HTSeq: A Python framework to work with high-throughput sequencing data

[Simon Anders]

Plans, yes -- but I'm so overwhelmed with other things that it might take a while till I get to that. Sorry.

[GenoMax]

Hi Simon,

We have been encountering an error with htseq-count (v. 0.6.1p1) on alignment files that have SAM v.1.4 tags.

The specific error is

Unknown CIGAR code 'X' encountered

I found an old request about this error which does not appear to have been implemented in htseq-count yet : http://sourceforge.net/p/htseq/support-requests/22/

Are there plans to add support for SAM v.1.4 tags to htseq-count? For now we have been working around this by generating SAM v.1.3 tags.

Thanks.

[gringer]

From the looks of it, the read locations are zero-based and open-ended on the right, so don't include the "end" location in the list of base locations. For an end location of -2, that's a bit more concerning, otherwise it's just business as usual for how these things are done.

[patchper]

I don't know where to report bugs so I posted here.
I think the start_d and end_d feature of GenomicIntervals have bugs.
With a SAM file below as sample.sam:
read1 0 chr 1 40 7M * 0 0 ATGGCGT AAAAAAA
read2 16 chr 1 40 7M * 0 0 ATGGCGT AAAAAAA

and:
>>> read1,read2 = list(itertools.islice(HTSeq.SAM_Reader('sample.sam'),2))

>>> read1
<SAM_Alignment object: Read 'read' aligned to chr:[0,7)/+>

>>> read2
<SAM_Alignment object: Read 'read2' aligned to chr:[0,7)/->

>>> read1.iv.start,read1.iv.end,read1.iv.start_d,read1.iv.end_d
(0, 7, 0, 7)

>>> read2.iv.start,read2.iv.end,read2.iv.start_d,read2.iv.end_d
(0, 7, 6, -1)

the end_d of read2 ended with a negative coordinate! This behavior is mentioned in document, but I think it is a bug rather than a feature.

[zjrouc]

i just got a result from ht-seq. It showed that my interesting gene has 7 counts in the alignment. However, from the view of IGV, i could easily identify much more counts than 7 on this gene. My alignment is from STAR, and i used more stringent parameters to control the multiple alignment, which means there should not be any multiple aligned reads in the output. I am really confused about this.

Any suggestion?

[cdias]

Hi,
I'm having trouble installing HTSeq.
I pretty much followed the instructions, but when I try to run it, I get the following error:

.local/lib/python2.7/site-packages/HTSeq-0.6.1-py2.7-linux-x86_64.egg/HTSeq/_HTSeq.so: undefined symbol: PyUnicodeUCS2_DecodeUTF8

Any help is appreciated!

[Simon Anders]

Multiprocessing can work only with objects that can be pickled. SAM_Alignment cannot be pickled. I suspect this may be the reason it does not work.

I suppose you are right. makes perfect sense.

Objects must implement __getstate__ and __setstate__ functions in order to be pickled/unpickled. Would it be difficult to implement these functions?

I don't think so. I would just need to find the time to do it.

All one needs to do is take all the slots defined for the class in _HTSeq.SAM_Alignment, pack them into a tuple for __getstate__ and write them back for __setstate__.

[superpyrin]

Yes, the C code is machine generated, but if you look at the pyx files which it is generated from, it should be clearer. Have a look at:
http://www-huber.embl.de/users/ander...c/contrib.html

Multiprocessing can work only with objects that can be pickled. SAM_Alignment cannot be pickled. I suspect this may be the reason it does not work.

Objects must implement __getstate__ and __setstate__ functions in order to be pickled/unpickled. Would it be difficult to implement these functions?

[Simon Anders]

Yes, the C code is machine generated, but if you look at the pyx files which it is generated from, it should be clearer. Have a look at:

http://www-huber.embl.de/users/anders/HTSeq/doc/contrib.html

[superpyrin]

You asked me this before, but Ididn't reply, right? Sorry about that, I was a bit overwhelmed with mails.

The bad news is: I have no clue why it does not work; I have never worked with the multiprocessing package. But I agree that it would be nice if this worked.

Maybe somebody else here has some idea?

my guess is that it is something trivial, perhaps missing implementation of some part of dictionary/list interface in on of HTSeq data structures. I tried to look into HTSeq source, but it seems to be machine generated code, so I quickly gave up.

[Simon Anders]

You asked me this before, but Ididn't reply, right? Sorry about that, I was a bit overwhelmed with mails.

The bad news is: I have no clue why it does not work; I have never worked with the multiprocessing package. But I agree that it would be nice if this worked.

Maybe somebody else here has some idea?

[superpyrin]

Running HTSeq in parallel

Hello,

I am trying to process mapped reads in parallel. However, when using pool of workers (multiprocessing package), I get following error:

Traceback (most recent call last):
File "testmp.py", line 15, in <module>
out = pool.map(repr, iter(sa), chunksize=1)
File "/usr/lib/python2.7/multiprocessing/pool.py", line 228, in map
return self.map_async(func, iterable, chunksize).get()
File "/usr/lib/python2.7/multiprocessing/pool.py", line 531, in get
raise self._value
AttributeError: 'NoneType' object has no attribute 'name'


Running in serial fashion (using just built-in 'map' function) works fine.

Do you know what can be wrong here?

Thank you.

---

Here is a simple script to reproduce the error. I am using HTSeq ver 0.6.1, Python 2.7.3, 64bit ubuntu 12.04

import HTSeq
from multiprocessing import Pool
# this works for me
map(repr, sa)

# this does not work
pool = Pool(processes=1)
sa = HTSeq.SAM_Reader('test.sam')
out = pool.map(repr, sa, chunksize=1)
print list(out)

[emolinari]

Yep, that output looks good.

samtools sort -n gives the same result as Unix sort -k 1,1 on the SAM file.

That's reassuring!!!
thanks indeed

Manu

[fanli]

Yep, that output looks good.

samtools sort -n gives the same result as Unix sort -k 1,1 on the SAM file.

[emolinari]

This sorts your alignment by genomic position. You want to sort by read name:

Hi fanli,

thanks for your reply! Meanwhile I had done samtools sort -n on my .bam file, samtools view -h to create a .sam file and run HTSeq again.
I guess it worked, as the sterror file created was way way smaller, and contains this:
100000 sam line pairs processed.
200000 sam line pairs processed.
300000 sam line pairs processed.
500000 sam line pairs processed.
600000 sam line pairs processed.
700000 sam line pairs processed.
800000 sam line pairs processed.
900000 sam line pairs processed.
1000000 sam line pairs processed.
1100000 sam line pairs processed.
1200000 sam line pairs processed.
1300000 sam line pairs processed.
1400000 sam line pairs processed.
1500000 sam line pairs processed.
1600000 sam line pairs processed.
1700000 sam line pairs processed.
1800000 sam line pairs processed.
1900000 sam line pairs processed.
2000000 sam line pairs processed.
2100000 sam line pairs processed.
2200000 sam line pairs processed.
2300000 sam line pairs processed.
2500000 sam line pairs processed.
[...]
31936941 sam line pairs processed.

So I guess this is correct, right?
So I guess my question is: does it make any difference practically if you sort the .bam and then make it .sam or if you sort a .sam file with the command you suggested? I am too ignorant to appreciate the difference!

Thanks again!!!