4.fastq".
As a next step, I am going to run cufflinks in order to assemble
t1_lane1, t1_lane2, t1_lane3, t1_lane4 into timepoint1,
t2_lane1, t2_lane2, t2_lane3, t2_lane4 into timepoint2,
t3_lane1, t3_lane2, t3_lane3, t2_lane4 into timepoint3,
As a final step, I am going to run cuffdiff to see the differential expression across different timepoints.
Do you think I understand correctly the workflow of tophat, cufflinks and cuffdiff?
2. According to the manual of tophat, the command line looks like "tophat -o [output] -G [gff] [reference] read1.fastq,read2.fastq,...,readN.fastq".
I am so confused about when multiple reads are put together into one command line.
- When is "tophat -o [output] -G [gff] [reference] read1.fastq,read2.fastq,...,readN.fastq" used?
- When is "tophat -o [output] -G [gff] [reference] read1.fastq", ..., "tophat -o [output] -G [gff] [reference] readN.fastq" used?
It will be really helpful if you give some specific design of experiment to make clear understanding.
1.
Let me describe one example first.
timepoint1: lane1.fastq, lane2.fastq, lane3.fastq, lane4.fastq (all of data come from plant1.)
timepoint2: lane1.fastq, lane2.fastq, lane3.fastq, lane4.fastq (all of data come from plant2.)
timepoint3: lane1.fastq, lane2.fastq, lane3.fastq, lane4.fastq (all of data come from plant3.)
I am thinking of run the below commands.
"tophat -o [output] -G [gff] [reference] t1_lane1.fastq",
"tophat -o [output] -G [gff] [reference] t1_lane2.fastq",
"tophat -o [output] -G [gff] [reference] t1_lane3.fastq",
"tophat -o [output] -G [gff] [reference] t1_lane4.fastq",
"tophat -o [output] -G [gff] [reference] t2_lane1.fastq",
"tophat -o [output] -G [gff] [reference] t2_lane2.fastq",
"tophat -o [output] -G [gff] [reference] t2_lane3.fastq",
"tophat -o [output] -G [gff] [reference] t2_lane4.fastq",
"tophat -o [output] -G [gff] [reference] t3_lane1.fastq",
"tophat -o [output] -G [gff] [reference] t3_lane2.fastq",
"tophat -o [output] -G [gff] [reference] t3_lane3.fastq",
"tophat -o [output] -G [gff] [reference] t3_lane4.fastq".
As a next step, I am going to run cufflinks in order to assemble
t1_lane1, t1_lane2, t1_lane3, t1_lane4 into timepoint1,
t2_lane1, t2_lane2, t2_lane3, t2_lane4 into timepoint2,
t3_lane1, t3_lane2, t3_lane3, t2_lane4 into timepoint3,
As a final step, I am going to run cuffdiff to see the differential expression across different timepoints.
Do you think I understand correctly the workflow of tophat, cufflinks and cuffdiff?
2. According to the manual of tophat, the command line looks like "tophat -o [output] -G [gff] [reference] read1.fastq,read2.fastq,...,readN.fastq".
I am so confused about when multiple reads are put together into one command line.
- When is "tophat -o [output] -G [gff] [reference] read1.fastq,read2.fastq,...,readN.fastq" used?
- When is "tophat -o [output] -G [gff] [reference] read1.fastq", ..., "tophat -o [output] -G [gff] [reference] readN.fastq" used?
It will be really helpful if you give some specific design of experiment to make clear understanding.
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