Help about setting up TopHat

lewewoo · 11-14-2011, 03:36 PM

The sequencing people in house told us that the library fragments range from 200-500 bp with a major band at ~280bp, so for tophat setting, how could we set up -r? we have 100 cycles for read1 and 100 cycles for read2.

tophat -r 80 index/index_mm9 R1.fastq R2.fastq

thanks!

(previously, the sequencing results from Illumina just informed us 75bp for paired end reads, and total fragments are 300bp, so we set up -r as 150)

Jon_Keats · 11-14-2011, 04:57 PM

Perfectly correct way to do it. If you want to do it based on the actual read data, align a subset 1-5 million reads to a transcriptome reference with bowtie or bwa, run picard insert metrics to get the true mean, median, standard deviation of each library.

There is code for this on my introduction thread
(http://seqanswers.com/forums/showthr...?t=4589&page=4)

lewewoo · 11-14-2011, 06:52 PM

Thanks a lot!
Tophat said "-r" is an expected number, that means Tophat just requires a "-r" number to estimate the mean number and your script could do a better measurement for the mean, right?
Thanks for sharing!

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11-14-2011, 03:36 PM	#1
lewewoo Member Location: Moon Join Date: Apr 2011 Posts: 60	Help about setting up TopHat The sequencing people in house told us that the library fragments range from 200-500 bp with a major band at ~280bp, so for tophat setting, how could we set up -r? we have 100 cycles for read1 and 100 cycles for read2. tophat -r 80 index/index_mm9 R1.fastq R2.fastq thanks! (previously, the sequencing results from Illumina just informed us 75bp for paired end reads, and total fragments are 300bp, so we set up -r as 150) Last edited by lewewoo; 11-14-2011 at 03:38 PM.

11-14-2011, 04:57 PM	#2
Jon_Keats Senior Member Location: Phoenix, AZ Join Date: Mar 2010 Posts: 279	Perfectly correct way to do it. If you want to do it based on the actual read data, align a subset 1-5 million reads to a transcriptome reference with bowtie or bwa, run picard insert metrics to get the true mean, median, standard deviation of each library. There is code for this on my introduction thread (http://seqanswers.com/forums/showthr...?t=4589&page=4)

11-14-2011, 06:52 PM	#3
lewewoo Member Location: Moon Join Date: Apr 2011 Posts: 60	Thanks a lot! Tophat said "-r" is an expected number, that means Tophat just requires a "-r" number to estimate the mean number and your script could do a better measurement for the mean, right? Thanks for sharing!