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how to use bed file as a input for htseq-count

Hi,

htseq-count only accept BAM/SAM. I downloaded a bed format RNA-seq data (strand-specific, single end). it has around 19 million tags. Bam file is not provided (only sra) and when I converted bed by bedToBam and ran htseq-count:

no_feature    17528277
ambiguous    1977
too_low_aQual    0
not_aligned    0
alignment_not_unique    0

a lots of reads discarded!!!

bed format:

chr1    100002008       100002043       0       0       -
chr1    100002027       100002062       0       0       -

bedToBam

0       16  chr1        100002009       255     35M     *       0      0        *       *
0       16  chr1        100002028       255     35M     *       0      0        *       *

I really appreciate if somebody could please help me

rna-seq
• 3,601 views • link
updated by Ram Ram • written by R • R

What does 0 0 means between end and strand. Was it introduced by error when typing ? Can you paste a few lines (excluding header) may be 2-3 from the BAM file that was generated by bedtoBam.

• 1 views • link
updated by Ram Ram • written by Ashutosh Pandey Ashutosh Pandey

I added few lines from bed and bam

• 1 views • link
written by R • R

Try -s reverse and see if that improves things.

• 0 views • link
written by Devon Ryan Devon Ryan

I used it when I ran the command

• 0 views • link
written by R • R

Then try -s yes. It's likely that either the strandedness setting is incorrect or the annotation and reference against things were mapped don't match. It's usually easiest to just look at things in IGV.

• 1 views • link
written by Devon Ryan Devon Ryan

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