Question: SAM to BAM conversion problem after HISAT2.
0
gravatar for fmerkal
14 months ago by
fmerkal20
USA/Lowell/University of Massachusetts Lowell
fmerkal20 wrote:

Hello,

I recently used HISAT2 to align my paired-end reads to a reference genome. The command I used was hisat2 -q -p 4 -x path_to_index -1 reads_1.fastq -2 reads_2.fastq -S out.sam. This produced a SAM file that was ~14 GB and had headers that matched the ones I looked up online for this type of file.

I am having trouble converting my SAM output file to a BAM file using the following command samtools view -bS out.sam > out.bam. I should mention that the samtools version I am using is 1.4.1 and the syntax should be ok. The out.bam file is only 45 bytes and when I try to check it with samtools flagstat samtools flagstat out.bam, I get the following errors:

[W::sam_read1] parse error at line 1
[bam_flagstat_core] Truncated file? Continue anyway.

I have tried looking into this problem but couldn't find an answer that related specifically to my issue. Any help would be greatly appreciated. Cheers, Fjodor

rna-seq software error • 914 views
ADD COMMENTlink modified 14 months ago by eennadi0 • written 14 months ago by fmerkal20

Output if head out.sam and samtools view out.bam | head? Did you use something like nohup during alignment?

ADD REPLYlink modified 14 months ago • written 14 months ago by ATpoint35k

Hi, I did not use nohup. I am running hisat2 on a cluster and I received an output describing the alignment rate (overall alignment ~92%). I did not see other files, like the various .log files you would get if you ran Tophat. The output I get with head out.sam is listed below.

@HD     VN:1.0  SO:unsorted
@SQ     SN:MT   LN:16775
@SQ     SN:W    LN:6813114
@SQ     SN:Z    LN:82529921
@SQ     SN:1    LN:197608386
@SQ     SN:2    LN:149682049
@SQ     SN:3    LN:110838418
@SQ     SN:4    LN:91315245
@SQ     SN:5    LN:59809098
@SQ     SN:6    LN:36374701

When I run samtools view out.bam | head, I get

[W::sam_read1] parse error at line 1
[main_samview] truncated file.
ADD REPLYlink modified 14 months ago • written 14 months ago by fmerkal20

note how above you run

samtools view out.bam

which is empty so will, of course, raise an error. See what

samtools view out.sam

prints

I think your BAM file is empty, for whatever reason, you would need to run the conversion again and see why it failed. I think it ran of out "something" and truncated the BAM file but the SAM file should be fine.

ADD REPLYlink modified 14 months ago • written 14 months ago by Istvan Albert ♦♦ 84k

You mean samtools sort -o out_sorted.bam out.sam worked for you? My problem is this generated .sam file which contains some alignments but when i ran samtools view -bS out.sam > out.bam both the out.sam and out.bam seem to corrupt the alignments such that nothing is both files

Curious to know how you went straight from samtools sort -o out_sorted.bam out.sam Did you sort bam first and then used sort first? Can you please be more detailed in your solution?

ADD REPLYlink written 14 months ago by eennadi0

Please open a new question for this if you have trouble after using the search function and reading the manual. If opening a question, please make sure to include all commands you used.

ADD REPLYlink written 14 months ago by ATpoint35k
1
gravatar for fmerkal
14 months ago by
fmerkal20
USA/Lowell/University of Massachusetts Lowell
fmerkal20 wrote:

Hello, I think I might have solved the problem. I used samtools sort to sort the SAM file and output it to BAM format with the command samtools sort -o out_sorted.bam out.sam. This seems to have created a BAM file that has a reasonable size ~2.9 GB (relative to the SAM file) and that I can view with samtools view out_sorted.bam.

This post was particularly helpful - htseq-count: error when reading beginning of SAM/BAM file. I still don't understand why it wasn't working when I used samtools view -bS out.sam > out.bam, but it seems fine for now. Thank you for all your help :)

ADD COMMENTlink written 14 months ago by fmerkal20
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