bedtools questions ! can't sort/index by samtools after bedToBam command

Question

[zhaizhaoyu7]

Hi all ! After I use STAR to alignment , I got the BAM file , this BAM file could index successfully by samtools , but after converting this BAM file to BED with command

 bedtools bamtobed -i sample.bam > sample.bed

and then,I use bedtobam convert it to bam

 bedtools bedtobam -i /sample.bed -g genome > new_sample.bam

I want to index this new BAM ,so I run

samtools index new_sample.bam

But I got this error finally

[E::hts_idx_push] Unsorted positions on sequence #1: 248926319 followed by 127352

samtools index: failed to create index for "new_sample.bam"

Why? Is there any wrong that makes this happen? How I can fix this problem ?

And I try to use samtools view to see what happen,but I got this,here is the command and error

samtools view -h new_sample.bam | head -10

@HD VN:1.0 SO:unsorted

@PG ID:BEDTools_bedToBam

VN:Vv2.29.2 [main_samview] truncated file.

And samtools sort failed either

samtools sort: truncated file. Aborting

BTW:samtools version=1.9 bedtools version=2.29.2

I new to NGS ,any reply is greatly appreciated!

[Istvan Albert]

the command you posted does not look right, that slash in front of sample.bed indicates a file in the root of you computer, most likely it is not what you ran:

bedtools bedtobam -i /sample.bed -g genome > new_sample.bam

overall something is not quite right with your sam file. Investigate the SAM file you've created.

samtools view new_sample.bam

the follow the errors if you get any

[zhaizhaoyu7]

Thanks for your reply!

I cut my absolute path because it's too long ,sorry that I left a '/'.

Apart from this,I didn't got any SAM file , I only have the BAM file in coordinate after I ran STAR.And I can index this BAM successfully. That's what I'm confused,there are only two steps:bedtools bamtobed and bedtobam,then I got the wrong BAM .

Here is samtools view I got without -h

[main_samview] truncated file.

Any reply will be appreciated.