tophat parameter setting

[hollandorange]

Dear All,

I was using tophat to find the splicing junctions but could not find any. I used reads with 36nt in length and 50 x estimated coverage.

my command line:

tophat -r 128 -i 20 -I 40000 -o tophat36_200_50 gene_index read1.fastq read2.fastq .

In the result, I can see lots of potential junctions when visualizing coverage.wig file, but there was 0 junction detected. I was wondering why? I read the tophat paper saying the island extraction using D>=300 and user can change this parameter. which is the command line option to change it? or more suggestions?

Thanks in advance. I also attached a part of the coverage.wig file.

Yanju

chr5 14679 14680 8493
chr5 14680 14681 7339
chr5 14681 14682 6146
chr5 14682 14683 5051
chr5 14683 14684 3897
chr5 14684 14685 2755
chr5 14685 14686 1807
chr5 14686 14687 907
chr5 14687 14688 2
chr5 14688 16447 0
chr5 16447 16483 1
chr5 16483 19963 0
chr5 19963 19964 790
chr5 19964 19999 791
chr5 19999 20000 1
chr5 20000 21797 0
chr5 21797 21798 1
chr5 21798 21799 2
chr5 21799 21833 3
chr5 21833 21834 2
chr5 21834 21835 1
chr5 21835 22187 0
chr5 22187 22223 1
chr5 22223 22248 0
chr5 22248 22249 3
chr5 22249 22284 4
chr5 22284 22285 1
chr5 22285 25435 0
chr5 25435 25436 119
chr5 25436 25437 1072
chr5 25437 25438 2010
chr5 25438 25439 2969