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  • gigigou
    replied
    I have solved the problem.
    In step 2, the file's name should be specified as the pl says.
    But I think it is a little inconvenient, so I modified the pl to let it work more efficiently.

    Leave a comment:


  • gigigou
    replied
    Originally posted by GenoMax View Post
    Provided you have done what others have already suggested before ...

    (and this may be a stupid suggestion) but are you sure the data you have is from your samples (i.e. there was no mix-up at the place where got it sequenced).
    I think the data is not the problem cause I have used other align tools to do the alignment, and they all give perfect results. So I think the problem is BWA, maybe I didn't specify the parameters properly, I'll try to solve it.
    Thank you!

    Leave a comment:


  • gigigou
    replied
    Originally posted by westerman View Post
    Since we do not have access to your files it is hard to troubleshoot problems. But a couple of pieces of general advice in troubleshooting a problem:

    1) Do not use any non-standard options; e.g., try 'aln' and 'samse' without any parameters.

    2) Try a smaller input file. Especially one with a known-to-be-good sequence. In other words take a bit of your ecoli reference and make it into a color-space read.

    Good luck in solving this.
    Thank you.
    All the parameters are specified as the manual says.
    I think the input file is small enough, only 50000 reads.
    Thank you for your help.
    I'll look into it.

    Leave a comment:


  • GenoMax
    replied
    Provided you have done what others have already suggested before ...

    (and this may be a stupid suggestion) but are you sure the data you have is from your samples (i.e. there was no mix-up at the place where got it sequenced).

    Leave a comment:


  • westerman
    replied
    Since we do not have access to your files it is hard to troubleshoot problems. But a couple of pieces of general advice in troubleshooting a problem:

    1) Do not use any non-standard options; e.g., try 'aln' and 'samse' without any parameters.

    2) Try a smaller input file. Especially one with a known-to-be-good sequence. In other words take a bit of your ecoli reference and make it into a color-space read.

    Good luck in solving this.

    Leave a comment:


  • gigigou
    replied
    Originally posted by westerman View Post
    I agree with swbarnes, after creating the color-space index then give bwa the color-space input file and not a nucleotide file.
    In the fsatq file that converted from csfasta, the reads are still in color space, as above.

    Leave a comment:


  • gigigou
    replied
    Originally posted by swbarnes2 View Post
    You are making a color space index, but converting your reads to fastq? Are you sure that's right?
    The reads are converted into fastq format, but they are still in color space
    @SRR001354.lite.sra.1 461_28_1048
    T23331323333132332323133333332333232
    +
    %%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%

    I tried to input the original csfasta file, it processed 50000 reads, but still with no reads mapped.

    50000 + 0 in total (QC-passed reads + QC-failed reads)
    0 + 0 duplicates
    0 + 0 mapped (0.00%:-nan%)
    0 + 0 paired in sequencing
    0 + 0 read1
    0 + 0 read2
    0 + 0 properly paired (-nan%:-nan%)
    0 + 0 with itself and mate mapped
    0 + 0 singletons (-nan%:-nan%)
    0 + 0 with mate mapped to a different chr
    0 + 0 with mate mapped to a different chr (mapQ>=5)

    Leave a comment:


  • westerman
    replied
    I agree with swbarnes, after creating the color-space index then give bwa the color-space input file and not a nucleotide file.

    Leave a comment:


  • swbarnes2
    replied
    Originally posted by gigigou View Post
    What's the general steps to align a set of reads to ref?
    Is the flowing right?
    1.build index -> bwa index -c -a is ecoli.fa
    2.convert csfasta and correspond qual file into fastq
    3.Find the SA coordinates of the input reads -> bwa aln -n 3 -t 4 -M 3 -c ecoli.fa t_ecoli.fastq >map.sai
    4.Generate alignments in the SAM format given single-end reads -> bwa samse -n 1 ecoli.fa map.sai t_ecoli.fastq >map.sam
    Do as the above, I get a sam file with no reads mapped. All the 25000 items have flag 4.
    What's more, I have 50000 reads in the input fastq file, why does it only process 25000?
    If I delete one read form the fastq file, BWA will process 24999 reads. Why?
    And what is the bwasw for?
    Thank you !
    You are making a color space index, but converting your reads to fastq? Are you sure that's right?

    Leave a comment:


  • gigigou
    replied
    Originally posted by jimmybee View Post
    I think in step 3 you are missing the output character. It should be:

    bwa aln -n 3 -t 4 -M 3 -c ecoli.fa t_ecoli.fastq > map.sai

    See if that helps
    Oh, sorry that I missed the ">" in the post, but I did add it in the command line.
    So it can't be the reason.
    Actually I do get the align file, but in the file I find there is no mapped items. All the 25000 reads are reported unmapped.
    thank you all the same

    Leave a comment:


  • jimmybee
    replied
    I think in step 3 you are missing the output character. It should be:

    bwa aln -n 3 -t 4 -M 3 -c ecoli.fa t_ecoli.fastq > map.sai

    See if that helps

    Leave a comment:


  • gigigou
    started a topic why can't I get alignment with BWA?

    why can't I get alignment with BWA?

    What's the general steps to align a set of reads to ref?
    Is the flowing right?
    1.build index -> bwa index -c -a is ecoli.fa
    2.convert csfasta and correspond qual file into fastq
    3.Find the SA coordinates of the input reads -> bwa aln -n 3 -t 4 -M 3 -c ecoli.fa t_ecoli.fastq >map.sai
    4.Generate alignments in the SAM format given single-end reads -> bwa samse -n 1 ecoli.fa map.sai t_ecoli.fastq >map.sam
    Do as the above, I get a sam file with no reads mapped. All the 25000 items have flag 4.
    What's more, I have 50000 reads in the input fastq file, why does it only process 25000?
    If I delete one read form the fastq file, BWA will process 24999 reads. Why?
    And what is the bwasw for?
    Thank you !
    Last edited by gigigou; 09-06-2012, 05:54 AM.

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