Tophat's Mean Inner Distance between Mate Pairs

Question

[english.server]

Hi all,

In RNA-seq studies , how can I get the info needed for the calculation of "Mean Inner Distance between Mate Pairs" which is a parameter needed by Tophat?

[english.server]

Thank you Andrew for your response. I am sorry to ask this but how can I find read length? Is it equal to InsertSize in SRA database?

[andrew.j.skelton73]

run the fastq file through fastqc, or look at one of the entries in the fastq and figure out how long the line is, each entry is a single read (plus quality score, header, etc)

[english.server]

Thanks again

Answer 1

[andrew.j.skelton73]

I'd generally align to the transcriptome using bowtie2 for each sample, run picard tools (CollectInsertSizeMetrics.jar) on the resulting file. Get the number for MEAN_INSERT_SIZE, then work out MEAN_INSERT_SIZE - (2 x read length)

Answer 2

[Brian Bushnell]

I'd generally just run BBMap, which does not require that parameter, as it calculates it automatically. Of course, I'm somewhat biased, as I wrote BBMap. But I have yet to find a scenario where Tophat is a desirable program to run over the alternatives (BBMap or STAR).

[andrew.j.skelton73]

Agreed, I have to admit I've never used BBMap, but STAR, and HISAT don't require this parameter - It's usually computed internally.