[Xi Wang]

Hi guys,

I'm new to seqanswers and to alignment. So I started using bowtie as it seems to have a good reputation manual and tutorial.

I am working through the tutorial, I have got to the point where I am supposed to build a new index using the E. coli strain O157:H7 downloaded from ncbi. However when I run the command I get this error

could not open NC_002127.fna

I'm using terminal in Mac OSX 10.5.8. Anybody had the same problem? Am I not putting the NC-002127.fna file in the correct directory?

I would move on in the tutorial however I need to use the build option to create an index for the organism I work on.

Thanks in advance.

Is your file NC_002127.fna in the work directory, which means the current directory where you type the command. If not, please give the path to the file, then the program can find the file.

[varunkilaru]

Out of Memory allocating the offs[] array for bowtie index

Hi Ben
First of all,Bowtie is great. Thanks for that.I am running into a few problems when I was working with the human index.

The command I used was bowtie -C hg19_c reads/e_coli_1000.fq. This fails with the message : Out of Memory allocating the offs[] array for bowtie index.

I am using a 8GB Windows 64 bit processor with a 3 ghz quad core processor. I am not understanding why it is running out of memory. Can you kindly let me know what could be the problem? Thanks a lot and sorry for the inconvenience

[betty]

Bowtie for meta data

hello everyone,
I am using Bowtie for SOLiD based metagenomic mapping. The references are millions of 1kb-length fragments, I don't know wheather it is the reason that index-building spent quite a lot of time (Total time for backward call to driver() for mirror index: 01:56:46).

My former analysis using AB software indicated that most of the reads could aligned to many references, and the mappable reads were not considerable.

For my case, could you give some suggestion on index-building and alignment parameters setting?

Thanks a lot,

Betty

[Xi Wang]

hello everyone,
I am using Bowtie for SOLiD based metagenomic mapping. The references are millions of 1kb-length fragments, I don't know wheather it is the reason that index-building spent quite a lot of time (Total time for backward call to driver() for mirror index: 01:56:46).

My former analysis using AB software indicated that most of the reads could aligned to many references, and the mappable reads were not considerable.

For my case, could you give some suggestion on index-building and alignment parameters setting?

Thanks a lot,

Betty

I think it could be such long time. For the human genome (~ 3Gb and 25 chromosomes), it takes about 3-4 hours.

[Lien]

Ben,
I've tried to read all the posts, but I may have missed this answer if posted.

I'm having problem with running bowtie on a mouse genome dataset.
The error is a large number of reads giving the warning:

Warning: Exhausted best-first chunk memory for read .....

current command line: bowtie -S -p 1 --solexa1.3-quals --un unmapped.fq -m 10 --max maxmapped.fq -n 3 -X 600 /ccmb/CoreBA/Data/BowtieData/mm9 -1 ../s_7_1_sequence.txt -2 ../s_7_2_sequence.txt mm9_align.sam


version: bowtie --version
bowtie version 0.12.3
64-bit
Built on ccmb-comp1.umms.med.umich.edu
Tue Mar 2 12:33:36 EST 2010
Compiler: gcc version 4.1.2 20080704 (Red Hat 4.1.2-46)
Options: -O3
Sizeof {int, long, long long, void*, size_t, off_t}: {4, 8, 8, 8, 8, 8}


Any suggestions would be helpful as I feel that I'm not getting the level of alignment I should be seeing with this data.

Thanks !
Jim

Hello,

I have the same problems using a 64-bit computer ('Warning: Exhausted best-first chunk memory for read HWUSI_ ...; skipping read). My paired-end data are in .txt format. Could this have anything to do with the problem? Otherwise, as I'm only starting to work with these data, I have no clue to other things that could be causing this problem.

Thanks a lot!
Lien

[betty]

mismatches in color space

hi,
I have a doubt about color space mismatches, and I don't know whether my understanding is correct.

I set -C -n 2 -l 20 for SOLiD data alignment. So, it permits at most 2 color space mismatches in the first 20 characters of the read (trimming the tag "T" and the first base).
In the output file, the single mismatch was treated as system error and was ignored. Only the adjacent mismatches which could be correctly explained by SNP were reported.

So, there may be many many single mismatches ignored in Bowtie because of system error? Is there any other consideration besides single and adjacent mismatches?

Any suggestions would be appreciated.

Regards,

Betty

[Lien]

Hello,

I have the same problems using a 64-bit computer ('Warning: Exhausted best-first chunk memory for read HWUSI_ ...; skipping read). My paired-end data are in .txt format. Could this have anything to do with the problem? Otherwise, as I'm only starting to work with these data, I have no clue to other things that could be causing this problem.

Thanks a lot!
Lien

Apparently, it is normal that some reads are skipped because they can't align. Just hope the percentage that is skipped isn't too high!

[Lien]

Hi again,

I performed a paired-end run on the Illumina, with 100bp reads. The original DNA fragments are +/- 200 bp. I have a percentage of about 40% of the reads that failed to align. I think some of the original DNA fragments are smaller than 200 bp, so there would be an overlap between both paired reads. To solve this, I think I would have to change the minimum insert size. But this would mean that this number would become negative (for example -30). Is this possible?

Thanks!

[isharon]

I have a reference genome that is in fact a collection of many contigs whose lengths range between a few hundreds to a few thousands bps. Also most of my reads probably won't align to any of the contigs. Will bowtie work for such case?

Thanks,
Itai

[Subho]

Hi Ben, how different are hg18 and NCBI36 genomes? Genomic co-ordinates for quite a few single nucleotide mutations obtained from RNAseq data based on bowtie hg18 index are not mapping to the same bases on NCBI36.
Thanx,-s

[Ben Langmead]

I have a reference genome that is in fact a collection of many contigs whose lengths range between a few hundreds to a few thousands bps. Also most of my reads probably won't align to any of the contigs. Will bowtie work for such case?

Thanks,
Itai

Yes, it should.
Ben

[Ben Langmead]

Hi Ben, how different are hg18 and NCBI36 genomes? Genomic co-ordinates for quite a few single nucleotide mutations obtained from RNAseq data based on bowtie hg18 index are not mapping to the same bases on NCBI36.
Thanx,-s

My understanding is that they're hg18 and NCBI v36.* are the same. See, e.g., the first question on this faq:

http://genome.ucsc.edu/FAQ/FAQreleases.html

Thanks,
Ben

[guisinmm]

I'm new to NGS, and I'm wondering if Bowtie could be of use to me. I have Illumina reads, and I want to assemble chloroplast genomes. I have built an index using another chloroplast genome, and I am successful in running Bowtie using default parameters. Sequence differences should be relatively minor in coding regions, but they could be significant in non-coding regions. I'll need to tweek the parameters accordingly, I guess. So, here are my questions: Would Bowtie be useful for me? How would you suggest I set -n or -v to allow for between species sequence differences? Once I get the output, how can I map these to the reference to create contigs? I see the location of the alignment in the output, but what step do I take next to work with the output?

Thanks for the help!
guisinmm

[seq_GA]

Hi,
I have downloaded fastq file format from ncbi (Illumina reads) and trying to use Bowtie and I get the following error.

Error: reads file does not look like a FASTA file
terminate called after throwing an instance of 'int'

Let me know how to overcome this problem. Thanks.

[seq_GA]

I managed to figure out the problem. I was using -f option for input file instead of -q option for inputfastq format. Thanks.