[ieuanclay]

Hi Ben,

I have seen this in 0.9.9 and 0.9.9.1 (x64), m.musculus ncbi36 and 37 indices (offrates 2,3,4,5). Args were -q --solexa-quals -a --unfq ... -p 2/3/6 . Input was only ~1/2 Mb of fastq reads! Worrying because the real input will be >2Gb! In all cases the combination of -a and --nostrata seemed to be causing the problem, because with only -a the footprint was as expected.

I will try 0.9.9.2 today and get back to you - i checked yesterday and noticed you had improved the --best behaviour (thanks!) so i'll try it with that too.

Thanks again,

Ieuan

## update ##
0.9.9.2 does not have the same problem, and has roughly the rsaem footprint for both -a and -a --nostrata. Any idea what the change was? Either way I am happy!

[Ben Langmead]

Hi Ieuan,

Can you tell me which Bowtie version/index/reads/arguments you're using? Also, could you give the same experiment a try with version 0.9.9.2 (just released)? I'll take a look.

Thanks,
Ben

[ieuanclay]

Hi Ben,

Thanks for your advice earlier, post-processing of results is now working well!

However I have found that using --all isn't a problem, until i specify --nostrata as well which causes me to rapidly run out of memory (>32Gb) and get the std::bad_alloc error I think a few people mentioned earlier. I had built indices with smaller -o, but even using a high offrate, small footprint index has the same problem. Any suggestions (other than not using --nostrata...)?

THanks again,

Ieuan

[Ben Langmead]

Yes, that's correct. An ambiguous characters in the read is "charged" as a mismatch, which can affect whether the alignment is legal according to the alignment policy.

[SillyPoint]

Ben, your answer to What Da Seq's question a couple of days ago about ambiguity characters was:

A result of Bowtie's indexing strategy is that alignments involving one or more ambiguous reference characters (N, -, R Y, etc.) are considered invalid by Bowtie, regardless of the alignment policy. This is true only for ambiguous characters in the reference; alignments involving ambiguous characters in the read are legal, subject to the alignment policy.

Do I interpret "legal, subject to the alignment policy" to mean they are accepted, but counted as mismatches subject to the -n limit?

Thanks,

--SP

[tniranj1]

You're probably right... I'm using for too old an operating system. Hopefully I will have results-oriented questions for you in the future, rathe than technical installation stuff. ;-)
Thanks again,
-TiN

[Ben Langmead]

Darn! Sorry to waste your time.

I can testify that the gcc4 and gcc346 versions on Leopard (from the developer's tools) work fine for me, as do the various gcc4 versions I've tried on Linux. I'm sorry that that 3.4.6 doesn't seem to be working under 10.3. I will add it to my TODO list to address some of that problematic SeqAn code before the next release. In the meantime, since 3.4.6 didn't work, waiting for your Leopard computer is the option that has the least chance of wasting more of your time.

Ben

[tniranj1]

I just installed gcc 3.4.6 and changed the etc/profile $PATH to reflect the update. When I ran make again, significantly more SeqAn-1.1 errors popped up (too much to post). There is no suffix to the new g++ file. Should I shoot for gcc4.x or would it be more appropriate to wait until our Leopard computer comes in... I would prefer to start testing with this computer now, though.
Really appreciate the help!
-TiN

[Ben Langmead]

Well, the oldest g++ I've used is 3.4.6, which works without warnings. I just tried 3.2.3 and got a bunch of warnings and errors; mostly in the SeqAn headers. So, yes, if you happen to have a newer g++ version somewhere on your machine then please try that. E.g., try typing g++ then hitting tab to see if there's something called g++4 or g++34 or similar. If there is something called g++34, for example, then make bowtie using 'make GCC_SUFFIX=34'. Let me know if that doesn't work; I can try to fix this in a future version of Bowtie.

Thanks,
Ben

[tniranj1]

I'm using gcc version 3.3 with bowtie 0.9.9.1. Do I need version 4 or higher for g++ in order for installation of bowtie to work, or is 3 sufficient?
Thanks,
-TiN

[Ben Langmead]

What version of g++ do you have (try 'g++ -v') and what version of Bowtie are you trying to compile? Is there another g++ version installed besides the default? I'm not familiar with 10.3, but you can try running g++3 and g++4 and see if either of those work.

Thanks,
Ben

[tniranj1]

Help with installation

I'm new to next-gen sequencing and have started playing around with different alignment tools for data that will soon be coming in to my lab. From what I've heard, Bowtie sounds perfect, and I appreciate the speedy feedback that's been made available to the community.
I do have a slight installation problem. I get the following error during "Make".

SeqAn-1.1/seqan/basic/basic_generated_forwards.h:507: error: parse error before numeric constant
SeqAn-1.1/seqan/basic/basic_generated_forwards.h:761: confused by earlier errors, bailing out
make: *** [bowtie-build] Error 1

I installed the platform-independent version on my Mac (OS 10.3.9... yes, it's old I know, we're upgrading soon). Appreciate any help with resolving this.

-TiN

[Ben Langmead]

Yes, all bowtie-build options are identical in terms of the index's ability to generate alignments (except those that have slight, non-specifics effect like --ntoa or --oldpmap).

[What_Da_Seq]

Thanks Ben. I could not identify an option for "bowtie-build" that is geared towards maximum efficiency (not speed nor memory efficiency) in generating alignments (least amount of non-aligned reads) in the Bowtie alignment.
Your help is appreciated.

Thanks

[Ben Langmead]

The Bowtie indexing step elides stretches of ambiguous bases in the reference. As a result, alignments that overlap an ambiguous base in the reference are never considered "valid" by Bowtie and will not be reported.

This is explained in a couple of paragraphs in the manual that are new as of 0.9.9.1:

A result of Bowtie's indexing strategy is that alignments involving one or more ambiguous reference characters (N, -, R Y, etc.) are considered invalid by Bowtie, regardless of the alignment policy. This is true only for ambiguous characters in the reference; alignments involving ambiguous characters in the read are legal, subject to the alignment policy.

Also, alignments that "fall off" the reference sequence are not considered legal by Bowtie, though some such alignments will become legal once gapped alignment is implemented.